Physiol Rev 94: 859 907, 2014

doi:10.1152/physrev.00037.2013

INTERSTITIAL CELLS: REGULATORS OF SMOOTH

MUSCLE FUNCTION
Kenton M. Sanders, Sean M. Ward, and Sang Don Koh

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada

Sanders KM, Ward SM, Koh SD. Interstitial Cells: Regulators of Smooth Muscle

L
Function. Physiol Rev 94: 859 907, 2014; doi:10.1152/physrev.00037.2013.
Smooth muscles are complex tissues containing a variety of cells in addition to muscle
cells. Interstitial cells of mesenchymal origin interact with and form electrical connec-
tivity with smooth muscle cells in many organs, and these cells provide important
regulatory functions. For example, in the gastrointestinal tract, interstitial cells of Cajal (ICC) and

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PDGFR cells have been described, in detail, and represent distinct classes of cells with unique
ultrastructure, molecular phenotypes, and functions. Smooth muscle cells are electrically coupled
to ICC and PDGFR cells, forming an integrated unit called the SIP syncytium. SIP cells express a
variety of receptors and ion channels, and conductance changes in any type of SIP cell affect the
excitability and responses of the syncytium. SIP cells are known to provide pacemaker activity,
propagation pathways for slow waves, transduction of inputs from motor neurons, and mechano-
sensitivity. Loss of interstitial cells has been associated with motor disorders of the gut. Interstitial
cells are also found in a variety of other smooth muscles; however, in most cases, the physiological
and pathophysiological roles for these cells have not been clearly defined. This review describes
structural, functional, and molecular features of interstitial cells and discusses their contributions
in determining the behaviors of smooth muscle tissues.

I. INTRODUCTION 859
II. ICC IN THE GASTROINTESTINAL TRACT 860
III. DEVELOPMENT AND PLASTICITY OF ICC 885
IV. INTERSTITIAL CELLS IN THE URINARY... 890
V. INTERSTITIAL CELLS IN THE... 894
VI. SUMMARY AND CONCLUSIONS 895
I. INTRODUCTION
Interstitial cells is a morphological term denoting a variety
of cells of differing origins and phenotypes occupying spaces
within the interstitium between the cells most prominent in
defining a given tissue. In smooth muscle tissues fibroblasts,
mast cells, macrophages, and interstitial cells of Cajal meet this
definition. While mainly considered structural or immune cells
by many morphologists, interstitial cells have come into prom-
inence because they drive or contribute to the normal func-
tions of smooth muscle organs, and remodeling or loss of these
cells can lead to a variety of motor disorders. This review
describes the physiology of the fibroblast-like classes of inter-
stitial cells, which can include interstitial cells of Cajal (ICC),
ICC-like cells, Cajal-like cells, fibroblast-like cells and teleo-
cytes in various anatomical descriptions of smooth muscle
tissues (138, 213, 231, 292, 297, 322, 326, 342, 369, 389).
There is a continuum of morphology in this group of cells,
with some cells having abundant rough endoplasmic reticu-
lum (ER), no basal lamina, no caveolae, and assuming a mor-
phology attributed typically to fibroblasts. Other cells assume
a more muscle-like appearance with less rough ER, but abun-
dant smooth ER, prominent or even complete basal laminae,
and caveolae (318, 376). Interstitial cells can form gap junc-
tions with each other and with neighboring smooth muscle
cells and can generate and conduct electrical signals that reg-
ulate smooth muscle excitability. Interstitial cells serve as pace-
maker cells, propagation pathways for regenerative electrical
events that cannot be propagated actively by smooth muscle
cells, transducers of inputs from motor neurons, and stretch
receptors. For historical reviews of the morphology and func-
tions of interstitial cells, the reader is referred to a monograph
by Lars Thuneberg (369) that reviews more than 200 morpho-
logical studies of muscle-like or fibroblast-like interstitial cells
and a previous physiological review of electrical rhythmicity in
visceral smooth muscles and role of ICC as pacemakers (326).
Work on interstitial cells in gastrointestinal (GI) muscles has
dominated this field of investigation because there were im-
portant experimental opportunities that could be exploited.
Interstitial cells in the tunica muscularis of GI muscles have
distinct morphological features (98, 213, 369), mice with mu-
tations in the protooncogene Kit have reduced populations of
ICC in specific regions of the GI tract, and Kit mutants develop
striking functional phenotypes (45, 167, 250, 378, 404, 417).
Immunolabeling with antibodies against c-Kit has become a
standard means for identification of Cajal-like cells (404) in
a variety of organs. However, many studies of tissues outside
the gut have encountered difficulties in labeling a distinct class
of interstitial cells (other than mast cells) with this technique,
and Kit mutants neither lacked the cells suspected to be inter-

0031-9333/14 Copyright 2014 the American Physiological Society 859

 SANDERS ET AL.
stitial cells nor displayed functional defects. Thus progress in
understanding the functions of interstitial cells in non-GI mus-
cles has been slower. The discussion in this review will begin
by reviewing highlights of research on ICC in the GI tract and
then focus on progress made on this class of cells in other
smooth muscle organs. Recent progress on a second class of
interstitial cells, known for decades in the morphological liter-
ature as fibroblast-like cells, will also be discussed. These
cells are labeled specifically in several smooth muscles by an-
tibodies against platelet-derived growth factor receptor
(PDGFR) (174, 207), and this has provided an important
means of accessing these cells in physiological and genomic
investigations.
II. ICC IN THE GASTROINTESTINAL TRACT
A. Structural Features of ICC and PDGFR
Cells and Networks
An important feature of GI interstitial cells (ICC and
PDGFR cells; see FIGURE 1 and TABLE 1 for clarification of
the terminologies used to describe these cells and their specific
anatomical localizations) is electrical coupling to smooth mus-
860 Physiol Rev VOL 94 JULY 2014 www.prv.org
cle cells. ICC and smooth muscle cells express a variety of gap
junction proteins (68, 136, 337), but little is known about the
nature and regulation of the gap junctions between interstitial
cells and smooth muscle cells. Gap junctions facilitate commu-
nication between interstitial cells and smooth muscle cells
(FIGURE 2). Electrical connectivity between interstitial cells
and smooth muscle cells in GI muscles causes the tunica mus-
cularis to behave, in fact, as a multicellular electrical syncy-
tium. We have referred to this functional structure as the
smooth muscle cell/ICC/PDGFR cell (SIP) syncytium (209,
330; see FIGURE 3). Changes in electrical conductances in one
type of SIP cell affect the electrical excitability of the other
coupled cells. Spontaneous pacemaker activity generated by
ICC conducts to smooth muscle cells and drives electrical slow
waves and phasic contractions, and neural inputs to ICC and
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PDGFR cells can conduct to smooth muscle cells and mod-
ulate contractile behavior. The integrated behavior of SIP cells
defines what has been referred to classically as myogenic
regulation of GI motor function. However, with current
knowledge, the term myogenic is too limited to define a major
division of GI regulation, and we suggest replacement of this
term with SIPgenic to refer to the complex and integrated
regulatory processes operating beneath the level of neural and
hormonal regulation. It is now recognized that remodeling or
FIGURE 1. AC: c-Kit ICC-MY in mouse,
monkey, and human gastric antrums, re-
spectively. Note similarities in the struc-
tural organization of ICC in all three spe-
cies. DF: c-Kit ICC-MY (D, green) and
PDGFR-MY (fibroblast-like) cells (E, red)
are distinct populations of cells. These pan-
els show cells from murine colon. Merged
images from D and E are shown in F. GI:
intramuscular ICC (ICC-IM; G, c-Kit is green)
in the monkey gastric fundus. ICC-IM form
close associations with enteric motor nerve
processes (nNOS motor neuron processes
are shown in H, red). I: merged image of G
and H. Scale bar in F applies to DF, and
scale bar in I applies to GI. (Authors are
grateful to Dr. Masaaki Kurahashi for im-
ages in DF and Dr. Peter Blair for images in
B and GI.)
 INTERSTITIAL CELLS IN SMOOTH MUSCLES

Table 1. Nomenclature and localizations of interstitial cells in the tunica muscularis of the GI tract
PDGFR- PDGFR-
Organ Subregion ICC-MY ICC-IM ICC-SEP ICC-SM ICC-SS MY IM Reference Nos.

Esophagus Skeletal muscle ? ? ? ? 316

Smooth muscle ? ? ? ? ? 73
Lower esophageal ? ? ? ? 408, 432
sphincter
Stomach Fundus CM * 35, 36, 45
LM
Corpus CM * 44
LM *
Antrum CM * 35, 36, 44, 82, 174,
LM * 265, 279, 305
CM *

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Pylorus ? ? 234, 392, 396, 408
LM
Small intestine Duodenum DMP * ? ? 19, 298
CM *
LM
Jejunum DMP * 36, 167, 174, 317, 376,
CM * 378, 387, 404
LM
Ileum DMP * 44, 100, 113
CM *
LM
Ileo-cecal CM ? ? ? 266
sphincter LM
Colon Proximal CM * 29, 36, 44, 99, 174, 182,
LM 220, 243, 319, 323,
343, 375, 387
Distal CM * 220
LM
Internal anal * ? 61, 62
sphincter

ICC and PDGFR cells are as observed by c-Kit and PDGFR immunoreactivity, respectively. Presence and
relative abundance are denoted by or , respectively. References are representative examples, not an
exhaustive listing; see text for further details. ICC-MY are from a network of cells between the circular and
longitudinal muscle layer and have a pacemaker role in stomach, small bowel, and colon. ICC-MY have been
called ICC-AP or ICC-MP by some authors (see text); however, these terms are misleading because ICC-MY are
not physically within or a component of the myenteric (Auerbachs) plexus. ICC-IM are spindle-shaped intra-
muscular cells lying in muscle bundles between muscle cells and in close apposition to varicose processes of
enteric motor neurons. ICC-IM have also been called ICC-CM or ICC-LM to designate the muscle layer in which
the ICC-IM were observed. ICC-IM are referred to as ICC-DMP in small intestine due to the clustering of these
cells among the nerve process of the deep muscular plexus. Like ICC-IM, the ICC-DMP are closely associated
with varicosities of enteric motor neurons. ICC-SM lie along the submucosal surface of the circular muscle
layer. These have a pacemaker function in the colon. ICC-SS lie at the subserosa of the colon. *Species-
dependent distributions: presence, absence, or low abundance depending on species. Generally found in
larger animals including humans but not in lab rodents.

loss of any component of the SIP syncytium or loss of connec-
tivity between SIP cells can lead to abnormal motor behaviors
in GI organs.
While the activity of the SIP syncytium is highly integrated in
GI organs, knowledge about the functions of SIP cells has
benefitted from studies of specific cell types. Electrical coupling
makes it very difficult to deduce the specific functions of one
component in intact tissues. Even with mutant animals lacking
specific elements of the SIP syncytium, it is still difficult to
ascertain the specific functions of each cellular component.
Dispersion of the tunica muscularis with proteolytic enzymes
yields a mixture of cells. Smooth muscle cells are abundant and
relatively easy to identify by their shapes, but interstitial cells
are more rare and difficult to identify with certainty (typically
amounting to 10% of cells within a given volume of muscle)
and display morphologies that overlap with other cells in the
dispersion. Cultures derived from explants or cells dispersed
from smooth muscle tissues contain a variety of cells, and if the
cells contact each other, gap junctions can form. As in tissues,

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 SANDERS ET AL.
C1
N is 2 m. Inset: higher magnification of the gap
2 al. (183), with kind permission from Springer
it again becomes problematic to maintain voltage control in
voltage-clamp studies or to know which cell has generated a
particular response. Another serious problem is that ionic con-
ductances that define the functions of SIP syncytium cells are lost
in cell cultures (436). Therefore, it is difficult to see the value of cell
cultures for studies of the native behaviors of SIP cells.
Genetically engineered mice with cell-specific expression of
fluorescent reporters have become important tools for studies
of SIP cells (221, 311, 419, 436). SIP cells from reporter strains
of animals can be identified unequivocally for electrophysio-
logical and imaging studies, and fluorescent activated cell sort-
ing (FACS) cells can be used to collect purified populations of
cells for comprehensive sequencing of transcriptomes. Expres-
sion of inducible Cre recombinase in specific SIP cells has been
accomplished and provides a powerful new technique for fu-
ture studies of interstitial cells (131, 200).
Another suggestive anatomical feature of interstitial cells is
their close associations with nerve varicosities, identified orig-
inally by electron microscopy (160, 318, 397). In the case of
ICC, this relationship was noted by Santiago Ramn y Cajal
(47), and he suggested that ICC were a type of peripheral
neuron that connects motor neurons to local effector cells,
such as smooth muscle cells. We know now that ICC are not
neurons and of mesodermal origin (238, 379, 409, 430). Close
associations between neurons and ICC were subjects of note in
many classical electron microscopic examinations of these
cells (73, 98, 179, 318). PDGFR cells are also closely asso-
ciated with terminals of neurons (36, 221, 434); however, the
very close areas of apposition (20 nm) between nerve vari-
cosities and ICC described in many species have not been
described for PDGFR cells. Close associations between in-
tramuscular ICC (ICC-IM) are relatively easy to identify in
transmission electron micrographs in experimental animals
and human GI muscles and have been described as synapse-
like. Close contacts might indicate innervation of ICC by
motor neurons. If neurotransmitters are released into regions
of close association, very high concentrations might be
achieved in the tiny postjunctional volumes, and transduction
of neural inputs may tend to be focused at the sites of close
contact. High junctional concentrations might also increase
the rates of metabolism and/or reuptake of neurotransmitters
and metabolites.
862 Physiol Rev VOL 94 JULY 2014 www.prv.org
FIGURE 2. Close connections between nerve
varicosity and ICC (C1) and gap junction with
smooth muscle cell in rat gastric antrum (183).
Mitochondria, rough endoplasmic reticulum,
and caveolae (arrowheads) are plentiful in ICC.
Gap junction exists with adjacent smooth muscle
cell (arrow). Very close contact is formed be-
tween nerve varicosity (N) and the ICC. Scale bar
junction between ICC and smooth muscle cell.
Scale bar in inset is 0.2 m. [From Ishikawa et
Science and Business Media.]
Close connections have also been described between nerve
varicosities and smooth muscle cells (73, 264, 397), but
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these junctions appear either to be more rare or, at least,
have been deemphasized or gone unmentioned in many
morphological reports. The physical proximity of motor
neurons to interstitial cells led to much speculation about
the role of these cells in transducing neural inputs; however,
functional innervation is much more controversial and dif-
ficult to demonstrate (see sect. IIF).
ICC have an abundance of mitochondria, moderately well-
developed Golgi, caveolae, and rough and smooth ER. The
perinuclear region is often densely packed with mitochon-
dria and cisternae of ER running along the plasma mem-
brane. The spaces between ER and the plasma membrane
create tiny volumes in which localized Ca2 transients may
regulate plasma membrane ion channels and other func-
tions of interstitial cells, such as pacemaker activity and
responses to neurotransmitters. ICC have caveolae and a
basal lamina in many regions of the gut, but these features
are typically lacking in PDGFR cells. Microtubules and
thin and intermediate filaments are observed in ICC, but
myosin thick filaments are not often found (320). Another
difference between ICC and PDGFR cells is that the lat-
ter are typically less electron dense than ICC within the
same thin sections. A great abundance of rough ER is com-
monly found in PDGFR cells compared with ICC, and
this feature was considered fibroblast-like by electron
microscopists. As mentioned previously, gap junctions are
formed between ICC and PDGFR cells and smooth mus-
cle cells (158, 159). While PDGFR cells tend to display
similar ultrastructure features in various organs and in dif-
ferent species, ICC display a variety of structural and ultra-
structural characteristics between species and organs, par-
ticularly in the abundance of caveolae and completeness
and prominence of their basal laminae (211). However,
there are not clear differences in ultrastructure that can be
extrapolated to all regions. Some investigators have de-
scribed ICC as specialized smooth muscle cells (88, 98, 179,
376). For example, ICC in the deep muscular plexus of the
small intestine express enteric actin, an actin isoform as-
sociated with GI smooth muscle cells (334), smooth muscle
myosin light chains, and sparse distribution of thick fila-
ments (376). In other studies, ICC are described as more
 INTERSTITIAL CELLS IN SMOOTH MUSCLES

ICC-IM
NB
PDGFR

*
SMC *

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B Innervation of SIP syncytium

Interstitial cells

PDGFR+-IM Kit+ (ICC-IM) Varicosity

Enteric motor neuron

SMC

Gap junctions
FIGURE 3. Structures of the SIP syncytium. A: electron micrograph (originally provided by Professor Teru-
masa Komuro) showing three types of cells in close proximity to nerve bundles (NB) and varicosities of enteric
motor neurons (*): smooth muscle cells, ICC, and PDGFR cells. These cells are electrically coupled (gap
junctions not shown in this image), forming an electrical syncytium known as the SIP syncytium. In some cases,
very close contacts (20 nm; black arrow in inset) can be found between nerve varicosities and ICC or smooth
muscle cells. Scale bars are 0.5 m in main image and 0.2 m in inset. B: depiction of SIP syncytium (with
dotted line showing approximate cut of a thin section such as shown in A). Smooth muscle cells, ICC, and
PDGFR cells are arranged around projections of excitatory and inhibitory enteric motor neurons. [Redrawn
from Sanders et al. (329). Copyright 2010 The Authors. Journal compilation Copyright 2010 The Physiological
Society.]

fibroblast-like (66, 113, 212, 309, 314). Cells more like
smooth muscle have more complete basal laminae and
prominent caveolae; these features are more sparse or lack-
ing in cells with a fibroblast-like appearance.
Most studies of ICC have been performed on mammalian
species and limited primarily to common laboratory ani-
mals and humans. Recent, morphological reports describe
ICC in the GI tract of zebrafish (14). The morphological
features of these cells are similar in description to their
mammalian counterpart; however, caveolae were not ob-
served in either ICC or smooth muscle cells. ICC in ze-
brafish can also be identified by antibodies for c-Kit.
Ultrastructural characteristics are useful for identification
of ICC and PDGFR cells, but the technical demands of
transmission electron microscopy (TEM) make this tech-
nique suboptimal for efficient characterizations of intersti-
tial cell distributions and clinical assessments of the state of
interstitial cells in human muscles (where frequently ade-
quate fixation to preserve identifying features is difficult to
achieve).
1. Histological and immunomarkers for ICC
For many years interstitial cells were difficult to investigate
because these cells represent only a fraction of the total cells

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 SANDERS ET AL.
in the tunica muscularis, and there were no specific labels
for these cells. Several histological stains were used to high-
light ICC, such as methylene blue (366, 369), Champy-
Maillet zinc iodide-osmic acid (59, 206, 321), Golgi (silver
impregnation) staining (47, 206), rhodamine 123 (402),
and NADH diaphorase (95, 420). None of these techniques
was highly specific for interstitial cells, and successful stain-
ing was accomplished in only some regions of the gut and in
only certain species (315). These limitations led one author
to summarize various histological methods for identifying
interstitial cells as capricious (58). For many years, the
most reliable method for identification of interstitial cells
was TEM, and a major obstacle in morphological studies
was the inability to confirm that cells stained by histological
techniques and imagined with light microscopy were the
same cells identified by ultrastructural criteria with TEM.
The ability to image cells with light and electron microscopy
was an advantage of the NADH diaphorase technique
(420). A study of the dog small intestine explored the nature
and abundance of intermediate filaments in ICC within the
deep muscular plexus (ICC-DMP) and discovered that vi-
mentin filaments were predominant (376). Vimentin be-
came the first immunolabel for interstitial cells and was
used widely in many organs to identify populations of these
cells. However, vimentin may not distinguish between c-
Kit (ICC) and PDGFR cells.
Investigators studying hematopoietic cells and developmental
defects in melanocyte and germ cells in Kit (White spotting; W)
mutants (49, 118) observed that cells in a variety of tissues
express Kit. To study the functions of c-Kit more broadly,
neutralizing antibodies (ACK2) were administered to neonatal
mice (250). These studies found that a population of cells
within the small intestine expressed c-Kit-like immunoreactiv-
ity, and neutralizing c-Kit antibody injections produced severe
defects in intestinal motility described as paralytic ileus. Closer
inspection showed that ileal muscles lacked normal phasic
contractile activity, but instead displayed clusters of arrhyth-
mic contractions. Responses to agonists (bradykinin and ace-
tylcholine) were also altered. Abnormal contractile patterns
were also observed in ileal muscles of W/WV mice. Major
morphological defects were not apparent in enteric nerves or
smooth muscle cells of ACK2-treated mice. Immunolabeling
demonstrated c-Kit cells in the region surrounding the myen-
teric plexus and within the circular muscle layer. Loss of
mechanical rhythmicity and reduction in c-Kit cells af-
ter treatment with neutralizing antibodies suggested that
the cells expressing c-Kit might be ICC. However, tech-
nical difficulties, including the inability to utilize the an-
tibodies for immunocytochemistry or to identify the im-
munolabeled cells as ICC with other histological tech-
niques, made it impossible for these authors to identify
the c-Kit cells as ICC definitively (250).
Positive identification of c-Kit cells in the GI tract was even-
tually revealed by colabeling cells with methylene blue and by
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immunocytochemistry (378). Examples of ICC from mouse,
monkey, and human gastric antral muscles, labeled with anti-
bodies to c-Kit, are shown in FIGURE 1, AC. Treatment of
newborn mice with neutralizing c-Kit antibodies reduced or
abolished the number of c-Kit immunopositive cells, the num-
ber of cells labeled with methylene blue, and cells with ultra-
structural features of ICC (378). Additional studies showed
that the cells in the region of the myenteric plexus (i.e., in the
space between the circular and longitudinal muscle layers) that
labeled with c-Kit antibodies (ICC-MY) were reduced in intes-
tinal muscles of W/WV mice (167, 404). Mast cells are also
labeled with c-Kit antibodies. However, few mast cells are
present in the tunica muscularis of mice kept in clean animal
facilities. Labeling with c-Kit antibodies became the standard
technique for study and identification of ICC, and this tech-
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nique has been widely exploited in developmental studies,
pathological evaluations, and molecular and physiological
studies during the past two decades. Much progress in our
knowledge of ICC has occurred due to studies made possible
by the use of c-Kit antibodies. c-Kit labeling was also adopted
for diagnosis of gastrointestinal stromal tumors (GISTs) after
GISTs were found to express c-Kit and may develop from ICC
due to gain-of-function mutations (discussed further in sect.
IIJ) (149).
Progress on ICC has depended on development or novel
application of technology and reagents (TABLE 2). When
c-Kit antibodies directed at extracellular epitopes were
found to label ICC, we tried immunolabeling of live cells
after enzymatic dispersion. However, the proteolytic en-
zymes used to disperse cells appear to damage the extracel-
lular epitopes. Thus it became desirable to develop reporter
strains with constitutive expression of fluorescent reporters.
Unfortunately, the cell-specific promoters that drive expres-
sion of Kit in ICC are unknown, so it was not possible to
generate a reporter strain with a simple knock-in strategy.
The first strain developed utilized endogenous Kit promot-
ers with a novel green fluorescent protein, ZsGreen,
knocked into the first exon of the Kit gene, creating a null
Kit allele, W(ZsGreen)(418). Heterozygous [W(ZsGreen)/]
had tiny fluorescent dots in Kit expressing cells. This was an
exciting advance, but the expression of ZsGreen was low in
most ICC. Pacemaker ICC expressed only a few green dots
per cell. Another approach utilized a similar strategy to
exploit the endogenous cell-specific promoter and express a
bright green fluorescent protein (copGFP) as the knock-in
reporter (311, 436). Cells of Kit(copGFP/) mice were highly
fluorescent and visible in intact muscles and in cell disper-
sions. It should be noted that cells can even be visualized
readily in living Kit(copGFP/) mice using a fluorescence mi-
cro-endoscope inserted into the abdomen (unpublished ob-
servations). The reporter strain and cells dispersed from the
tunica muscularis have been used in many subsequent phys-
iological and molecular studies (discussed in sections be-
low) and offered, for the first time, an efficient means of
isolating and purifying living ICC.
 INTERSTITIAL CELLS IN SMOOTH MUSCLES

Table 2. Milestones in interstitial cell research in the GI tract

Year(s) Major Investigation and Advances Key Reference Nos.

Before 1985 Morphological studies of ICC; controversies with staining techniques; 47, 98, 113, 179, 309, 314,
EM considered the gold standard for identifying ICC. 369, 434, 435
19801994 Whole muscle experiments; dissection to remove areas rich in 17, 29, 90, 137, 247, 354,
specific ICC populations; use of selective poisons to destroy ICC 355, 370, 402
function.
1989 Isolation of fresh ICC and identification of cells by morphological 235
features; demonstration of intrinsic electrical rhythmicity in
isolated ICC.
1992present Use of c-Kit mutants and c-Kit neutralization to study physiological 45, 167, 250, 377, 378, 400,
functions of ICC. 403, 404
1994present Recognition of Kit immunohistochemistry as specific means to 44, 45, 167, 378, 404
identify and characterize ICC networks.
1994present Demonstration of the role of ICC in electrical rhythmicity. 44, 45, 82, 125, 167, 279,

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378, 404
1996present Demonstration of the role of ICC in motor neurotransmission. 22, 24, 45, 178, 324, 400,
408
1996present Association of ICC loss or growth with pathophysiological conditions. 205, 249, 252, 279, 282
1998 Discovery that c-Kit is a marker for gastrointestinal stromal tumors 78, 145, 149, 151, 269
(GIST); GIST may arise from gain-of-function mutation in c-Kit of
ICC.
19982009 Culturing of ICC for more detailed physiological studies. 208, 368, 410
2004present Use of flow cytometry to quantify and FACS to purify immunolabeled 52, 53, 161, 276, 277
ICC.
2004present Following activation of interstitial cells and propagation of pacemaker 146, 241243, 283, 423
activity with digital imaging of Ca2 transients.
2005 Demonstration of the role of ICC as stretch receptors. 214, 417
2008 Isolation of progenitor cells capable of generating ICC. 16, 248
2009 Reporter strains of mice in which freshly dispersed interstitial cells 221, 311, 418, 436
can be isolated and positively identified in mixed populations of
cells.
20072009 Recognition of the importance of Ano1 (encoded by Tmem16a) in 52, 122, 172, 436
ICC and its role in pacemaking.
2009 Identification of PDGFR as a specific marker for fibroblast-like cells. 174, 177
2011 Isolation of PDGFR cells using a reporter strain and physiological 221
studies suggesting their role in inhibitory neurotransmission.
2013 Cell-specific iCRE strains that allow inducible knockdowns of genes/ 131, 200
proteins in interstitial cells.

References are representative examples to help the reader into the literature and not meant to be an
exhaustive listing; see text for further details.

Despite the progress made with c-Kit immunohistochemis-
try, the search for molecular reagents to identify and ma-
nipulate ICC populations goes on. A striking advance oc-
curred when antibodies developed from GIST tumors (dis-
covered on GIST-1; DOG-1) were found to label GIST
tumor cells and ICC (93, 415). It was later discovered that
DOG-1 corresponded to the gene product of ANO1 (aka
TMEM16a). A gene array screen of transcripts expressed
by small intestinal ICC showed Ano1 to be one of the
most highly expressed genes in ICC (52), and the gene
product of Ano1, a chloride channel known as ANO1, is
expressed robustly and exclusively by ICC throughout
the GI tract (35, 122, 172). An advantage of labeling ICC
in GI muscles with ANO1 antibodies is that mast cells are
not labeled, and therefore, this method may be superior
to c-Kit labeling in conditions in which mast cells infil-
trate the muscle layers or, as in human GI muscles, where
they are often abundant.
2. Histological and immunomarkers for PDGFR
cells
PDGFR cells were referred to as fibroblasts or fibro-
blast-like cells (FLC) by morphologists (213). While TEM
provided a description of the ultrastructural features of
FLC and close appositions made by these cells to other
cellular constituents of the muscularis, it was difficult to
get a full appreciation of the extent and distribution of
these cells throughout the GI tract, the major anatomical
niches they occupy, and the relationships of these cells to
other cells from the limited number of images in the
published literature. Histological techniques were unable

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 SANDERS ET AL.
to identify FLC specifically, and few studies considered a
role for these cells in regulating the behavior of the
smooth muscle tissues.
Two immunomarkers were initially found to label FLC.
The discovery that many GIST express c-Kit (149) was ac-
companied by reports that GIST cells and ICC also express
CD34 (312). Others reported that CD34 cells were adja-
cent to ICC, but represented a distinct population of cells
(289, 387, 388). Cells with CD34-like immunoreactivity in
the human small GI tract were identified as c-Kit FLC.
Thus CD34 became a first immunolabel for FLC, but it was
also recognized that CD34 is not a specific label in the gut
wall, and other mesenchymal cell types express this antigen
(289, 387). The guinea pig GI tract was found to express
small-conductance Ca2-activated K channels (SK2 and
SK3), and SK3 cells were found to be c-Kit cells in the
myenteric region and within the circular muscle layers of GI
muscles (202). Two additional studies reported labeling of
c-Kit cells with antibodies to SK3 in human and mouse GI
muscles (108, 386). SK2 expression was dominant in
smooth muscle cells, but transcripts of SK3 were also found
in these cells (310). These data are consistent with immu-
nohistochemical findings showing punctate SK3 immuno-
reactivity in smooth muscle cells (202). Thus labeling with
antibodies to SK3 is also nonspecific under some circum-
stances.
Significant progress occurred when FLC throughout the GI
tracts of adult animals were found to express PDGFR
(174, 177). PDGFR cells were shown to be FLC by im-
munocytochemistry and robust expression of SK3.
PDGFR cells are distinct from c-Kit ICC and unaffected
in Kit mutants in which ICC are greatly reduced (FIGURE 1,
DI). PDGFR cells form networks adjacent to ICC in the
region of the myenteric plexus and are intertwined with ICC
and processes of enteric neurons in muscle layers (174,
221). Similar distributions of PDGFR cells and inter-
relationships with ICC and enteric motor neurons have
been observed in several mammalian species including hu-
mans (35, 36, 50, 220, 221). Thus PDGFR antibodies
have become a reliable marker for FLC, and most investi-
gators have adopted the terminology of PDGFR cells to
refer to these cells. As more is learned about the phenotype
(chemical coding) of this class of interstitial cells, more spe-
cific terminology may become advantageous, because
PDGFR is expressed by many cells in the body and at
various stages of development. At the present time, specific
markers to distinguish cells within the region of the myen-
teric plexus (PDGFR-MY; FIGURE 1, DF) or within the
muscle bundles of the circular and longitudinal muscle lay-
ers (PDGFR-IM; FIGURE 1, GI) have not been identified.
Developing specific labels for subclasses of PDGFR cells
will allow finer resolution of their phenotypes and functions
in the future.
866 Physiol Rev VOL 94 JULY 2014 www.prv.org
In addition to the obvious advantages of a robust and selec-
tive immunolabel for morphological studies and patholog-
ical evaluations of PDGFR cells, a mouse engineered to
express a histone 2B-eGFP fusion protein driven off the
endogenous promoter for PDGFR (135) was found to
have bright eGFP fluorescence in nuclei of cells immuoreac-
tive for PDGFR antibodies (221). Cells with eGFP recapit-
ulated the distribution of PDGFR cells in the gut with
high fidelity. Constitutive labeling of PDGFR cells
opened the door to molecular phenotyping and physiolog-
ical studies of these cells (as discussed below).
B. Notes on Nomenclature for Interstitial
Cells
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Interstitial cell nomenclature has been somewhat of a bat-
tleground for many years, and it is not yet resolved. Thus it
is important to provide some background on this topic and
a key for readers to understand the varied terminologies
found in the literature to describe these cells. Cajal de-
scribed cells within villi, in the deep muscular plexus of the
small intestine, and in the region of the myenteric plexus
(Auerbachs plexus in the terminology of the day) that had
fusiform cell bodies and long, thin and branching, axonlike
processes. The latter may have been the visual cue to Cajal
suggesting that ICC were a class of primitive neuron. When
Cajals staining techniques were repeated in more recent
studies, it appears that neither glial cells nor fibroblast-like
cells (including ICC) displayed this exact morphology. It
has been suggested that the cells Cajal described may have
been a chimeric structure consisting of glial cells or intersti-
tial cells and contiguous nerve fibers (206). In spite of a
possible disparity between the structures described by Cajal
and the cells now identified as ICC, widespread usage of this
term in the literature makes altering this terminology coun-
terproductive.
Electron microscopy and histochemical techniques made it
clear that ICC occupied several distinctive niches in the GI
tract. Immunolabeling with c-Kit antibodies (e.g., Refs. 35,
44, 183, 377, 378) and ANO1 antibodies (35, 122, 172)
clarified the distribution of ICC in specific locations and
showed that ICC occupy most of the same locations in all
mammals, including humans. Together with functional
studies (described below), certain populations of ICC, dis-
tributed within specific planes in the tunica muscularis, can
be grouped and thus linked by more specific terminology.
We have called ICC within the region between the longitu-
dinal and circular muscle layers, ICC-MY, while others re-
fer to these cells as ICC-AP (while Auerbachs plexus was
still in common usage) or ICC-MP (since myenteric plexus
has come into preferential usage). We favor the term
ICC-MY because these cells are not a component of the
myenteric plexus (as the terms ICC-AP or ICC-MP might
imply). ICC-MY lie between the muscle layers of the myen-
teron and surround myenteric ganglia, but they are not
 INTERSTITIAL CELLS IN SMOOTH MUSCLES
intrinsic to ganglia or the tertiary plexus. Therefore, a label
implying that they are part of the myenteric plexus is mor-
phologically incorrect. Cells within muscle bundles have
been referred to as intramuscular ICC or ICC-IM. Some
authors have used specific terms for ICC-IM within the
circular muscle (ICC-CM) or longitudinal muscle (ICC-
LM); however, no distinctive morphological or functional
data exist to suggest differences between these ICC. A spe-
cialization of ICC-IM is found in the small intestine in
which ICC are densely distributed and intimately associated
with neurons of the deep muscular plexus, and therefore
these cells are referred to as ICC-DMP. Cells at the inner
aspect of the circular muscle layer in the colon and more
sparsely in this location in the stomach are in contact with
the submucosa and therefore called ICC-SM. Cells along
the submucosal surface of the circular muscle layer are
sometimes referred to as ICC-SMP, but these cells are dis-
tinct and well removed from the submucous plexus, so this
designation is misleading. Cells within the septa that sepa-
rate muscle bundles are referred to as ICC-SEP. TABLE 1
provides localizations of the different classes of ICC
throughout the GI tract and attempts to also summarize the
alternative terminologies that have been used in the litera-
ture.
HUMAN MURINE
A Gastric antrum
B Small intestine
10 s 1s
C Colon
Physiol Rev VOL 94 JULY 2014 www.prv.org
When it became possible to immunolabel fibroblast-like
cells with PDGFR antibodies (174), it was recognized that
this population of interstitial cells occupies most of the same
anatomical niches as ICC, and therefore, similar terminol-
ogy was proposed to designate these cells: 1) PDGFR
cells were utilized because identification by chemical coding
is more specific than the vague term fibroblast-like cells,
2) PDGFR-IM was applied to cells within muscle bun-
dles, and 3) PDGFR-MY was applied to cells in the plane
of the myenteric plexus between the circular and longitudi-
nal muscle layers (FIGURE 1, DI; TABLE 1).
C. Role of Interstitial Cells in Pacemaking
and Generation of Electrical Rhythmicity
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In many, but not all, regions of the GI tract, motor patterns
are intrinsically phasic in nature. Spontaneous pacemaker
activity (termed, slow waves; FIGURE 4) drives and times the
phasic contractile behavior. Slow waves occur in continu-
ous rhythmic depolarization/repolarization cycles, and dif-
ferent regions of the bowel display distinct intrinsic slow
wave frequencies (FIGURE 4, AC). Slow waves also vary in
duration from one to several seconds. Interslow wave trans-
CANINE FIGURE 4. Electrical slow waves recorded from
smooth muscle cells in various regions of the gas-
trointestinal (GI) tracts of 3 species. Slow waves
-30
consist of a relatively abrupt upstroke depolarization
followed by a stable or slowly changing plateau
phase. Although frequencies and amplitudes of slow
mV waves vary in different tissues, note the regularity of
duration and frequency in muscles from each region
and species. The activity shown is integrated electri-
-70 cal behavior: a summation of slow waves generated
by ICC, conducted to smooth muscle cells, and re-
1 min sponses of smooth muscle cells. Depolarization of
smooth muscle cells can activate voltage-dependent
Ca2 channels and generate Ca2 action potentials.
0 Ca2 action potentials are elicited in muscles of the
small intestine and colon during periods of enhanced
excitation. Action potentials are not typical in gastric
muscles, except in the terminal portion of the an-
mV
trum and pylorus. Note differences in resting mem-
brane potentials (most negative potentials between
slow waves) in different regions. Regulation of mem-
-55
brane potential sets the excitability of these muscles
10 s and the response elicited by slow wave activity. Dot-
ted lines were drawn at 40 mV, the approximate
threshold for appreciable activation of L-type Ca2
currents and excitation-contraction coupling. Note
that slow waves are membrane potential fluctua-
0 tions from negative potentials where open probability
of voltage-dependent Ca2 channels is very low to
potentials where open probabilities increase and
generate Ca2 entry. Thus slow waves are an intrin-
mV
sic mechanism organizing the motor output of GI
muscles into phasic contractions. Propagation of
slow waves in ICC networks generates segmental
-80 and peristaltic contractions. [From Sanders et al.
20 s (331).]
867
 SANDERS ET AL.
membrane potentials (typically referred to as resting mem-
brane potential or RMP) of GI smooth muscle cells range
between 80 and 40 mV and are generally somewhat
negative to the range of potentials required for substantive
activation of voltage-dependent Ca2 channels. Thus
smooth muscle cells in the SIP syncytium do not generate
Ca2 action potentials at the relatively negative potentials
(RMP) between slow waves.
Activation of Ca2 channels provides the switch for excita-
tion-contraction (E-C) coupling in the tunica muscularis.
Slow waves are oscillations in membrane potential from
base to peak of 10 60 mV. Muscles with quite negative
resting membrane potentials display larger amplitude slow
waves, but these events never overshoot 0 mV. When slow
waves are recorded from smooth muscle cells, they rise from
RMP at a peak velocity of 1 V/s during the upstroke
depolarization and then dwell in a plateau phase for vari-
able durations. At completion of the plateau phase, the cells
repolarize to RMP.
The primary purpose of slow waves is to depolarize smooth
muscle cells sufficiently to activate Ca2 influx. Like vascu-
lar muscles, smooth muscle cells of the GI tract express
voltage-dependent (L-type) Ca2 channels (86, 191, 236,
263). During slow wave depolarizations, the open proba-
bility of smooth muscle Ca2 channels increases and suffi-
cient Ca2 entry occurs to trigger excitation-contraction
coupling (281, 391). The rhythmic pattern of slow waves
naturally organizes the contractile activity of GI muscles
into phasic contractions. In the corpus and antrum of the
stomach, slow wave depolarization alone initiates peristal-
tic contractions, and modulation of slow wave amplitude
and duration regulates the force of contractions. Depolar-
ization for several seconds during the plateau phase of slow
waves is sufficient for enough Ca2 entry to initiate con-
tractions. In the pyloric sphincter, small bowel, and colon,
slow waves bring membrane potentials of smooth muscle
cells to a threshold for Ca2 action potentials, and trans-
membrane potential recordings from muscles of these or-
gans typically show one or more Ca2 action potentials
superimposed upon the peaks of slow waves (92). In these
regions Ca2 action potentials are the smooth muscle cell
response to slow wave depolarizations.
The source and mechanism of slow waves in the GI tract
have been under investigation for decades. Slow waves were
considered to be nonneurogenic and referred to in the liter-
ature typically as myogenic in origin because blocking
nerve action potentials with tetrodotoxin did not abolish
slow wave activity. For many years it was assumed that
smooth muscle cells generated the spontaneous slow wave
activity. The literature before the mid 1980s contained
many studies designed to investigate the mechanism of slow
waves. Some of the more detailed experiments came from
the laboratories of C. Ladd Prosser and Tadao Tomita,
868 Physiol Rev VOL 94 JULY 2014 www.prv.org
using a variety of electrophysiological approaches including
voltage clamping via sucrose gap (65, 275, 374). These
studies, and much of the earlier work, were summarized in
detail by Professor Tomita (373).
After development of single-cell voltage-clamp techniques,
experiments on smooth muscle cells began in several labs,
led by Thomas Bolton, Hiroshi Kuriyama, and the team of
Josh Singer and John Walsh (25, 27, 28, 180, 222, 223,
349, 350). None of these studies, or studies performed in
many other labs, recorded intrinsic electrical activity with
the characteristics of slow waves from smooth muscle cells
(96). Smooth muscle cells generated Ca2 action potentials;
in some cases these occurred spontaneously, or they could
be elicited by depolarization (26). In one study action po-
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tentials with plateau components were recorded from co-
lonic muscle cells of the dog (293); however, these events
were blocked by dihydropyridines, making them dissimilar
to the electrical slow waves of the canine colon (411). The
general conclusion from a multitude of studies was that
smooth muscle cells do not have the ionic apparatus re-
quired to generate slow waves or to be the pacemaker cells
in GI muscles. Thus the term myogenic is incorrect. Another
cell type in GI muscles, ICC, was suggested by morpholo-
gists (98, 369) to be the pacemaker cells because these cells
are electrically coupled to smooth muscle cells. However,
physiological verification of this hypothesis was needed.
Dissection experiments performed on several regions of the
GI tract suggested that discrete regions of tissue provide
pacemaker activity for the bulk of the circular and longitu-
dinal muscle layers. Prosser and colleagues (64, 65) noted
that separation of circular and longitudinal muscles of the
small intestine left circular muscles inactive, but the longi-
tudinal muscle layer retained pacemaker activity. They sug-
gested that slow waves originate in longitudinal muscles
and are amplified and propagated by circular muscles. Later
studies, in which specific strata of the circular and longitu-
dinal muscle layers were isolated, found that slow waves
were generated in the region between the circular and lon-
gitudinal muscle in the stomach and small intestine (17,
137, 185). Similar studies on colon muscles isolated two
pacemaker regions in the dog: the region between the cir-
cular and longitudinal muscle generated a 17 cycle/min elec-
trical rhythm and a thin layer of tissue at the submucosal
surface of the circular muscle layer generated a 6 cycle/min
rhythm (354, 355). Pacemaker activity extended into the
circular muscle layer along the septa that separated the
circular muscle into bundles (412). ICC networks popu-
lated all of the specialized pacemaker regions (29, 30, 412).
The next tasks were to disrupt ICC networks and determine
effects on slow waves and to isolate ICC to determine
whether these cells were capable of pacemaker activity.
Methylene blue can be taken up by ICC in living tissues (a
supravital dye), and the cells are damaged after exposure to
 INTERSTITIAL CELLS IN SMOOTH MUSCLES
high-intensity illumination (370). Damage to ICC by meth-
ylene blue exposure was associated with inhibition of slow
wave activity. Methylene blue also has pharmacological
effects on a variety of cells and causes, among other effects,
significant depolarization of smooth muscle cells (328) and
inhibition of nitric oxide synthase (416). Sufficient depolar-
ization (e.g., with elevated external K to the level pro-
duced by methylene blue) blocked slow waves (328). Thus
the link between methylene blue labeling of ICC and the
hypothesized pacemaker role of ICC was not clarified by
experiments using methylene blue (370). Another supravi-
tal dye, rhodamine 123, accumulates in mitochondria, and
ICC were considered to be a potential target for this dye
because of their abundance of mitochondria. Rhodamine
123 is cytotoxic in some cells, so the effect of this dye on
pacemaker activity was tested. Rhodamine 123 labeled ICC
of the canine colon and blocked slow wave activity without
the problem of substantial depolarization as observed with
methylene blue (402). However, all cells have mitochon-
dria, so cytotoxic effects in cells besides ICC may have been
responsible for the effects of rhodamine 123 on pacemaker
activity.
Dispersion of colonic smooth muscle tissues yielded smooth
muscle cells and a few cells with an odd morphology. These
cells had multiple short processes and a prominent nuclear
region. Ultrastructural examination of the cells with pro-
cesses showed them to have morphological features com-
mon to ICC (i.e., abundance of mitochondria, caveolae, and
lack of thick filaments). Patch-clamp recordings under cur-
rent-clamp conditions showed these cells generated sponta-
A B
C D
-35
mV
-65
2s
Physiol Rev VOL 94 JULY 2014 www.prv.org
neous depolarization with characteristics similar to slow
waves in colonic muscles (235). This was the first direct
evidence that ICC of GI muscles have intrinsic pacemaker
capability.
1. ICC as pacemakers in the small intestine
Erratic contractions of intestinal muscles from mice treated
with neutralizing c-Kit antibodies and in W/WV mutants
suggested defects in electrical pacemaker activity (250).
Further examination of the intestinal muscles of animals
treated with c-Kit antibodies showed that ICC-MY, the
network of ICC in the region identified as the pacemaker
area in small intestine, were largely missing (378). Loss of
ICC correlated with the absence of slow wave activity in
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animals treated with c-Kit antibodies. Small intestinal mus-
cles of W/WV animals also lacked most ICC-MY. Electrical
recordings from these mice showed that normal slow wave
activity occurred in wild-type and heterozygotes, but were
absent in small intestinal muscles of W/WV mice (FIGURE 5)
(167, 404). Additional studies utilizing mutants, such as
Sl/Sld mice, in which the ligand for c-Kit (steel or stem cell
factor) is deficient, also displayed loss of ICC-MY and pace-
maker activity (262, 403). Collectively, these studies, ex-
ploiting several paradigms of ICC loss (e.g., loss of ICC
after development and developmental failure), established
the role of ICC as intestinal pacemakers.
Other than the loss of ICC-MY, no other structural or de-
velopmental defects were noted in small intestines of c-Kit
mutants or animals treated with c-Kit neutralizing antibod-
FIGURE 5. Loss of ICC-MY in small intes-
tine of W/WV mice (404). A: networks of
ICC-MY and ICC-DMP of the small intestine
are shown in this whole-mount labeled with
anti-c-Kit antibodies. B: loss of ICC-MY, but
sparsely labeled ICC-DMP, running parallel
to the circular muscle layer, remain in
W/WV muscles. C: omnipresent slow
wave activity recorded from wild-type mus-
cles. D: loss of slow waves in small intesti-
nal muscles of W/WV mice. Note also the
significant depolarization that occurs in
W/WV muscles. Recordings in C and D
were from circular muscle cells.
-55
2s
869
 SANDERS ET AL.

ies. It is also important to note that ICC in the region of the
deep muscular plexus (ICC-DMP) appeared normal in
W/WV and Sl/Sld mice. Intestinal motility was altered in
c-Kit and stem cell factor mutants, but responses to stimu-
lation of intrinsic enteric motor neurons were retained and
smooth muscle tissues responded to neurotransmitters and
generated Ca2 action potentials (80, 251, 403). Complete
loss of motor activity would prohibit intestinal transit,
amounting to a fatal pseudo-obstruction of the bowel.
However, W/WV mice live into adulthood without obvious
loss of nutritional assimilation (56). A normal pattern of
segmentation and peristaltic waves, observed in the small
intestines of wild-type mice, was not present in W/WV mice.
Intestinal muscles of these animals displayed action poten-
tials and contractions, but these events were more random
nels is reduced, and the intrinsic excitability of smooth mus-
cle cells is therefore suppressed between slow waves. Slow
waves, superimposed upon the influence of ICC-MY on
membrane potential, organize the generation of muscle ac-
tion potentials and phasic contractions into the phasic pat-
tern characteristic of the small intestine. The conductances
responsible for the negative membrane potentials of ICC
have not yet been determined.
2. ICC as gastric pacemakers
ICC-MY are present in the gastric corpus and antrum of
W/WV mice, but with application of neutralizing antibod-
ies, lesions in gastric ICC-MY networks developed after
9 40 days in organotypic cultures (279). Slow waves were

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and less coordinated, leading to weakened propulsive activ-
ity (80).
Survival and apparent nutritional sufficiency in W/WV mice
raises the question of the importance of ICC and slow
waves. Loss of ICC-MY in W/WV mice resulted in a 10- to
17-mV depolarization in the smooth muscle syncytium
(378, 403, 404). Depolarization alone was not responsible
for loss of slow waves because this level of depolarization,
as tested with elevated external K, did not block slow
waves (378). A second function of ICC-MY, therefore, ap-
pears to be setting membrane potential to more negative
levels than would be accomplished by the intrinsic ionic
conductances of smooth muscle cells. Thus ICC-MY in the
small intestine shift the membrane potential of the inte-
grated SIP syncytium from a range in which smooth muscle
cells generate spontaneous action potentials to more nega-
tive potentials in which the open probability of Ca2 chan-
not recorded in regions of muscle where ICC-MY were lost
but persisted in areas where ICC networks remained. Elec-
trophysiological recording from guinea pig stomach also
supported the hypothesis that ICC-MY are pacemakers in
the stomach (82). When intracellular impalements were
made, slow wave activity was recorded from circular
smooth muscle cells, positively identified by dye filling dur-
ing recording. More rarely, cells were impaled with much
faster and larger amplitude slow wave events (termed driver
potentials by these authors). The cells exhibiting driver po-
tentials were between the muscle layers and had morphol-
ogies consistent with cells labeled with c-Kit antibodies.
Simultaneous recording from ICC and nearby smooth mus-
cle cells showed that the slow waves in ICC preceded the
events in smooth muscle cells (FIGURE 6). These investiga-
tors also recorded from longitudinal muscle cells, and the
slow waves in these cells were similar in waveform to events
in ICC but of reduced amplitude. The slow waves recorded

1
20 mv
2
3 sec

LM

ICC-MY

CM bundle

ICC-IM Enteric motor neuron

FIGURE 6. Propagation of slow waves from ICC-MY to circular smooth muscle cells in the guinea pig stomach
(trace was provided by Professor David Hirst). In the experiment illustrated, an ICC-MY was impaled (electrode
1) and a circular smooth muscle cell was impaled simultaneously. Traces above show slow wave events of large
amplitude in ICC-MY and attenuated amplitude in smooth muscle cell. Note slight delay in activation of slow
wave in smooth muscle cell.

870 Physiol Rev VOL 94 JULY 2014 www.prv.org

 INTERSTITIAL CELLS IN SMOOTH MUSCLES
from longitudinal muscle were termed follower potentials
by these authors. Their results supported the hypothesis
that ICC-MY generated slow waves and these events con-
ducted to both circular and longitudinal muscle layers.
3. ICC as colonic pacemakers
ICC are reduced in the colons of W/WV mice and in genet-
ically similar Ws/Ws mutants in rats. However, the loss of
ICC in the colon is incomplete, and therefore comparisons
of activities in wild-type and mutant animals are not as
clear-cut as in other regions of the GI tract. Proximal colons
of wild-type rats and mice display a pattern of slow depo-
larization with superimposed clusters of action potentials
(4). Action potential clusters were also observed in colonic
muscles of Ws/Ws rats, but these events were irregular and
slower in frequency than in wild-type colons.
D. Mechanism and Modeling of Pacemaker
Activity
Studies to deduce the slow wave mechanism in whole GI
muscles were frustrated by the inability to record selectively
from ICC. This problem was solved by the elegant work of
groups led by David Hirst and Hikaru Suzuki. As men-
tioned above, Dickens and co-workers (82) recorded from
gastric pacemaker cells and confirmed that the recordings
were made from ICC-MY by filling cells with fluorescent
dyes during recording. Similar recordings of pacemaker ac-
tivity were made from gastric and small intestinal ICC-MY
(196, 198, 199), and the mechanism of slow waves was
investigated using membrane potential recordings. Experi-
ments using Itpr1/ mice showed that loss of IP3 receptor
type 1 blocked gastric slow waves (364). The IP3 receptor
blocker 2-aminoethoxydiphenyl borate (2-APB), although
not entirely specific, also blocked slow waves in ICC-MY
(196). However, this effect was accompanied by significant
depolarization, and sufficient depolarization alone can re-
duce or block slow wave generation. Treatments with a
variety of pharmacological antagonists and ion channel
blockers confirmed the general concept that slow waves
consist of two components: the initial component (up-
stroke) was sensitive to Ni2 and reduced by extracellular
Ca2, and the second (plateau) was sensitive to caffeine,
cyclopiazonic acid, and Cl channel blockers (155, 196,
198, 199). Reducing extracellular Cl also reduced the pla-
teau potential. Multiple ionic conductances are at play in
cells of the SIP syncytium. Blocking conductances or Ca2
handling mechanisms in one type of cell or altering ionic
gradients might impose effects on ionic mechanisms in cou-
pled cells. Thus definitive explanations for the pacemaker
mechanism were limited until ICC could be isolated for
single-cell voltage-clamp studies.
1. Studies of pacemaker activity in cultured ICC
c-Kit cells developed in cell culture, and therefore cul-
tured cells were used in many studies to investigate the
Physiol Rev VOL 94 JULY 2014 www.prv.org
mechanism of slow wave rhythmicity. After a few days in
primary culture, single cells or networks of cells with
c-Kit immunoreactivity can be identified, and these cells
generated spontaneous inward currents under voltage-
clamp conditions (208, 368). The inward currents re-
versed 10 20 mV positive to 0 mV, and the currents were
not blocked by dihydropyrdines. In current-clamp mode,
spontaneous slow wave-like depolarization transients
were observed that corresponded to the frequency of in-
ward currents in voltage-clamp. These events displayed
waveforms and mimicked some of the pharmacology of
slow waves in intact muscles; however, the frequency was
lower in cultured cells. Several labs have studied the ionic
conductances expressed and the mechanism of spontane-
ous electrical rhythmicity in cultured ICC models. The
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inward current was generally linked to a nonselective
cation conductance; however, one group suggested the
inward currents were more consistent with a Ca2-acti-
vated Cl conductance on the basis of reversal potentials
and block by SITS (372). Ion channels expressed in cul-
tured c-Kit positive cells and concepts of electrical rhyth-
micity were summarized in a previous review (331), and
a mathematical model was developed based on these
ideas (101, 102).
Problems developed with cultured cell models of ICC. ICC
make up 10% of the cells in the tunica muscularis, so
enzymatic dispersion of GI muscles results in a relatively
small percentage of ICC in the myriad of cells obtained.
Gene or protein expression studies on these cells are likely
to be no more specific than expression studies on whole
muscle extracts. The relative abundance of c-Kit cells after
various periods of time in culture and the effects of growth
media on retention of c-Kit expression have not been deter-
mined, so it is unknown whether the c-Kit cell population
increases or decreases relative to other cells as a function of
time in culture. Some investigators have tested growing c-
Kit cells on a layer of cells expressing stem cell factor, and
this promising approach enhanced the number and growth
of c-Kit cells (308). However, these studies included few
evaluations of the molecular and functional phenotype of
ICC. Most investigations of cultured c-Kit cells have been
performed without the benefit of a reporter molecule ex-
pressed in ICC for the purpose of unequivocal identifica-
tion. Initial labeling with antibodies to confirm the presence
of c-Kit immunopositive cells (208, 368) is not performed
on all cells sampled for electrical recording. Cells were se-
lected for recording on the basis of gross morphology. Thus
it is possible that the cells from which molecular evaluations
or physiological recordings were made were confused with
other cell types in mixed cell populations from which the
cultures were grown. Some investigators have performed
purification steps before culturing, such as immunoselec-
tion or FACS (276); however, tests to certify cell purity have
been rather limited.
871
 SANDERS ET AL.
As networks of c-Kit cells develop in culture, they experi-
ence changes in phenotype within a few days. However, few
tests verifying the fidelity of the native phenotype have been
performed on cultured c-Kit cells. A genomic study of
freshly dispersed ICC from the murine small intestine pro-
duced a catalog of highly expressed genes that could pro-
vide a standard for validation of the ICC phenotype (52).
Our own experience with cultured ICC was that the con-
ductances responsible for slow waves in situ were lost
within a few days in primary culture (436). Therefore, we
have concluded that the mechanisms of rhythmicity re-
viewed previously (331) may largely be artifacts of cell cul-
ture. This experience should raise caution about conclu-
sions based on studies of cultured c-Kit cells that are not
accompanied by careful verification that the mechanisms
and pathways observed are equivalent in the native pheno-
type. For this reason, little space in this review is devoted to
the many ion channels, agonist responses, and slow wave
mechanisms that have been reported from studies of cul-
tured c-Kit cells.
2. Studies of pacemaker activity in freshly isolated
ICC
A main concern about the activity recorded from cultured
ICC-like cells was that they lacked the ability to be paced by
application of current pulses. This was a fundamental and
important difference between the behavior of cultured cells
and whole muscles, which can be paced (296). Due to skep-
ticism about the behavior of cultured cells, Akinori Noma
and colleagues (125) sought to study ICC from the myen-
teric region (pacemaker region) of the mouse small intes-
tine. These authors developed a unique dispersion tech-
nique that did not disrupt the anatomical orientation of the
cells from myenteric plexus region but liberated cells that
could be voltage clamped. ICC-MY, verified by immu-
nolabling of unfixed tissues, had an average whole cell ca-
pacitance of 25 pF. ICC had resting potentials of 72 mV
and generated spontaneous transient depolarizations under
current clamp and large inward currents under voltage
clamp. The inward currents were termed autonomous cur-
rents, because once these currents were initiated by depo-
larization, they displayed an autonomous time course of
500 ms. Autonomous currents were attributed to a non-
selective cation conductance. Autonomous currents dis-
played what appeared to be inactivation properties, but
inactivation was characteristically different from the
Marcovian behavior of voltage-dependent ion channels. It
was concluded that activation and deactivation of autono-
mous currents depended on intracellular mechanisms that
responded to depolarization and regulated channel open-
ings. It was also shown that replacing the extracellular so-
lution with one that was nominally free of Ca2 caused
gradual reduction in the amplitude of autonomous cur-
rents, suggesting that Ca2 stores were an important com-
ponent in activation of autonomous currents. Demonstra-
tion of autonomous currents in freshly isolated ICC was an
872 Physiol Rev VOL 94 JULY 2014 www.prv.org
important step in understanding the cellular mechanism of
pacemaker activity and a potent indication of the loss of
function occurring via remodeling of ICC in cell culture.
Production of a reporter strain of mice in which ICC were
labeled constitutively by expression of a green fluorescent
protein (Kit/copGFP) made it possible to identify ICC in
mixed cell dispersions of the tunica muscularis and perform
experiments soon after enzymatic dispersion (311, 436).
Tmem16a (official name now is Ano1) had previously been
identified as one of the most highly expressed genes in ICC
by a gene array screen of the ICC transcriptome (52), but
the function of the protein encoded by Tmem16a was un-
known at the time. In 2008 the gene product of Tmem16a
was found to be a Ca2-activated Cl channel (Anoctamin
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1 or ANO1) (48, 336, 426). Immunohistochemical studies
showed that ANO1 was highly expressed in ICC of several
species, including humans (122, 163, 172).
ICC freshly isolated from Kit/copGFP mice display a large-
amplitude inward current that was activated by depolariza-
tion and followed a time course similar to the current pre-
viously identified as autonomous current (125). How-
ever, the reversal potential of the current in freshly isolated
ICC indicated that a Cl conductance was responsible for
the large-amplitude inward currents activated by depolar-
ization (436). The Cl conductance displayed unusual ac-
tivation properties: a very sharp increase in current was
noted with depolarizations in the range of 70 and 50
mV and a voltage-dependent delay in activation was ob-
served at near-threshold potentials. At potentials positive to
the activation threshold, the current developed to maxi-
mum amplitude and was linearly dependent on the Cl
gradient. The voltage dependence of activation was not fit
by a Boltzmann function, and therefore, the underlying con-
ductance was not likely to be voltage dependent. Activation
of the conductance was blocked by Ni2, replacement of
extracellular Ca2 with Ba2, or exclusion of Ca2 from
the extracellular solution. These and other experiments sug-
gested that activation of the inward current was due to an
initial transient influx of Ca2, and the delay in activation
with near-threshold depolarizations may have been due to
minimal Ca2 influx at more negative steps. ICC also ex-
pressed a single-channel, Ca2-activated Cl conductance
of 7.8 pS, which is similar to the conductance of ANO1
channels expressed in HEK cells (426). ICC currents were
blocked by niflumic acid with an IC50 of 4.8 M. Under
current clamp, ICC discharged spontaneous depolariza-
tions that mimicked the properties of slow waves in intact
muscles. The spontaneous depolarization events were also
blocked by niflumic acid. As described in the next para-
graph, the Ca2-activated Cl conductance appears to be
responsible for slow waves in GI muscles, so the current
activated in single ICC was referred to as the slow wave
current.
 INTERSTITIAL CELLS IN SMOOTH MUSCLES
Experiments on intact muscles suggested that a component
of slow waves was due to a Ca2-activated Cl conduc-
tance (e.g., the plateau phase of slow waves was blocked by
treating tissues with membrane permeable Ca2 buffers,
niflumic acid, and by removal of extracellular Cl) (153,
198). Cl channel blockers were also found to reduce the
frequency and block slow waves in murine, monkey, and
human small intestine and stomach (172). However, it
should be noted that the concentration sensitivity for these
effects varied between organs and species with gastric mus-
cles of monkeys being the most sensitive to niflumic acid.
ICC express several splice variants of Ano1 (172), and it is
possible that the variable sensitivity to Cl blockers and/or
the differences in the waveforms of slow waves might be
due to variations in the complement of Ano1 isoforms ex-
pressed in different regions of the GI tract. The role of
ANO1 channels in slow waves was further tested using
Tmem16/ (Tmem16atm1Bdh/tm1Bdh) mice (313). Previous
studies had shown that 80% of mice with congenital de-
activation of ANO1 channels died within a week after birth.
Experiments were performed, therefore, on newborn ani-
mals and after slow waves developed further in organotypic
cultures (406). All mice of several litters were assayed for
electrical slow waves by intracellular recording and then
genotyped later for Tmem16atm1Bdh alleles. Newborn
Tmem16a/ mice displayed no slow wave activity while
normal slow wave activity was recorded from mice with one
or both wild-type alleles. After 6 days in organotypic cul-
ture, slow waves developed and increased in amplitude in
mice with wild-type alleles but were absent in muscles of
Tmem16a/ mice. Mice of some litters were allowed to
develop for up to 23 days after birth. No slow waves were
recorded from gastric and small intestinal muscles from
Tmem16a/ mice, but normal slow waves were recorded
from siblings with wild-type alleles. These data strongly
support the hypothesis that Cl channels, encoded by Ano1
(Tmem16a), are responsible for slow waves in GI muscles
and justify the term slow wave current to describe the large-
amplitude inward currents initiated spontaneously and by
depolarization of single ICC.
3. The clock driving the pacemaker mechanism in
ICC
Insight into the clock mechanism driving slow waves was
provided by studies on gastric muscles from the guinea pig
(155). The intervals between slow waves were tabulated
and found to vary around a fairly tight mean value, and the
interval was not dictated by neural inputs to the muscle.
The interval between slow waves was tightly linked to the
duration of slow waves, and the probability of initiating the
next slow wave increased from low values immediately af-
ter repolarization of the past slow wave until the next event.
The increase in excitability was linked to the discharge of
small-amplitude spontaneous transient depolarizations
(STDs, or unitary potentials, as termed by the authors).
The increase in probability of STDs was associated with
Physiol Rev VOL 94 JULY 2014 www.prv.org
activation of a full-amplitude slow wave. STDs are com-
monly recorded from cells within intact networks of ICC in
situ (155, 196 199, 383) and can be conducted to and
recorded from smooth muscle cells in small strips of muscle
(21, 45, 155, 197). STD activity is lost from smooth muscle
recordings when ICC are absent (45). Single ICC also gen-
erate STDs, and these events are due to transient activation
of Ca2-activated Cl currents (436, 437). Stochastic gen-
eration of STDs in ICC within networks of pacemaker cells
(155) leads to activation of the voltage-dependent process
linked to activation of slow wave currents (436). There is
still speculation about the nature of the voltage-dependent
process. Our group has favored voltage-dependent Ca2
entry, mainly via T-type Ca2 channels, as the voltage-
dependent mechanism (194, 433) because reduced extracel-
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luar Ca2, Ni2, and mibefradil can block slow wave gen-
eration and slow or block the propagation slow waves in
intact muscles and generation of slow wave currents in sin-
gle ICC (20, 405, 411, 413, 436). The 1H isoform
(Cacna1h) has been suggested as the T-channels responsible
for slow wave propagation (120). Others have suggested a
mechanism involving voltage-dependent activation of IP3
formation or voltage-dependent sensitization of IP3 recep-
tors (153, 273, 383). More experiments will be needed to
resolve this controversy.
It is important to note that every type of ICC from which
electrical recordings have been made displays STDs; how-
ever, only some ICC are capable of generating slow waves
(196 199, 436, 437). This suggests that the basic pace-
maker mechanism (i.e., STDs) is an intrinsic feature of ICC;
however, some ICC lack the voltage-dependent mecha-
nism(s) required for STDs to summate into slow wave cur-
rents or to propagate slow waves actively (see section be-
low). The simplest explanation for the inability of some
classes of ICC to generate slow waves is that these cells lack
the voltage-dependent mechanism(s) responsible for initia-
tion of slow wave currents (see FIGURE 7).
Ca2 handling mechanisms that control activation of
ANO1 channels are not well understood at present. It seems
clear that release of Ca2 from Ca2 stores via IP3 receptors
is required for generation of slow waves (364), but sources
of Ca2 for filling of stores, initiation, or amplification of
Ca2 transients by Ca2 entry or ryanodine receptors, and
the role of Na/Ca2 exchange are controversial. The Ca2
sensor that is either intrinsic to ANO1 or provided by an
accessory protein has also not been identified. Finally, the
role of excluded volumes that might occur because of close
apposition between stores and the plasma membrane are
not understood. At present, our best concept of the slow
wave mechanism is illustrated in FIGURE 7. In spite of many
uncertain elements, there have been numerous recent at-
tempts at modeling pacemaker behavior in ICC (42, 101,
102, 384, 428). Simulations of pacemaker activity of ICC
are improving, and the models and refinements provided by
873
 SANDERS ET AL.

1. Generation of STICs (All ICC)

FIGURE 7. A model of electrical rhyth-

ANO1 micity based on studies of freshly isolated
ICC of the murine small intestine (433,
Plasma membrane 436, 437). Figures show portion of
plasma membrane in close association
Cl Ca2+
with Ca2 release mechanisms of the en-
Ca2+ Excluded doplasmic reticulum (ER). 1: The basic
ATP ADP, Pi volume event of electrical rhythmicity is spontane-
ous transient inward currents (STICs) elic-
RyR IP3R ited by localized release of Ca2 from
stores. IP3-gated channels (IP3R) are the
SERCA main source of Ca2 driving activation of
Ca2+ Ca2+ Ca2-activated Cl channels (ANO1) in the
Ca2+ plasma membrane to generate STICs, but

Downloaded from http://physrev.physiology.org/ by 10.220.33.5 on November 8, 2017

Endoplasmic reticulum ryanodine receptor (RyR) may also contrib-
ute. Ca2 is recovered into stores via ac-
tive Ca2 pumping into the ER. STICs gen-
erate spontaneous transient depolariza-
tions (STDs), the voltage response to the
inward currents. 2: The second phase of
rhythmicity is activation of T-type voltage-
2. Generation of slow wave currents (Pacemaker ICC)
dependent Ca2 channels (VDCC) in re-
Ca2+ Ca2+ sponse to the STDs caused by STICs. Ca2
ANO1 VDCC ANO1 VDCC entry synchronizes Ca2 release in other
regions of membrane, leading to synchro-
nized opening of ANO1 channels through-
out the cell. This generates slow wave cur-
Cl Cl Excluded rents. Depolarization also activates VDCC
Ca2+
volume in adjacent electrically coupled cells, lead-
Ca2+
Ca2+ Ca2+
ATP ADP, Pi
Ca2+
Ca2+ Ca2+
ing to cell-to-cell active propagation of slow
waves in ICC networks. 3: In the 3rd phase
RyR IP3R RyR IP3R of rhythmicity, binding of chronotropic ago-
nists (such as ACh in this example) to G
SERCA protein-coupled receptors (type 3 musca-
rinic receptor, M3, in this example) leads
Ca2+ Ca2+ Ca2+ Ca2+
Ca2+ to IP3 formation and increased frequency
and amplitude of STICs. More frequent
Endoplasmic reticulum STICs and greater amplitude depolariza-
tions increase the likelihood of activation of
whole cell slow wave currents. The steps in
this mechanism are deduced from studies
on ICC of the murine small intestine. Phar-
3. Chronotropic enhancement of pacemaker current (ICC-IM or ICC-MY) macological data suggest that the general-
ized mechanism proposed applies to other
Ca2+ ACh Ca2+ GI muscles (e.g., dependence upon Ca2-
activated Cl currents to generate STICs
ANO1 VDCC M3 ANO1 VDCC and slow wave currents); however, the
mechanism for regulation of frequency is
poorly understood and appears to vary in
Cl Cl ICC from different regions of the GI tract
Ca2+
Ca2+ Ca2+ Excluded and the mechanism for voltage-dependent
ADP, Pi volume Ca2 entry and/or release, necessary for
ATP
Ca2+ IP3 Ca2+ IP3 slow wave propagation, appears to vary.
For example, slow waves recorded from
RyR IP3R RyR IP3R different regions of the murine GI tract and
in other species display a range of sensitiv-
SERCA ities to extracellular Ca2, Ni2, and dihy-
Ca2+ Ca2+ Ca2+ Ca2+ dropyridines. The ionic and molecular ex-
Ca2+ planations for the diversity in the slow wave
Endoplasmic reticulum mechanism and for regulation of frequency
are not fully understood.

874 Physiol Rev VOL 94 JULY 2014 www.prv.org

 INTERSTITIAL CELLS IN SMOOTH MUSCLES
further experimentation will be very useful in planning fu-
ture experiments and in predicting the actions of therapeu-
tic agents on electrical rhythmicity in the gut.
E. Role of ICC in Propagation of Slow Waves
1. Extracellular electrical recording from visceral
smooth muscles
A great deal of the information about GI pacemaker activity
used by gastroenterologists to make diagnoses, biomedical
scientists in testing hypotheses and drugs, and instructors
for teaching GI physiology has been deduced from studies
using electrical recordings made with extracellular metal
electrodes laid against or fastened to the serosal or mucosal
surfaces of GI organs or applied to the abdomen (i.e., elec-
trogastrograms). Studies of this sort have been performed
for almost 100 years; however, a recent report raises ques-
tions about the validity of results obtained with this method
(18). Activity was recorded from murine gastric sheets in an
attempt to relate electrical activity to the aberrant motility
patterns in mutant mice with dysmotility phenotypes. These
studies utilized monopolar or bipolar recording via silver
electrodes arranged into arrays, as used commonly in many
recent studies (e.g., Refs. 87, 227, 274, 390). Biopotentials
detected by these electrodes were monitored by standard
techniques, and it was noted that the frequency of gastric
slow waves was significantly lower than the frequencies
observed when intracellular electrical recording were made
from intact gastric sheets (103, 104). When contractions
were blocked by dihydropyridines or wortmannin (both
drugs chosen to block contractions downstream of the
mechanism that generates slow waves), extracellular biopo-
tentials were abolished. Impalement of cells in muscles,
however, showed that the drugs had no effect on slow wave
amplitude or frequency. By combining imaging of muscle
movements with extracellular recording, it became appar-
ent that very small muscular movements (50 m) were
capable of generating voltage transients several tens of mi-
crovolts in amplitude (i.e., equivalent to the events typically
considered to be slow waves). Thus extracellular electrical
recording from visceral smooth muscles may be largely ar-
tifacts of contractile movements. In exploring the lengthy
literature in which extracellular recording has been used to
monitor the electrical activity of the GI tract, it is apparent
that controls for movement artifacts have been neglected.
Thus it is reasonable to be skeptical about the results of
classic studies that have based major conclusions upon this
technique. Future investigations using extracellular record-
ings will need to demonstrate the validity of these record-
ings by control experiments in which recording fidelity is
maintained after rigorous stabilization of movement. A re-
cent study claiming maintenance of extracellular recording
fidelity after infusion of a dihydropyrdine into pig intestine
in vivo relied only upon visual inspection to ensure that
movement was blocked (10). This study, while clearly seek-
Physiol Rev VOL 94 JULY 2014 www.prv.org
ing to prove the fidelity of extracellular recording, falls well
short of a suitable control study for extracellular recording,
because: 1) dihydropyridines dont block all contractile
movements in many species, 2) no concentration/effect data
for dihydropyridines were provided, 3) movements of the
viscera due to respiration and pulsatile blood flow could not
possibly have been absent in a living animal, and 4) more
rigorous monitoring of movement than visual inspection
would be needed to detect the small movements capable of
eliciting electrical artifacts.
Difficulties in recording authentic electrical activity in GI
muscles with extracellular electrodes are likely due to the
structure of ICC networks, the fact that ICC capable of
regenerative propagation are buried within muscle layers,
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and the magnitude and kinetics of transmembrane currents
responsible for slow waves. In contrast to the heart, in
which cardiac myocytes actively regenerate action poten-
tials along a coherent wave front, the gut has a thin and
relatively sparse network of ICC generating the transmem-
brane currents responsible for slow waves. The velocity of
the upstroke phase of slow waves is 100-fold less than the
upstroke velocity of cardiac action potentials, indicating
much lower current densities during slow wave depolariza-
tions. Field potentials resulting from slow waves are far
weaker than the field potentials generated by cardiac and
skeletal muscles or neurons and may be lost in the noise of
recording.
2. Propagation of slow waves in intact muscle strips
Motility patterns in the gut depend on coordination of mil-
lions of smooth muscle cells. For example, in gastric motil-
ity, slow waves originate in the proximal corpus and peri-
staltic contractions spread over many square centimeters of
the stomach. Timing of contractions is accomplished by
active propagation of slow waves around and along the
tubelike GI organs. GI muscles display clear properties of
active slow wave propagation, and several studies have
characterized propagation using intracellular recordings to
determine propagation velocities of slow waves initiated
from known sites of pacing. These studies have typically
used muscle strips or sheets from larger animals to obtain
cable lengths sufficient to resolve temporal separation of
slow waves recorded from two points. In the canine antrum,
for example, slow wave propagation velocity was measured
at 20 60 mm/s in the circumferential direction (long axis of
circular muscle fibers) and 10 mm/s in the longitudinal
direction (long axis of longitudinal muscle fibers) (17, 20).
The precise explanation for the anisotropy in propagation
velocities is unknown. Slow waves display refractory prop-
erties, as do excitable events other in excitable cells and cell
networks. In the canine antrum, the period between slow
waves required for complete restoration of slow wave up-
stroke velocity, amplitude, and duration is at least 10 s
(296), demonstrating the long latencies required for reset-
ting Ca2 entry/release mechanisms and ion channels re-
875
 SANDERS ET AL.
quired for slow wave generation and propagation. In fact,
ion channel recovery from inactivation is categorically
much quicker than the long refractory period of slow
waves, suggesting that other mechanisms, such as reloading
of intracellular Ca2 stores, is likely to be the rate-limiting
step in slow wave frequency and responsible for long peri-
ods of refractoriness.
ICC have a critical role in slow wave propagation because
these cells provide the pathway for active propagation in GI
muscles. Smooth muscle cells lack the ionic apparatus nec-
essary to regenerate slow waves. Thus a coupled network of
ICC is required for coherent propagation of slow waves and
coordination of contractions (FIGURE 8). This point was
demonstrated by dissection experiments in which bits of
tissue containing pacemaker cells were removed from mus-
cles of the canine colon. In intact muscles, slow waves of
uniform amplitude were recorded from cells along the sub-
Single ICC
1.
2.
ICC network
3.
ICC network
coupled to SMCs
876 Physiol Rev VOL 94 JULY 2014 www.prv.org
mucosal surface of the circular muscle layer, and active
propagation of slow waves occurred in this region. Slow
wave activity was lost if a thin band of tissue along the
submucosal surface (containing pacemaker ICC-SM) was
removed, and slow waves decayed within a few millimeters
from a region with the pacemaker cell network intact (332).
A similar phenomenon was observed in gastric muscles
treated with neutralizing c-Kit antibodies, slow waves were
generated in areas where clusters of ICC were retained, but
these events did not propagate to regions devoid of ICC
(279). Furthermore, after loss of ICC, gastric muscles could
not be paced by application of current pulses.
Recordings of slow waves in ICC and from nearby smooth
muscle cells demonstrate how slow waves conduct via elec-
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trical coupling into smooth muscle cells. Large-amplitude
slow waves were recorded in ICC, and much attenuated
slow waves were recorded from nearby smooth muscle cells
FIGURE 8. Active propagation of slow
waves. 1: The initiating step occurs by lo-
calized Ca2 transients (elevated Ca2 de-
picted by green color in all cells; arrow),
initiating STICs in an ICC. 2: Depolarization
caused by STICs activates Ca2 entry and
Ca2-induced Ca2 release raising Ca2
throughout the ICC and activating whole cell
slow wave current. 3: Depolarization causes
active propagation of slow waves through the
ICC network (horizontal black arrow shows
direction of propagation), and slow wave cur-
rents are activated cell to cell, as the wave-
front spreads. 4: As slow waves propagate
through the ICC network, they conduct pas-
sively into electrically coupled smooth muscle
cells (SMCs). Slow waves depolarize SMCs
and activate L-type Ca2 channels. Ca2 en-
try (green) triggers SMC contraction. Spread
of slow waves in the ICC network leads
spread of contractions necessary for seg-
mental and peristaltic contractions.
 INTERSTITIAL CELLS IN SMOOTH MUSCLES
(82, 155, 196, 198, 199). Hirst and colleagues (68) de-
scribed the electrical coupling between ICC and smooth
muscle cells as weak and showed that conduction of slow
waves is similar to conduction of electrotonic potentials
between these cells. Taken together, these studies suggest
that slow waves, generated by the ICC, conduct passively
into the smooth muscle syncytium and are not actively re-
generated by smooth muscle cells. Thus active (regenera-
tive) propagation of slow waves by ICC-MY is another
central function of these cells. Unlike the heart that is paced
from discrete clusters of pacemaker cells and action poten-
tials propagate via the regenerative mechanisms in cardiac
myocytes, the gut requires a continuum of ICC for regener-
ative (active) propagation of slow waves. This feature has
important consequences in pathophysiological circum-
stances where loss of some ICC might uncouple regions of
ICC networks or reduce the safety factor for propagation,
thus preventing normal coherent spread of slow waves and
causing loss of contractile coordination.
A comment should be included about the form of electrical
coupling between ICC-MY and smooth muscle cells be-
cause some authors have claimed that gap junctions be-
tween these cells cannot be found by electron microscopy
and have proposed alternative methods of electrical cou-
pling, including structures called peg-and-socket junctions
(371) or electrical field coupling (360). Whether some form
of ephaptic communication could be effective in conducting
slow waves from ICC-MY to smooth muscle cells seems
unlikely and has not been demonstrated. Gap junctions be-
tween these cells may be rare and small in size, and these
structures have been observed between ICC-MY and
smooth muscle cells in some species (331). Direct electrical
measurements have shown that electrical coupling between
ICC-MY and smooth muscle cells is weak (68), and this
may result from few and small gap junctions.
ICC-IM in corpus and antral muscles also appear to have
regenerative capabilities that can sustain circumferential
propagation of slow waves. Hirst and colleagues (152, 154)
noted that ICC-MY formed a dense network along the
greater curvature, but this population of ICC was greatly
diminished or missing near the lesser curvature in rodent
stomachs. In spite of the decrease in ICC-MY, slow waves
propagated from the greater curvature to the lesser curva-
ture with little decrease in amplitude (152). These authors
also found that ICC-IM in the corpus were capable of a
relatively fast rate of spontaneous slow wave generation
and suggested that these cells are the source of the higher
frequency slow waves in the corpus. They viewed activation
of the gastric musculature in the following manner: 1)
ICC-IM generate corporal slow waves, and these events
determine the dominant frequency in the stomach; 2) slow
waves from ICC-IM activate slow waves in the ICC-MY
network, and these events spread down the stomach; 3)
slow waves conduct into bundles of smooth muscle cells,
Physiol Rev VOL 94 JULY 2014 www.prv.org
elicit depolarization, and initiate contractions; and 4) depo-
larization also initiates active responses in ICC-IM (referred
to as regenerative potentials) which spread rapidly along
the long axis of these cells in circumferential bands (81). In
order for this mechanism to work, ICC-IM in the corpus
and antrum must have a voltage-dependent mechanism that
is primed to provide cell-to-cell regeneration of slow waves.
Cellular studies of ICC-IM have been limited, but a voltage-
dependent mechanism intrinsic to ICC-IM has not yet been
identified. Such a mechanism could be present in ICC-IM or
in the septal ICC (ICC-SEP) that run between bundles of
smooth muscle cells because small bundles of gastric mus-
cle, that likely contain both ICC-IM and ICC-SEP, are ca-
pable of regenerative propagation (383).
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There may be significant differences between the ICC-IM in
different parts of the stomach. In laboratory animals ICC in
fundus muscle bundles do not display voltage-dependent
regenerative mechanisms (21), whereas the ICC within bun-
dles in the distal stomach can generate active responses to
depolarization or upon break from hyperpolarization (153,
383). There are clearly many things to learn about the in-
teractions between ICC and the specific ionic properties of
different classes of ICC before slow wave propagation is
fully understood or can be modeled accurately.
3. Ca2 imaging to study mechanism and
propagation of pacemaker activity
Intracellular Ca2 transients are responsible for the spon-
taneous transient inward currents (STICs) that generate
STDs in ICC. Depolarization from STDs triggers global
Ca2 transients that activate whole cell slow wave currents
(FIGURE 8). Imaging of Ca
2
transients with Ca2 sensitive
dyes has been used to characterize propagation of pace-
maker activity in networks of ICC in situ (146, 283). ICC
take up Ca2 indicators, such as fluo 4, making it possible
to resolve cyclical Ca2 transients in ICC that correspond to
slow waves, and simultaneous recording of Ca2 transients
and slow waves showed 1:1 correlation between these
events. Ca2 transients provide a window and cell-specific
detail on pacemaker activity that is not possible with elec-
trical recording. Studies using ICC-specific expression of
molecular probes, such as GCaMP, will be a powerful new
technique for studying slow wave propagation in intact GI
muscles and organs.
Ca2 imaging studies on guinea pig and mouse tissues in
vitro reinforced the concept that slow wave generation is
stochastic in nature (155), because events within a network
of ICC in sheets of muscle are generated from variable
points and propagation within a network occurs via often
changing pathways (146, 283). Activation of a network of
ICC is directed from the first cell to reach threshold, but this
position of leadership changes from cycle to cycle. Creation
of spatiotemporal maps from video images allows visual-
ization of direction and velocity of activity fronts. Ca2
877
 SANDERS ET AL.
imaging also reinforced the pacemaker role for ICC-MY
because Ca2 transients in closely spaced ICC-MY and
smooth muscle cells occur sequentially (FIGURE 9), as would
be expected if slow waves, generated in ICC-MY, initiated
depolarization and activation of voltage-dependent Ca2
channels in neighboring smooth muscle cells (146, 147,
283). Average propagation velocities of wave fronts in ICC
networks in the mouse were between 2 6 mm/s, and there-
fore consistent with the velocities of slow wave propagation
in murine muscles. The propagation velocity of Ca2 tran-
sients associated with slow waves greatly exceeds the rates
of Ca2 waves that result only from diffusion (10 50 m/s)
(367), supporting the idea that slow wave propagation de-
pends on a voltage-dependent mechanism.
The mechanism of propagation was investigated by imag-
ing of Ca2 dynamics in ICC networks, and these studies
A
LM
ICC-MY
0 ms 100 m 64 ms 128 ms 192 ms
B propagating through ICC networks. Drugs, such as cyclo-
LM
ICC-MY
100
Iu
0.5 s
Ca2+ Transients
C propagation in larger animals and human muscles, and
Circumferential
100
m
1s
see frames in A
FIGURE 9. Spread of Ca2 transients in the ICC network of murine
small intestine. ICC were loaded with fluo 4. Sequence of video
images shows development of Ca2 transient in ICC-MY and spread
of wave front from upper left quadrant to whole image. Streaks of
fluorescence are longitudinal smooth muscle cells (LM) that are
above the ICC-MY network. Note that ICC fire Ca2 transients prior
to events in LM. Ca2 transients in regions of interest in ICC-MY and
LM cells are shown as traces as a function of time in the bottom
right of figure. Spatiotemporal maps were constructed from repet-
itive Ca2 transients sweeping through the ICC-MY network. [Re-
drawn from Hennig et al. (147). Copyright 2010 Blackwell Publish-
ing Ltd.]
878 Physiol Rev VOL 94 JULY 2014 www.prv.org
showed that electrical coupling between cells was funda-
mental to the organization of propagating Ca2 transients
(283). Treatment of ICC-MY networks with 18-glycyr-
rhetinic acid, a gap junction uncoupler, did not block Ca2
transients in individual cells, but blocked the spread of tran-
sients through the network. After 18-glycyrrhetinic acid,
individual cells paced at their intrinsic frequencies, which
varied from cell to cell, and failed to be entrained by a
dominant pacemaker frequency in the network. By tabulat-
ing the time course of Ca2 transients in multiple cells
within a network, it was possible to characterize the cellular
sequence of activation and the time course of cellular tran-
sients event to event. This analysis showed that the activa-
tion sequence (i.e., spread of slow waves through a net-
work) varies even when the direction of propagation re-
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mains constant. The time lag in activation between adjacent
cells also varied from cycle to cycle, likely reflecting the
dynamics of refractoriness and excitability of individual
cells. These properties are consistent with the cablelike
properties of a complex two- or three-dimensional network
of excitable cells.
Imaging studies also support the hypothesis that the volt-
age-dependent mechanism for propagation is Ca2 entry
through voltage-dependent, possibly T-type Ca2 channels
(283). Nicardipine did not affect Ca2 transients in ICC-
MY, but greatly reduced or blocked Ca2 transients in
smooth muscle cells. Ni2 (100 M) severely disrupted
Ca2 transients, and mibefradil blocked Ca2 transients
piazonic acid (10 M) and 2-APB (50 M), which affect
intracellular Ca2 handling mechanisms, reduced the fre-
quency of ICC-MY Ca2 transients, and ryanodine (10
M) did not affect frequency but reduced the amplitude of
these events. These data are consistent with the idea that
Ca2 entry entrains the discharge of localized Ca2 release
events, generates whole cell Ca2 transients, and facilitates
propagation of slow wave currents in ICC networks.
Imaging has also been used to study pacemaker activity and
these studies have allowed generalizations to be made about
the role of ICC in pacemaker activity and the mechanism
for pacemaking. For example, studies on muscles of the
human jejunum showed Ca2 transients in ICC-MY with
characteristics similar to the events in murine muscles
(241). The Ca2 transients in human ICC-MY were bipha-
sic in nature, and the initial component was sensitive to
Ni2 and mibefradil. Ca2 transients in human jejunal
ICC-MY were not affected by dihydropyridines, but were
reduced or blocked by cyclopiazonic acid and 2-APB. Be-
cause slow waves spread passively (i.e., with decrement)
into smooth muscle bundles, electrical events cannot pene-
trate very far into the depth of thicker muscles of larger
animals and humans (412). However, ICC lying within the
septa (ICC-SEP) between muscle bundles in these tissues
 INTERSTITIAL CELLS IN SMOOTH MUSCLES
were found to activate sequentially as a function of distance
from ICC-MY in the human small intestine (242). Thus
propagation of slow waves through ICC-SEP drives depo-
larization of smooth muscle cells deep within smooth mus-
cle bundles.
There are two pacemaker regions in the canine colon, each
producing different frequencies of pacemaker events (354).
Interesting differences were observed in the activation and
propagation of Ca2 transients in ICC in the submucosal
pacemaker area (ICC-SM), that produce slow waves, and
ICC-MY, that produce faster oscillations, called myenteric
potential oscillations. Rhythmic Ca2 transients (5 8/min)
spread without decrement through the ICC-SM network,
but Ca2 transients in ICC-MY (16/min) were typically
unsynchronized in adjacent cells. Excitatory neural inputs
synchronized the Ca2 transients in ICC-MY. These find-
ings demonstrate the diversity of behaviors and pacemaker
frequencies found in ICC from different parts of the GI
tract. Why ICC with different pacemaker frequencies, volt-
age dependencies, and synchronization between cells de-
velop in different regions of the GI tract and what factors
are responsible for pacemaker diversity are unknown at the
present time.
4. Relating Ca2 transients in ICC to motor patterns
of GI organs
Comparing the activation and propagation of Ca2 tran-
sients in wild-type and W/WV mutants provided a means of
determining the role of ICC-MY in regulating motor pat-
terns in the small intestine (FIGURE 9) (147). In the mouse
Ca2 transients in ICC-MY precede transients in adjacent
longitudinal muscle fibers by 66 127 ms. The frequency of
Ca2 transients in muscle fibers (30 cpm), variability in the
intervals between transients (7%), and propagation ve-
locities (4 mm/s) in the longitudinal muscle layers of wild-
type mice were similar in flat sheets of tunica muscularis,
isolated intestinal segments, or exteriorized loops of intes-
tine. In exteriorized loops Ca2 transients were observed
around the circumference of the intestine and were con-
strained within a band 1.5 mm in length. Ca2 transients
in longitudinal muscle cells preceded longitudinal shorten-
ing and circular contraction by 1 s. Contractions in wild-
type mice were stable, most prominent in the longitudinal
axis, and originated at the oral ends of preparations. Force-
ful contractions were also observed in muscles of W/WV
mice; however, contractions were abrupt in onset and un-
coordinated compared with the contractile patterns of wild-
type mice. The velocity of propagation of Ca2 transients in
longitudinal muscles was about six times faster than prop-
agation in wild-type small intestine, and origination sites of
Ca2 transients were highly unstable. Ca2 transients prop-
agated over smaller areas, and frequent collisions of Ca2
transients were apparent.
Physiol Rev VOL 94 JULY 2014 www.prv.org
ICC-MY facilitate coherent propagation of electrical activ-
ity which provides organization for muscle depolarization,
Ca2 transients, and contractions (147). When ICC are
reduced in number, smooth muscle cells escape from the
discipline of slow waves and revert to intrinsic excitability
mechanisms. After loss of slow waves, smooth muscle cells
generate spontaneous action potentials, and there is little
segment-wide regulation of the timing of action potentials.
Thus the motor behavior in the absence of ICC-MY differs
considerably from normal motility patterns. Contractions
are less circumferential, directional organization of con-
tractions decreases, and action potentials and contractions
generated from random sites often collide.
Slow waves can propagate for long distances through ICC
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networks, providing organization of contractile activity at
the organ level. The time constants of the SIP syncytium do
not favor propagation of action potentials, and therefore,
regenerative propagation of action potentials tends to be
limited to a small cluster of cells (2). Smaller regions of
muscle, synchronized only by action potentials, produce
less organized and weakened contractions observed in small
intestines of W/WV mice (80). Thus, while muscles and
organs lacking ICC-MY still generate spontaneous electri-
cal activity and contractions, the loss of the rhythmic depo-
larization-repolarization cycle provided by clock-like slow
waves leads to abnormal and less efficient intestinal motil-
ity.
F. Role of Interstitial Cells in
Neurotransmission
An early idea proposed by Cajal (47) was that interstitial
cells are primitive neurons providing connectivity between
autonomic nerve fibers and effector tissues. We now know
that ICC are not neurons and do not develop from precur-
sors from the neural crest as do the neurons and glia of the
enteric nervous system (238, 379, 430). However, the idea
that ICC mediate inputs from enteric motor neurons has
developed dramatically in recent years. This idea has been a
controversial topic in ICC research because several investi-
gators have reported that contractile responses to nerve
stimulation are retained in animals lacking some or most of
the ICC thought to mediate neural inputs (ICC-IM). The
debate about the role of ICC in neurotransmission was
discussed in recent reviews (126, 329) and is summarized in
this review. Resolving this controversy is important because
regulating motor input to visceral smooth muscle organs
may provide a means for therapeutic regulation of motor
activity. Understanding the postjunctional mechanisms for
transduction of neural inputs and how these signals are
linked to excitation-contraction coupling are fundamental
to understanding the reasons for many motor defects in the
GI tract.
879
 SANDERS ET AL.
Some types of ICC (and PDGFR cells, as discussed below)
are distributed in a manner that would facilitate innervation
by motor neurons (FIGURE 1, GI). The cells termed ICC-IM
(or ICC-DMP in small intestine) and PDGFR-IM lie along
the projections of excitatory and inhibitory motor neurons
within bundles of muscle fibers in most regions of the gut.
These interstitial cells form gap junctions with smooth mus-
cle cells. Close associations with varicosities of motor neu-
rons and electrical connectivity with smooth muscle cells
are suggestive, but not proof, of a role for these cells in
neurotransmission or neuromodulation. Morphologists
noted very close contacts between ICC and varicosities of
enteric motor neurons with electron microscopy, and Imai-
zumi and Hama (179), for example, suggested that the
interstitial cell may play a role in transmitting stimuli re-
ceived from the axon to surrounding smooth muscle cells by
an electrotonic response. This comment, and the original
idea of Cajal, stimulated further investigation of ICC in
neurotransmission. A morphometric study of the lower
esophageal sphincter performed by Edwin Daniel and Vir-
ginia Posey-Daniel revealed an abundance of extremely
close contacts (20 nm) between varicosities and ICC and
far fewer of these contacts between varicosities and smooth
muscle cells (73). These authors suggested that if interca-
lation of ICC between motor nerve terminals and smooth
muscle cells was prevalent, then attempts to define the
characteristics of nonadrenergic, noncholinergic junction
potentials in smooth muscle and compare them with effects
of putative mediators may be misleading. If the smooth
muscle junction potential is produced after electrotonic
transmission from interstitial cells and its characteristics are
determined by events in the interstitial cells, then there is no
reason to expect coincidence of effects of nerve-released and
exogenously added mediators. The first may act directly on
interstitial cells and the second on smooth muscle cells as
well as on interstitial cells, with the action on smooth mus-
cle cells likely to be predominant. This comment turned
out to be an important signpost in the course of study of
enteric motor neurotransmission.
1. Loss of excitatory and inhibitory
neurotransmission in muscles of W/WV mice
In most regions of the GI tract, ICC-IM are closely associ-
ated (20 nm gaps) with varicosities of motor neurons and
form gap junctions with adjacent smooth muscle cells.
ICC-IM are greatly reduced in numbers in gastric muscles of
W/WV mice (45). Electrical field stimulation of wild-type
muscles evokes excitatory and inhibitory junction poten-
tials in the fundus, and both nitrergic and cholinergic junc-
tion potentials were reduced significantly in muscles of
W/WV mice with reduced ICC-IM (45, 365, 400). Loss of
these responses was not due to failure of motor neurons to
develop and innervate muscle bundles or to release neu-
rotransmitters, and responses were not lost because smooth
muscle cells lost sensitivity to ACh and nitric oxide (NO).
Contractile responses to exogenous transmitters were sim-
880 Physiol Rev VOL 94 JULY 2014 www.prv.org
ilar in wild-type and W/WV muscles. The membrane hyper-
polarization response to NO donors was greatly reduced in
W/WV muscles, but muscle relaxation caused by NO do-
nors was of similar magnitude in wild-type and W/WV mus-
cles. Thus different mechanisms appear to mediate nitrergic
responses in ICC-IM and smooth muscle cells. The impor-
tance of one mechanism might be emphasized in wild-type
muscles and compensated for by redundant mechanisms in
muscle cells that develop in the absence of ICC-IM. ICC-IM
were also greatly reduced in numbers, and inhibitory and
excitatory junction potentials were reduced in muscles of
Steel mutants (Sl/Sld) that lack membrane-bound stem cell
factor (403). Lower esophageal and pyloric sphincter mus-
cles of W/WV mice (408) and small intestinal muscles
treated with neutralizing antibodies to c-Kit (407) also dis-
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played reduced populations of ICC-IM (ICC-DMP in small
intestinal muscles) and reduced postjunctional responses to
stimulation of enteric motor neurons. From these studies, it
was reasoned that loss of ICC-IM results in reduced con-
nectivity between enteric motor neurons and smooth mus-
cle cells, and ICC-IM have an important role in transducing
motor neurotransmitters in GI muscles.
2. Close contacts between nerves and effector cells
in visceral motor neurotransmission
There have been many reports about the close connectivity
between enteric motor nerve varicosities and ICC (see sect.
IIA; FIGURES 2 AND 3); however, few quantitative studies
on this subject have been provided. Clearly, very close (20
nm) connections with varicosities are not exclusive to ICC,
and a few studies (73, 110, 264) have described similar
connections between neurons and smooth muscle cells. One
of these studies, using a morphometric approach, con-
cluded that close connections were more prevalent between
varicosities and ICC (73). A quantitative understanding of
the structural features of motor innervation is important,
because this may provide spatial parameters necessary for
modeling the dynamic profiles of neurotransmitters re-
leased from motor neurons. Basic questions regarding en-
teric motor neurotransmission, such as the nature and mo-
lecular specializations of neural active zones (i.e., neu-
rotransmitter release sites), probabilities of vesicle or
neurotransmitter release from varicosities per impulse,
number of transmitter molecules released upon vesicle fu-
sion, effects of frequency on types and quantities of neu-
rotransmitters released, prejunctional modulation of trans-
mitter release, metabolic and uptake pathways involved in
deactivation of neurotransmitter signals, and dynamics of
neurotransmitter concentrations in postjunctional volumes
are poorly understood (329). Thus discussions about
whether volume or synaptic-like neurotransmission
dominate in muscles of the GI tract are speculative at pres-
ent. Even if volume transmission seems likely from rigorous
consideration of morphological details (110), the extent to
which effective neurotransmitter concentrations reach
 INTERSTITIAL CELLS IN SMOOTH MUSCLES
postjunctional receptors depends on the many factors listed
above.
3. Expression and function of receptors and
effectors of neural responses in interstitial cells
Use of immunohistochemical techniques and isolation of
smooth muscle and interstitial cells has allowed evaluations
of the expression of receptors and effects of neurotransmit-
ter responses and comparative responsiveness of specific
populations of postjunctional cells. In some cases an immu-
nohistochemical approach (i.e., functional immunohisto-
chemistry) can be also useful in determining which cells
respond to neurotransmitters released from motor neurons
because specific changes in postjunctional signaling pro-
teins (e.g., phosphorylation, generation of second messen-
gers, and protein translocations) can be determined.
Several studies have shown that soluble guanylate cyclase
(sGC), the physiological receptor for NO, is highly expressed
in ICC. The inhibitory effects of NO and NO-dependent en-
teric inhibitory neurotransmission in GI muscles are blocked
by inhibitors of sGC as shown in several studies by the effects
of 1H-(1,2,4)-oxadiazolo-(4,3-)quinoxaline-1-one (ODQ)
(106, 164, 429). In the rat small intestine, sGC (-subunit) is
highly expressed in ICC-DMP and colocalized with type 1
cGMP-dependent protein kinase (cGK-1) (324). NO syn-
thase (NOS)-immunoreactive neurons were found adjacent
to ICC-DMP. cGK-1 expression was also resolved in
smooth muscle cells, but very low levels of sGC-like immu-
noreactivity were found restricted to the innermost layer of
circular muscle cells. Expression of sGC (- and -subunits
probed) is also found in interstitial cells throughout the
guinea pig GI tract (175, 176). Both ICC-IM and
PDGFR cells express these proteins. Expression of
sGC- in ICC-IM and PDGFR cells was confirmed by
immunocytochemistry. NOS neurons were in close associ-
ation with cells expressing sGC. As in the rat, smooth mus-
cle cells show little or no immunoreactivity for sGC. This
observation was controlled by clear expression of sGC in
smooth muscle cells of the muscularis mucosae and in blood
vessels, confirming that the techniques used were capable of
detecting sGC in smooth muscle cells. These authors con-
cluded that sGC expression is low in smooth muscle cells in
the tunica muscularis. Functional immunohistochemical
studies showed that stimulation of nitergic neurons in GI
muscle strips or treatment with NO donors enhanced
cGMP in ICC (348, 431).
During muscarinic stimulation, PKC isozymes are translo-
cated from the cytoplasm to the membrane to elicit re-
sponses. PKC is expressed in the intramuscular ICC of the
murine small intestine (ICC-DMP), and immunohistochem-
istry was performed before and after muscarinic stimula-
tion of small intestinal muscles (398). The antibody chosen
for these studies did not recognize cytoplasmic PKC, but
after exposure to ACh or electrical field stimulation of in-
Physiol Rev VOL 94 JULY 2014 www.prv.org
trinsic motor neurons, PKC was detected in ICC-DMP.
Translocation was verified by showing a shift in PKC from
the soluble to the particulate fractions of extracts of intes-
tinal muscles. Translocation of PKC was also initiated by
phorbol ester, and muscarinic responses and neurally
evoked translocation were blocked by atropine or TTX.
These experiments directly demonstrated innervation of
ICC-DMP by cholinergic neurons.
ICC-DMP also express neurokinin 1 (NK1) receptors (127,
178, 357, 361), and binding of agonists to these receptors
on ICC causes internalization (237). NK1 antibodies were
used to test whether membrane internalization could be
elicited by stimulation of enteric motor neurons (178). Ex-
citatory motor neurons, which release both ACh and tachy-
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kinins in the GI tract, were shown to be closely associated
with ICC-DMP expressing NK1 receptors. Stimulation of
muscle strips with either exogenous substance P or by elec-
trical field stimulation led to a shift from diffuse immuno-
labeling of NK1 receptors around the periphery of cells
before stimulation to small granular structures within cells
after stimulation. Internalization of NK1 receptors in re-
sponse to field stimulation was blocked by a selective NK1
antagonist (WIN62577) or TTX.
Internalization of NK1 receptors was also exploited as a
means to separate ICC-DMP from ICC-MY in the small
intestine. Muscles were incubated with substance P labeled
with Oregon green (OG-488) before dispersing cells. Cells
were sorted for c-Kit and then again for Oregon green. Cells
with both labels were considered to be ICC-DMP, as con-
firmed by fluorescence microscopy. The cells were probed
for numerous genes known to be involved in mediation of
postjunctional responses to enteric motor neurotransmit-
ters. Genes including Chm2 and Chm3 (cholinergic re-
sponses), Tacr1 (neurokinin responses), Gucy1a3 and
Gucy1b3 (nitergic responses), P2ry1 and P2ry4 (purinergic
responses), and Vipr2 (responses to vasoactive intestinal
peptide) were expressed by ICC-DMP. Further work is nec-
essary to determine whether receptor expression by intra-
muscular ICC in various regions of the GI tract is linked to
ion channels and other effectors that contribute to motor
nerve responses.
ACh released from motor neurons in the gastric fundus
activates different postjunctional mechanisms than are ac-
tivated by muscarinic agonists added to solutions bathing
muscles (33). In this study phosphoproteins involved in
Ca2 sensitization mechanisms were analyzed before and
after addition of carbachol to bathing solutions or after
stimulation of cholinergic motor neurons. Neural release of
ACh activated only phosphorylaton of protein kinase C
(PKC)-potentiated phosphatase inhibitor protein (CPI-17),
but bath application of carbachol activated phosphoryla-
tion of CPI-17 and myosin phosphatase targeting subunit 1
(MYPT1). When acetylcholine esterase was inhibited by
881
 SANDERS ET AL.
neostigmine, activation of cholinergic neurons caused phos-
phorylation of CPI-17 and MYPT1. Stimulation of cholin-
ergic neurons in W/WV mice also caused phosphorylation
of CPI-17 and MYPT1. These data suggested that ACh
released from neurons is compartmentalized, possibly
within the tiny volumes created by the close apposition of
nerve varicosities and ICC. Due to this compartmentaliza-
tion, ACh released from neurons does not appear to reach
smooth muscle receptors linked to activation of Rho kinase
and MYPT1 phosphorylation. Inhibiting ACh metabolism
or reducing contacts between ICC and motor neurons and
the small junctional volumes formed as a result of these
contacts appears to enhance the availability of ACh and
exposure of the neurotransmitter to smooth muscle recep-
tors.
Taken together, there is significant evidence suggesting di-
rect innervation of ICC by enteric motor neurons. Studies
summarized above show spatial proximity of ICC to nerve
varicosities, loss of postjunctional responses in the absence
of ICC, expression of receptors for enteric motor neu-
rotransmitters, translocation of signaling molecules, and
internalization of receptors in ICC upon nerve stimulation.
Of course, parallel neurotransmission to other SIP syncy-
tium cells is quite possible; smooth muscle cells and
PDGFR cells also lie in close proximity to varicosities of
motor neurons. The involvement of PDGFR cells will be
discussed below. At this point it seems important to note
that there is little evidence in the literature of direct motor
innervation of smooth muscle cells before removal of ICC.
4. Studies reporting normal neuromuscular
responses in W mutants
There are reports in the literature demonstrating sustained
responses to nitrergic and cholinergic neurotransmission in
W/WV mice and Ws/Ws rats (165, 432). These findings have
led some authors to conclude that ICC are not important or
necessary for enteric motor neurotransmission. However, a
better question to ask might be whether responses to neu-
rotransmitters and other bioagonists are normal in the
absence of ICC. Even though contractile responses to neu-
rotransmitters are apparent in the absence of ICC, it is clear
that responses differ from wild-type responses. In the case
of the gastric fundus, loss of ICC led to recruitment of a
Ca2 sensitization mechanism not normally activated by
ACh released from neurons (33), thus increasing the gain on
smooth muscle contractile responses. This may help pre-
serve (or even amplify) cholinergic contractile responses in
muscles lacking ICC-IM, but it would also tend to alter
responses to all other physiological agonists, such as para-
crine substances and hormones. Such an effect might ad-
versely affect integrated motor responses in the stomach.
Ws/Ws rat fundus muscles developed significantly less
spontaneous tone than wild-type rats, and responses to
nerve stimulation were variable (165). Nerve stimulation
882 Physiol Rev VOL 94 JULY 2014 www.prv.org
resulted in contraction or relaxation under circumstances in
which relaxation responses were elicited in wild-type mus-
cles. Tone developed spontaneously in wild-type muscles,
but phasic contractions were noted in W mutants. Noncho-
linergic excitatory responses appeared elevated in Ws/Ws
rat muscles. Differences in motor patterns and variability in
the dominance of excitatory or inhibitory regulation in dif-
ferent animals indicate remodeling of neuromuscular re-
sponses in animals with mutant W alleles.
Postjunctional nitrergic responses were also reported to be
normal, or at least preserved, in a study of neuromuscular
responses in the lower esophageal sphincter (LES) of W/WV
mice (432). Some animals displayed nitrergic responses
while muscles of other animals of the same genotype did
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not. It was also reported that ICC-IM were retained in some
regions of the LES; however, the presence or absence of cells
labeled with c-Kit antibodies did not appear to correlate
with nitrergic responses. Although some muscles displayed
nitrergic components during inhibitory junction potentials,
most of the electrical responses displayed in this paper (e.g.,
see FIGS. 3 6 of Ref. 432) were similar to the responses of
LES muscles from W/WV mice (e.g., reduced nitrergic com-
ponents of IJPs and absence of membrane STDs) that had
been described previously (408).
In other studies, responses monitored by manometry were
compared in the LES of W/WV and nNOS/ mice (352).
These authors reasoned that if ICC-IM are required for
nitrergic responses, then pressure responses in the LES of
W/WV and nNOS/ mice should be very similar. How-
ever, the motor behaviors of W/WV and nNOS/ mice
were different: tone was elevated in the LES of nNOS/
mice and relaxation responses were abnormal; tone was
greatly depressed in W/WV mice and relaxation responses
were considered normal. This study seems to have neglected
previous reports showing that ICC-IM are involved in me-
diating responses to excitatory and inhibitory neural inputs
(45, 400). Therefore, one might predict that responses in
W/WV and nNOS/ mice would be quite different. W/WV
mice would be expected to have reduced LES tone due to
reduced cholinergic input. If only nitrergic inputs are com-
promised, as in nNOS/ mice, LES tone may be elevated
and relaxation responses would be greatly decreased. It
should also be noted that it is problematic to compare the
magnitudes of responses in muscles with different levels of
basal tone, and the conclusions that nitrergic responses
were normal in the LES and pyloric sphincter of W/WV mice
were based on such comparisons (351, 352).
Lesions in ICC are not homogeneous throughout the bowel
in W mutants (45, 404). At present, the relationship be-
tween loss of ICC and loss of motor function has not been
quantified, and in the case of inhibitory regulation of con-
tractions, the relationship might be very complicated. For
example, regulation of contraction by membrane potential
 INTERSTITIAL CELLS IN SMOOTH MUSCLES
is highly nonlinear and might be accomplished in the gut by
small membrane potential changes. Membrane potentials
of GI muscles, at rest in tonic muscles or at the peaks of slow
waves in phasic muscles, lie near or within the range of
potentials in which the open probability of L-type Ca2
channels is steeply dependent on voltage. Therefore, small
hyperpolarization responses can greatly reduce the open
probability of Ca2 channels and reduce Ca2 entry to
levels that do not sustain contraction. In fact, typical hyper-
polarization responses in wild-type muscles take membrane
potential far negative to the range in which the open prob-
ability of Ca2 channels is sufficient to elicit contraction.
Therefore, hyperpolarization responses might be greatly at-
tenuated before contractile responses are inhibited. Regions
of the gut with reduced ICC or reduced ICC function might
not show motor effects without a substantial reduction in
ICC. Studies have been conducted on muscles of W mutants
from regions of the gut with partial reduction in ICC. For
example, ICC were reduced but not abolished in colons of
Ws/Ws rats (4). Nitrergic components of IJPs were recorded
from approximately half of cells impaled for electrical re-
cording, and nitrergic responses were reduced or not re-
solved in the remaining cells. Tabulation of ICC at or near
each impalement was not provided, so it is unknown
whether loss-of-function correlated with a localized reduc-
tion in ICC. Other studies of this type were reviewed previ-
ously (329). It should also be noted that within a given
region of muscle (e.g., gastric fundus) there is variation in
the numbers of c-Kit cells from animal to animal. Thus
studies in the future using W mutants should include assess-
ments of ICC populations in regions from which neural
responses are recorded.
5. Cell specific inactivation of neurotransmitter
receptors or effectors
Two recent reports have utilized the Cre/loxP recombina-
tion approach to target activation or deactivation of specific
genes in ICC. c-KitCreERT2 mice were generated to target
inducible activation of iCre recombinase in ICC (200). By
crossing these mice with a strain with conditional expres-
sion of diphtheria toxin A, it was possible to reduce ICC in
adult mice. Depletion of ICC caused significant distur-
bances in GI motility. Membrane potentials of small intes-
tinal muscles were depolarized, slow wave activity was dis-
rupted, and there were significant slowing of gastric emp-
tying and intestinal transit. Mice with disrupted ICC
exhibited no excitatory or inhibitory junction potentials in
the small intestine, suggesting that reducing the number of
ICC effectively denervated the smooth muscle. In the colon,
excitatory junction potentials were greatly reduced by loss
of ICC, but effects on inhibitory junction potentials were
less affected. Targeted deletion of Prkg1, which encodes a
major effector for nitrergic neurotransmission, PKG1,
caused significant reduction in nitrergic responses and de-
layed colonic transit. In another study using ICC and
smooth muscle specific iCre strains, the 1 subunit of guan-
Physiol Rev VOL 94 JULY 2014 www.prv.org
ylate cyclase (Gucy1b3) was targeted in ICC, smooth mus-
cle cells or in both types of cells (131). Knocking down
soluble guanylate cyclase (sGC) in fundus smooth muscle
cells caused reduction in responses to a bath-applied NO
donor, but responses were not greatly affected with knock-
down of sGC in ICC. Responses to NO released from neu-
rons was not greatly affected in mice with cell-specific
knockdown of sGC in smooth muscle cells or in ICC, but
knocking down sGC in both cell types blocked most of the
nitrergic response in the fundus. These authors attributed
nitrergic responses, therefore, to combined effects on ICC
and smooth muscle cells, suggesting parallel neurotransmis-
sion to both cell types.
Animal models in which specific genes can be inactivated
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selectively in one type of cell will be powerful tools for
future exploration of the role of interstitial cells in enteric
motor functions. One must be cautious in interpreting the
responses of iCre-dependent gene inactivation models,
however, because this approach seldom results in quantita-
tive inactivation of floxed alleles, particularly when crosses
are made with homozygous mice with two floxed alleles in
the targeted cells (15). In such cases, recombination of both
floxed alleles must occur for knockout of gene transcription
in a given cell. Leaving one functional allele results in partial
depletion of gene products, as in heterozygotes. It is well
known that heterozygotes often display functions equiva-
lent to the wild-type and are frequently used as control
animals in gene inactivation studies. Assessment of the de-
gree of gene inactivation in the cells targeted for expression
of Cre or iCre is important information to include in future
studies using this technology.
6. PDGFR cells in enteric motor
neurotransmission
ICC are not the only cells in the postjunctional environ-
ment that respond to enteric motor neurotransmitters.
PDGFR cells are also closely associated with varicos-
ities of motor neurons and intermingled with ICC around
nerve processes (174, 177, 221). Isolation of colonic
PDGFR cells from Pdgfratm11(EGFP)Sor/J, a reporter
strain with eGFP expressed in cell nuclei driven off the
endogenous Pdgfra promoter (135), allowed positive selec-
tion of cells for molecular and functional studies (13, 221).
Cells from colon and stomach sorted by FACS displayed
high expression of Pdgfra, P2ry1, and Kcnn3 (encoding
SK3 channels). Under voltage clamp, PDGFR cells dia-
lyzed with 500 nM Ca2 displayed robust outward currents
that reversed at approximately EK. The outward currents
were blocked by apamin. Held at physiological resting po-
tentials, some cells generated spontaneous transient out-
ward currents (STOCs) that were blocked by apamin. Sin-
gle-channel studies showed that PDGFR cells express 10
pS Ca2-activated K channels, which is consistent with
expression of SK3.
883
 SANDERS ET AL.
Purines, such as ATP, -nicotinamide adenine dinucleotide
(-NAD), and ADP, activated Ca2-dependent K currents
under voltage clamp, and these responses were blocked by
apamin and the P2Y1 receptor antagonist MRS2500 (221).
In current-clamp mode, purines activated rapid responses
that hyperpolarized cells to EK. In contrast, responses of
colonic smooth muscle cells held at physiological resting
potentials were minimal, or small inward currents were
activated. Smooth muscle cells had small depolarization
responses to ATP in current clamp. These results show that
PDGFR cells mimic the purinergic neural responses of
whole colonic muscles (i.e., hyperpolarizations inhibited by
apamin and MRS2500; Refs. 116, 173), and these re-
sponses do not occur in smooth muscle cells at physiological
potentials. ICC also show little or no response to purines
that can explain purinergic responses in situ. Thus
PDGFR cells are the most likely postjunctional cells to
mediate purinergic motor neurotransmission. PDGFR
cells may also contribute to tonic inhibition in the colon.
PDGFR cells generate STOCs, and this could provide a
net stabilizing effect on the excitability of smooth muscle
cells. It should also be noted that PDGFR cells express
sGC (175, 176), but a role for NO/cGMP-dependent mech-
anisms in these cells has not been identified.
Activation of SK3 currents in PDGFR cells would require
dynamic cytoplasmic Ca2 signaling. The occurrence of
STOCs in these cells suggests that periodic, localized Ca2
release from intracellular stores is an important mechanism.
Ca2 signaling mechanisms were investigated in PDGFR
cells of gastric fundus muscles in situ (13). Cells in fundus
muscles of Pdgfratm11(EGFP)Sor/J mice were loaded with fluo
4 and identified by nuclear eGFP. Spontaneous localized
Ca2 transients were observed in PDGFR cells. Purines
(ATP, ADP, UTP, and -NAD) and the P2Y1 agonist MRS-
2365 enhanced Ca2 transients, and these responses were
blocked by MRS-2500, a specific P2Y1 antagonist. ADP,
MRS-2365, and -NAD failed to elicit Ca2 transients in
cells of P2ry1/ mice, but responses to ATP persisted.
Phospholipase C (U-73122), IP3 receptor (2-APB), ryano-
dine receptor (ryanodine), and SERCA pump (cyclopia-
zonic acid and thapsigargin) inhibitors blocked Ca2 tran-
sients evoked by purines. Activation and regulation of Ca2
transients are likely to mediate the openings of SK3 chan-
nels, generation of STOCs and responses to purines, and
purinergic inhibitory junction potentials in GI muscles.
7. Neuromuscular transmission and integration in
the SIP syncytium
As discussed above, the extent to which motor neurotrans-
mission is volume-like or more focalized (e.g., synaptic-
like due to close appositions between varicosities and
postjunctional cells or restrictions on transmitter diffusion
due to metabolism or uptake) is controversial at present.
What seems not debatable, based on current knowledge, is
that at least three postjunctional cell types are clustered
884 Physiol Rev VOL 94 JULY 2014 www.prv.org
within 100 nm of nerve varicosities in GI muscles, and each
of these cells expresses specialized receptors and effectors
that respond to enteric motor neurotransmitters. Binding of
neurotransmitter molecules to ICC and PDGFR cell re-
ceptors activate or modulate ionic conductances, as de-
scribed above, and these cells are electrically coupled to
smooth muscle cells by gap junctions. Thus the neuroeffec-
tor junction in GI muscles is more accurately conceptual-
ized as the SIP syncytium, composed of electrically coupled
smooth muscle cells, ICC, and PDGFR cells (FIGURE 3)
(209, 330). Changes in the conductance of any type of SIP
cell can affect the input resistance of the syncytium. To-
gether SIP cells not only determine the integrated excitabil-
ity of muscle layers and responses to neuotransmitters, but
it is quite likely that responses to hormones and paracrine
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mediators are also integrated through the responses of the
SIP syncytium. Classically, terms such as myogenic,
neurogenic, and neuroeffector junction used to de-
scribe different regulatory mechanisms in GI tissues have
not been inclusive of the important responses elicited by
interstitial cells.
G. ICC as Stretch Receptors in GI Muscles
The walls of GI organs go through dramatic distortions
while filling and during the digestion and movement of food
and wastes. Stretch-dependent mechanisms have been de-
tected in neurons (84, 219, 358, 359) and smooth muscle
cells (97), but interstitial cells may also have an important
sensory role in transducing mechanical events and pro-
viding immediate feedback affecting motility. Freshly iso-
lated ICC from human intestine displayed a Na current in
patch-clamp experiments (362). The conductance was
blocked by QX-314 but not by nifedipine or Ni2. Tran-
scripts for SCN5A, which encode subunits of cardiac
TTX-resistant Na channels, were found by single-cell
PCR. Stretch of intestinal muscle increased the frequency of
slow waves, and QX-314 or lidocaine reduced slow wave
frequency. These data suggest that pacemaker ICC express
a mechanosensitive Na conductance that regulates slow
wave frequency.
Another study investigated the chronotropic effects of
stretch in the murine gastric antrum (417). Slow waves were
recorded from antral muscles during computerized applica-
tion of muscle stretch at a rate of 6.0 m/s to a maximum
tension of 5 mN. This increased muscle length by 27%,
which is within what might be expected physiologically
during antral contractions. Stretch of antral muscles caused
4 mV of depolarization and increased slow wave frequency
from 2.5 to 3.6 cpm. The magnitude of the response to
stretch was positively related to the rate of muscle stretch.
Stretch responses were blocked by reduced extracellular
Ca2 and by Ni2, but unaffected by nifedipine. Responses
to stretch were not neurogenic because they were not
blocked by TTX. Acceleration of antral slow waves dis-
 INTERSTITIAL CELLS IN SMOOTH MUSCLES
rupted corpus to antrum slow wave propagation temporar-
ily. Stretch responses were not recorded in muscles of
W/WV mice, suggesting that ICC-IM may mediate stretch-
dependent chronotropic effects. It was also found that in-
domethacin blocked stretch-dependent chronotropic re-
sponses, and these responses were also not present in mice
lacking cyclooxygenase-2 (Ptgs2/). These findings sug-
gest that stretch may activate phospholipase A2 and gener-
ate arachidonic acid. Synthesis of PGE2, a product of
COX-2, may drive stretch-dependent responses in ICC-IM,
since this prostanoid has been shown to have positive chro-
notropic effects in antral muscles of several species (103,
327, 333).
H. Interactions With Afferent Nerve Fibers
There are at least two types of mechanoreceptor structures
arising from extrinsic afferent neurons in smooth muscles of
GI organs: intraganglionic laminar endings (IGLEs) and
intramuscular arrays (IMAs). The morphology and intercel-
lular associations of IMAs have been carefully described in
recent years; however, the function of these structures is still
poorly understood (294). IMAs arborize into a complex
array of terminal processes that run in parallel to the
smooth muscle fibers and are associated with ICC-IM in the
stomach (32, 295). IMAs are also associated with three
species of efferent fibers, distinguished by antibodies to
nNOS (inhibitory motor neurons), vesicular acetylcholine
transporter (excitatory motor neurons), and tyrosine hy-
droxylase (sympathetic neurons). Calcitonin gene-related
peptide (CGRP)-immunopositive afferent fibers (presumed
to be collaterals of visceral afferent neurons) are also asso-
ciated with IMAs (294). The varicosities of IMAs form
synaptic-like contacts with ICC and display vesicles and
prejunctional densities (166, 294). The ICC-IMA complex
has been likened to a functional correlate of the muscle
spindle organ of striated muscles. The function of ICC in
relation to IMAs has not been clarified. There is evidence
that vagal afferents can engage in axon reflexes (414),
which is accomplished by branches of peripheral axons that
contain both sensory and effector functions without the
benefit of any synapses or an integration center. In the case
of ICC-IMA complexes, the numerous lamellar varicosities
of IMA neurites distributed around ICC and close associa-
tions with CGRP-immunopositive processes may, in fact,
innervate ICC, providing an integrative sensorimotor func-
tion to supplement the main sensory activities of afferent
neurons. Physiological tests of this hypothesis have not been
reported.
There is also a report of innervation of ICC-MY by intrinsic
primary afferent neurons (IPANs) in the small intestine.
IPANs are myenteric neurons that send processes to the
lamina propria to sense the mechanical and chemical state
of the gut lumen. These neurons provide the afferent limb of
intrinsic GI reflexes (109). IPANs are Dogiel type II neurons
Physiol Rev VOL 94 JULY 2014 www.prv.org
that have a characteristic electrophysiology signature of
after-hyperpolarization (AH) following action poten-
tials. Therefore, IPANs are also known as AH neurons.
Identified AH neurons were described as making close con-
tacts with ICC-MY, and depolarization of the neurons cor-
related with modulation of Ca2 transients in ICC-MY
(438). These authors concluded that direct inputs from AH
neurons regulate ICC-MY function.
III. DEVELOPMENT AND PLASTICITY OF
ICC
A. Role of c-Kit in Development of ICC
Networks
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ICC are of mesodermal origin (238, 379, 430) and de-
rived from cells within the gut. Cells expressing tran-
scripts of Kit appear on about embryonic day 9 (E9) in
mice (280), and c-Kit protein expression is detected by
immunohistochemistry at E12 (379). Initially c-Kit cells
form a layer of undifferentiated mesenchymal cells
around the periphery of the intestine (at E12). c-Ret
cells, which are precursors of enteric neurons and glia,
form a distinct layer at the inner surface of the c-Kit
cells. Circular muscle cells are apparent at E15, but at
this stage the intestine lacks a longitudinal muscle layer.
Within the circular muscle layer, smooth muscle anti-
gens, such as enteric actin and desmin, are prominent,
and the cells have typical ultrastructural features such as
thin and thick myofilaments, intermediate filaments,
dense bodies, caveolae, and well-developed endoplasmic
reticula. c-Kit cells are largely immunopositive for vi-
mentin, but a few cells begin to develop immunoreactiv-
ity for desmin at E15, suggesting that cells of the longi-
tudinal muscle layer may develop from c-Kit precursors
(379). Others reached a similar conclusion by showing
with in situ hybridization that cells at the periphery of the
small intestine that expressed Kit mRNA coexpressed
myosin heavy chain transcripts (203). By E18, a clear
longitudinal muscle layer is formed and c-Kit cells de-
veloped processes and formed a lose network in the re-
gion of the myenteric plexus (ICC-MY). Longitudinal
muscle cells express desmin, and ICC retain expression of
vimentin. The other population of ICC in the small bowel
(ICC-DMP) is not found in murine embryos and develops
within the first week after birth.
The heterogeneity of ICC lesions in W/WV mice (404) led to
speculation that c-Kit is not required for development of all
ICC. Some populations of ICC fail to develop with the loss
of function in W/WV mutants, but other populations of ICC
develop, albeit usually in reduced density and with reduced
immunoreactivity to c-Kit antibodies. These observations
suggest that ICC from different regions of the GI tract and
even within specific regions have different sensitivities to
885
 SANDERS ET AL.
c-Kit signaling. Another possible difference might be in sur-
vivability of different ICC in the absence of c-Kit. One study
investigated the development of ICC in animals with Kit
alleles deactivated by knock-in of LacZ into the Kit locus.
Homozygotes for the transgene (KitLacZ/) expressed c-Kit
and ICC normally, but KitLacZ/LacZ mice lacked c-Kit but
cells identified as ICC were as abundant as in KitLacZ/ mice
(31). It is possible, however, that the cells identified as ICC
were precursors for ICC identified in development by E15
(379). Whether the cells identified as ICC (at about E18)
were functional was not determined. Another group inves-
tigated the distribution of ICC in Wbanded (Wbd) mice (203).
Wbd contains a genomic rearrangement of chromosome 5
that results in inversion of the c-kit gene and ectopic expres-
sion of Kit (204). These mice displayed normal distributions
of cells identified as ICC at D5, but adult animals lacked
ICC. The authors concluded that ICC did not require sig-
naling via c-Kit for development, but functional c-Kit was
required for maintenance of the ICC phenotype as animals
matured. Identification of cells as ICC was performed by
methylene blue staining, but there was no verification that
the cells labeled with methylene blue were ICC. Electrical
recordings failed to demonstrate slow wave activity in Wbd/
Wbd mice, suggesting that the ICC pacemaker phenotype
never developed. Thus the question is whether ICC devel-
oped in Wbd/Wbd mice or whether another cell type, labeled
by methylene blue, was identified as ICC in these mutants.
For example, it has never been determined whether meth-
ylene blue labels ICC exclusively or whether PDGFR
cells are also labeled by this dye.
The question of the role of c-Kit in development of func-
tional ICC during the late embryonic period was addressed
in another study by treating muscles with neutralizing c-Kit
antibodies from E15 through birth. Functional ICC develop
in small intestinal segments in organotypic cultures started
at E15; however, inclusion of c-Kit neutralizing antibodies
blocked development of c-Kit positive cells and pacemaker
activity (406). It was also observed that ICC-MY and slow
waves developed normally in WV/ and in W/ heterozy-
gotes, but fail to develop between E17 and birth (postnatal
day 0; P0) in W/WV embryos (23). Similar results, including
loss of ICC and failure of pacemaker development, oc-
curred in muscles treated for several days with the tyrosine
kinase inhibitor imatinib mesylate (STI157). These data
support the concept that ICC precursors cells might emerge
in animals with compromised c-Kit function, but subse-
quent development into mature and functional ICC re-
quires c-Kit signaling.
The reasons why ICC develop within specific planes in the
tunica muscularis (e.g., ICC-MY and ICC-SM) or in asso-
ciation with specific structures, such as enteric neurons
(ICC-DMP or ICC-IM), are poorly understood, but the pat-
terns of ICC localization are conserved, with minor varia-
tions, in mammalian species and fish (14, 210, 307). ICC
886 Physiol Rev VOL 94 JULY 2014 www.prv.org
require signaling via c-Kit for development (23, 306, 404),
so one explanation for their localization within muscles
might be that they are concentrated near cells expressing
stem cell factor, the ligand for c-Kit. Experiments on trans-
genic animals with lacZ linked to the promoter for stem cell
factor showed that enteric neurons express stem cell factor
(380), but others have reported that smooth muscle cells
also express stem cell factor transcripts and protein (161,
238, 422). Expression of stem cell factor by neurons might
direct positioning of ICC, because either nerve cell bodies or
processes occupy most of the same niches as ICC. However,
it is unclear how smooth muscle expression of stem cell
factor might direct the localization of ICC with such precise
patterning. In fact, experimental evidence suggests that neu-
rons, and thus trophic factors expressed by neurons, are not
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obligatory for development or localization of ICC, because
ICC developed in muscles taken for culture from mamma-
lian and bird intestines before colonization of the muscles
with neural crest cells (238, 430). Functional ICC also de-
veloped in c-Ret/ mice that lack enteric neurons below
the proximal stomach (409). Therefore, significant ques-
tions remain about the signals and sources of signals direct-
ing the positioning, architecture, and connectivity of inter-
stitial cell networks.
Significant development of ICC occurs in mice during the
postnatal period. At P0, ICC-MY form a loose network
with cells displaying a few short and branching processes.
The density of ICC-MY at P0 was 750 mm2. The de-
mands of maintaining the structure of ICC networks as
bowel diameter expands require proliferation of ICC. An
age-dependent proliferation of ICC-MY, estimated to be
15-fold from neonates to adulthood, maintained the rel-
ative density of these cells in adult animals (259). The pro-
liferative potential of ICC was studied by incorporation of
5-bromo-2-deoxyuridine (BrdU). A large number of cells
within ICC networks were labeled with BrdU shortly after
birth, but the number of cells incorporating BrdU, 24 h after
loading, decreased as a function of age. Cells with short
processes, but apparently already integrated into ICC-MY
networks, incorporated BrdU within 1 h after injections,
and pairs of cells with BrdU were noted after 24 h. It was
difficult to find proliferating cells after P24. The fraction of
total cells that incorporated BrdU was also determined by
daily injections, and this number increased to 64% by P24.
The expansion of ICC-MY appeared to be due to prolifer-
ating cells within ICC networks (259), as opposed to pro-
liferation from a rare population of KitlowCD44
CD34IGF-1 cells, proposed by others as the progenitors
of ICC (248). It should be noted that most cells within the
ICC-MY network were CD44, and some cells showed
weak labeling with antibodies to CD34 and insulin-like
growth factor I (IGF-I) (259). These findings tend to suggest
that KitCD44CD34IGF-1 cells are normal compo-
nents of ICC-MY networks and do not necessarily represent
a specialized population of ICC progenitors.
 INTERSTITIAL CELLS IN SMOOTH MUSCLES
Early in human development (7 wk), c-Kit cells can be
found along the entire length of the esophagus in the region
of the myenteric plexus, but this distribution changes with
age such that ICC were far more abundant in the distal
one-third versus the proximal esophagus by weeks 1316
(300). After 20 wk, ICC were extremely rare in the proxi-
mal (skeletal muscle) portion of the esophagus and had
developed into spindle-shaped ICC-IM in the circular and
longitudinal muscle layers in the distal third (smooth mus-
cle) portion of the esophagus. The middle third of the
esophagus displayed ICC in septa between smooth and skel-
etal muscle fibers. ICC developed over approximately the
same window of time in the stomach, forming first a layer of
c-Kit precursors around the periphery of the organ adja-
cent to the developing myenteric plexus (301). By 20 wk,
ICC can be found in all of the adult niches: ICC-MY, ICC-
IM, and ICC-SEP. ICC develop in the human midgut and
hindgut somewhat later than in the proximal GI tract. At 9
wk, c-Kit labeling is present at low levels within the circular
muscle (393). By week 11, c-Kit labeling is present along the
inner and outer aspects of myenteric ganglia. At 14 wk, ICC
displayed an approximately adultlike distribution. In the
distal colon, c-Kit cells emerged during weeks 10 11.
Two parallel bands of cells were found within the myenteric
region between the circular and longitudinal muscle layers
and near the submucosal edge of the circular muscle (299).
The proximal to distal delay in the development of ICC in
the human GI tract is also a feature of the development of
ICC and pacemaker activity in the mouse (379, 406).
B. Development of Electrical Rhythmicity in
ICC
A lineage decision is made at about E15 in the murine
intestine, and cells that continue to express c-Kit develop
pacemaker activity (379). Electrophysiological studies of
jejunal muscles during the late embryonic period found no
electrical slow waves at E17, but small-amplitude fluctua-
tions in membrane potentials (STDs) were observed. Net-
works of ICC were well-articulated at E17, and multiple
processes linking cells together were observed (23, 379,
406). At E18, slow waves, resistant to block by dihydro-
pyridines (a signature feature of slow waves vs. action
potentials), were recorded by intracellular microelectrode
techniques (23). The organization, frequency, and ampli-
tude of slow waves continued to develop through birth and
the first week after birth, reaching mature amplitude and
frequencies by about P10. Other investigators set the onset
of electrical rhythmicity in the proximal small intestine later
in development, finding weak electrical activity in unfed
neonates and strong reduction in the electrical activity by
verapamil (246). These authors concluded that ICC-MY
were not functional at birth, and pacemaker activity and
slow waves did not develop until about P2. ICC-MY were
identified with methylene blue in this study, but this dye is
Physiol Rev VOL 94 JULY 2014 www.prv.org
less reliable than immunohistochemical techniques (see
sect. IIA1).
Consistent with the role of ICC as pacemakers, there is a
proximal-to-distal delay in the developmental of pace-
maker activity (406). Slow waves were recorded from the
proximal GI tract (stomach and antrum) during the late
embryonic period (as described above), but developed after
birth in the ileum and colon. Slow wave activity with clus-
ters of action potentials superimposed, characteristic of the
proximal colon, developed in colonic muscles by P6-P8;
however, this activity was infrequent, with long periods of
quiescence between events. Regular, rhythmic slow waves
and action potential clusters were not fully developed in the
proximal colon until about P10.
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Development of ICC and slow waves occurs unfettered in
organotypic cultures of GI muscles (23, 406), and this ap-
proach has been utilized to correlate the timing of ICC-MY
development and the development of electrical rhythmicity
and to evaluate the impact of c-Kit signaling on develop-
ment. Inclusion of neutralizing c-Kit antibodies in muscles
of neonatal animals abolished ICC networks and blocked
pacemaker activity within a few days of exposure. At birth,
as observed in animals, ICC were much more susceptible to
effects of neutralizing c-Kit antibodies than after developing
in animals for several days before being put into organ
culture (406). Longer periods (e.g., 30 days) of exposure
to c-Kit antibodies were required to lesion ICC-MY net-
works and block pacemaker activity in muscles from ma-
ture (P20) animals. Since the dependence on c-Kit signal-
ing for initial development of ICC-MY was challenged by
others (31, 203), experiments were also performed to deter-
mine whether functional ICC developed with c-Kit blocked
or disabled genetically. ICC-MY and slow waves developed
in jejunal muscles of wild-type, W/, and WV/ mice by
E18, but failed to develop in muscles of W/WV muscles.
ICC-MY failed to develop in jejunal muscles removed from
mice at E17 (i.e., before slow waves develop) and cultured
with neutralizing c-Kit antibodies (23). Similar results were
obtained if muscles were put into organ culture after slow
waves developed (E18-P0). Imatinib mesylate (STI157), a
c-Kit inhibitor, had no acute effect on slow waves, but ex-
posure to this compound blocked postnatal growth and
development of ICC-MY and slow waves. Thus c-Kit sig-
naling during development is requisite for establishment
and maintenance of the pacemaker function of ICC.
C. Fate of ICC When c-Kit Is Blocked
Loss of ICC has been observed in human GI muscles from
patients with a variety of motility disorders (11, 43, 143,
168, 205, 385). Dysmotilities seem an obvious consequence
of defective ICC or loss of ICC due to the important func-
tions of these cells. In spite of the clinical significance, the
factors responsible for ICC loss in patients are poorly un-
887
 SANDERS ET AL.
derstood. Attempts have been made to develop animal
models of ICC loss with the idea of establishing the cause
and effect of ICC loss in motility disorders and to study the
plasticity of ICC.
ICC are vulnerable to block of c-Kit signaling in neonatal
and adult animals; however, the time required for c-Kit
block increases from the postnatal period to adulthood.
Neutralizing c-Kit antibodies injected into neonatal mice
and for several days after birth cause loss of ICC within a
week (250, 378), and all classes of ICC are affected. The
c-Kit inhibitor imatinib mesylate also lesions ICC networks
in neonatal murine intestinal muscles in organ culture (23).
In adult guinea pigs, imatinib caused a time-dependent re-
duction in ICC-MY of the small intestine, shortening of cell
processes, and generalized thinning of the network (258).
The number of ICC-MY was reduced by about one-third
after 16 days of treatment. ICC-DMP were also affected,
but effects were less dramatic and slower in onset. No evi-
dence of apoptosis was noted in ICC after treatment with
imatinib; instead, remaining cells coexpressed -smooth
muscle actin, suggesting that ICC-MY undergo a pheno-
typic change toward a smooth muscle or myofibroblast phe-
notype when c-Kit is blocked. BruD incorporation was ob-
served in ICC-MY after withdrawal of imatinib, and pairs
of cells with highly developed processes that were well-
incorporated in ICC-MY networks demonstrated that cell
division of mature ICC was at least partially responsible for
repopulation of ICC-MY networks after ending c-Kit
blockade (258).
Downstream cell signaling initiated by stem cell factor bind-
ing to c-Kit includes contributions from phosphatidylinosi-
tol 3-kinase (PI3-kinase), phospholipase C- (PLC-), defects in ICC are described below.
phospholipase D, SRC family kinases, p21ras GTPase-acti-
vating protein, and mitogen-activated protein kinase
(MAPK) (244). There are multiple classes of PI3-kinase, but
growth factor receptors typically activate class IA. Binding
of stem cell factor leads to autophosphorylation and
dimerization of c-Kit and generation of high-affinity bind-
ing sites for signaling molecules, including PI3-kinase.
Phosphorylation of c-Kit leads to binding of the p85 regu-
latory subunit of PI3-kinase and subsequent activation of
the catalytic subunit p110. The effects of PI3-kinase inhib-
itors, wortmannin and LY 294002, were tested on develop-
ment of ICC and pacemaker activity in mice. The amplitude
of slow waves increases after birth, and this development
also occurs in organotypic cultures (406). Inclusion of wort-
mannin or LY 294002 in organ cultures of intestinal mus-
cles taken from animals at birth caused loss of ICC-MY and
blocked slow waves within a few days (401). Pacemaker
activity was also blocked in adult muscles by PI3-kinase
inhibitors, but up to a month of exposure was required to
develop these effects. Another study employed a transgenic
mouse with a tyrosine 719 to phenylalanine (Y719F) mu-
tation in Kit (119). No loss of ICC function was observed in
888 Physiol Rev VOL 94 JULY 2014 www.prv.org
these mice, and it was concluded that signaling through
PI3-kinase is not required for development and function of
ICC. There are two means of activating PI3-kinase, how-
ever, by direct binding of PI-3kinase to the phorphorylated
tyrosine residue of c-Kit and indirectly by binding to the
tyrosine phosphorylated accessory protein GAB2 (271).
Thus the Y719F mutation in Kit may not block all PI3-
kinase signaling.
ICC-MY networks and slow wave activity can be reestab-
lished after removal of conditions or reagents that block
c-Kit. This point will be more completely illustrated in the
next section, but model studies of this concept have been
performed on muscle strips in organ culture. As above,
treatment of P0 muscles with neutralizing c-Kit antibodies
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or imatinib mesylate for 3 days caused loss or dramatic
reduction in ICC-MY and loss of pacemaker activity. After
removal of these reagents, ICC networks and slow waves
were restored within 9 days (23).
D. Animal Models of ICC Loss and
Pathophysiology
In addition to Kit mutants and animals in which c-Kit is
blocked, ICC loss has been reported in several animal mod-
els of GI motility disorders. These are important because
parallel examples of ICC loss occur in similar or equivalent
diseases in human patients. It should be noted that if a
common factor is responsible for ICC loss in various disease
states, it has not been identified. Furthermore, a debate
exists about whether functional ICC are lost through cell
death or by remodeling of the ICC phenotype. Examples of
animal models with predictable, and in some cases reversal,
1. Bowel obstruction
Partial obstruction of the bowel leads to marked distension,
hypertrophy, and hyperplasia of smooth muscle cells, and
thickening of the muscle layers of the bowel proximal to the
obstruction (111, 112). Studies of a rat model of partial
obstruction showed remodeling of enteric neurons and re-
duced numbers of ICC in the hypertrophic region of bowel
(91). In mice, partial obstruction resulted in hypertrophy in
a long segment of small intestine and a lesion in ICC net-
works that was nearly complete a few millimeters above the
obstruction but decreased in severity over 100 mm of bowel
(51). In specific planes of the tunica muscularis normally
occupied by ICC, cells with ultrastructural features more
typical of fibroblasts were observed, but there was no evi-
dence of cell death or necrosis (e.g., shrinking, condensed or
fragmented nuclei, indicative of apoptosis, or cell swelling
or bursting) was observed in any region where ICC were
lost. Slow waves were greatly reduced in amplitude or ab-
sent in the section of bowel with the most extensive loss of
ICC and responses to neural inputs were also greatly re-
 INTERSTITIAL CELLS IN SMOOTH MUSCLES
duced in these regions. ICC, slow wave generation, and
neural responses recovered in 1 mo if the obstruction was
relieved.
2. Diabetes
Reduction in ICC in diabetes and the importance of this
phenomenon in diabetic gastroparesis has been a topic of
interest since defects in ICC networks were noted in a
mouse model of type I diabetes. Non-obese diabetic mice
(NOD/LtJ) mice were shown to have delayed gastric emp-
tying and reduced and arrhythmic slow wave activity in the
antrum (278). Fundus muscles from NOD/LtJ mice were
less responsive to activation by enteric motor neurons,
which might be associated with reduced gastric accommo-
dation during ingestion of food. Functional defects were
associated with reduced ICC in the antrum and loss of close
connectivity between motor neurons and ICC in the fundus.
Another study confirmed the link between loss of ICC and
development of delayed gastric emptying, and showed that
loss of Kit expression, the marker for ICC, was the most
consistent correlate for defects in gastric emptying (57).
These authors noted that hemeoxygenase-1 (HO1) expres-
sion, which normally is expressed at low levels in the tunica
muscularis, increased as diabetes developed. Mice that
maintained expression of HO1 were protected from devel-
opment of delayed gastric emptying, and mice that failed to
maintain levels of HO1 developed emptying delays. It was
suggested that the benefit of HO1 in diabetes may be to
protect ICC against enhanced oxidative stress. An exciting
finding was that induction of HO1 expression by hemin
rescued mice that had developed delayed gastric emptying.
Resident macrophages, which lie in close proximity to ICC,
were identified as the cells with enhanced expression of
HO1 in diabetes. The protective effects of HO1 on gastric
emptying appear to be due to the production of carbon
monoxide (CO) (190). Inhalation of CO by NOD/LtJ mice
with delayed gastric emptying, reduced oxidative stress, en-
hanced Kit expression, and reversed delayed gastric empty-
ing.
The density of ICC in the distal stomach was also reduced in
streptozotocin-induced diabetes in rats, and loss of connec-
tivity between nerves and ICC-IM was observed in the fun-
dus (395). These changes were associated with decreased
gastric emptying in diabetic animals. Others have reported
similar finding for streptozotocin-induced diabetes in rats
and found significant loss of ICC in the antrum (267). These
authors also found an association between retention of ICC
and upregulation of HO1 expression. For example, HO1
expression was enhanced in the corpus and ICC networks
were essentially unchanged in that region. Antral ICC were
depressed, and this was associated with no enhancement in
expression of HO1. Loss of ICC was reversed by induction
of HO1 expression by cobalt protoporphyrin.
Physiol Rev VOL 94 JULY 2014 www.prv.org
The reasons for loss of ICC in NOD/LtJ mice have been
investigated. Reduced IGF-I and insulin signaling, and not
hyperglycemia, were found to be responsible for the loss of
ICC in diabetes (162). Expression of insulin and IGF-I re-
ceptors were localized to enteric neurons and smooth mus-
cle cells, and transcripts for these receptors were not re-
solved in ICC purified by FACS (161). The same cells that
expressed IGF-I and insulin receptors also expressed stem
cell factor. Loss of stem cell factor was found to be the cause
of ICC loss in diabetic tissues. Therefore, signaling via IGF-I
and insulin does not occur directly through ICC, but ICC
maintenance depends on these growth factor receptors, as
they are important in maintaining expression of stem cell
factor in cells adjacent to ICC.
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Type II diabetes is by far the most prominent form in human
patients. C57BL/KsJdb/db mice, a monogenic model of type
II diabetes, were examined for gastric emptying and damage
to ICC networks (421). c-Kit cell density and expression
of stem cell factor were also evaluated. Compared with
db/ mice, db/db had delayed gastric emptying as well as
prolonged gut transit times, and contractile activity was
irregular in frequency. Cells with c-Kit immunoreactivity
were reduced in antrum, small intestine, and colon, as was
expression of stem cell factor. These authors concluded that
loss of ICC contributes to the motility defects in type II
diabetes.
Reduced ICC numbers have also been reported in samples
of antral muscles from full thickness gastric biopsies from
human patients symptomatic of diabetic and nondiabetic
gastroparesis (105, 132, 133, 184). An interesting finding in
human samples was a shift in splice variants of ANO1 in
ICC from patients with gastroparesis (252). A novel splice
variant that lacked exons 1 and 2 and a portion of exon 3 of
ANO1 was observed in patients gastroparesis. ANO1 en-
codes the ion channel responsible for pacemaker currents in
ICC (see sect. IID2), and a shift in splice variant expression
might affect the kinetics or frequency of slow waves. Ex-
pression of the unique splice variant from diabetic tissues in
HEK cells resulted in decreased conductance and slower
kinetics than displayed by full-length ANO1 channels.
3. Surgical resection of the bowel
Acute inflammatory responses to surgery, such as bowel
resection, have also been linked to adverse effects on ICC
and their associated functions. Resection of murine small
intestine caused loss of c-Kit-like immunoreactivity, loss of
slow waves, attenuated neural responses, and abnormal re-
sponsiveness to carbachol (425). The loss of c-Kit immuno-
reactivity was shown to be due to loss of ICC via electron
microscopy. The lesion in ICC and loss of function de-
creased as a function of distance (5 cm) above and below the
point of the anastomosis. Loss of ICC was acute and par-
tially recovered within 24 h of surgery. Recovery was more
rapid when muscles were treated with an iNOS inhibitor,
889
 SANDERS ET AL.
6
L-N -(1-iminoethyl)lysine hydrochloride (L-NIL). Animals
treated preoperatively with iNOS inhibitors experienced re-
duced pacemaker activity only near the site of the anasto-
mosis, and transgenic animals lacking iNOS were protected
against most of the postsurgical defects in ICC networks
and pacemaker activity (424).
E. Cancer
Kit is a protooncogene encoding a receptor tyrosine kinase.
Gain-of-function mutations in the juxtamembrane domain
of Kit are found in 75 80% of GISTs, the most common
form of human sarcoma, and the idea that GISTs derive
from ICC has been considered (149, 150, 195, 272). Jux-
tamembrane domain mutations discovered in GISTs render
c-Kit constitutively active, and expression of the mutated
isoforms in cells resulted in unregulated growth (149). Im-
munostaining with antibodies to c-Kit has been an impor-
tant advance in categorizing mesenchymal tumors, and re-
alizing that c-Kit drives the growth of GISTs has afforded a
therapeutic approach to controlling GISTs. Activating mu-
tations in PDGFR have also been identified in GISTs. Thus
it is also possible that cells of the PDGFR cell lineage
could contribute to development of GISTs. Inhibition of
c-Kit or PDGFR with tyrosine kinase inhibitors reduces
progression of GISTs, but these drugs need to be maintained
for indefinite periods for effectiveness. This has suggested
that sustained growth of GISTs may be due to proliferation
of cancer stem cells that are not dependent on signaling via
c-Kit for survival.
Mice with a familial mutation in c-Kit, KitV558/, develop
GISTs and die prematurely from pathology of the GI tract.
These mice were used to determine the fate of ICC and ICC
functions during GIST development (224). Most classes of
ICC developed marked hyperplasia throughout the GI
tracts of KitV558/ mice. Normal pacemaker activity and
responses to enteric motor neurons were conserved in tis-
sues during development of GISTs. If GISTs develop from
mature ICC, one would think that hyperplasia of these cells
would result in enhanced frequency and amplitude of STICs
and slow waves due to an expansion in the pacemaker pop-
ulation. The fact that rhythmicity was not enhanced in
KitV558/ mice suggests that either the hyperplasia associ-
ated with development of GISTs is accompanied by reduc-
tion in the spontaneous rhythmicity or that GISTs develop
from c-Kit cells that are not spontaneously active (i.e.,
immature ICC or ICC precursor cells).
Although the lineage of ICC has been suggested to be the
source of GISTs, the exact cells from which GISTs are de-
rived is controversial (312). Cells characterized as Kitlow
have been suggested as progenitor cells for ICC (248). These
cells also express CD44, CD34, insulin receptors, and
IGF-I. Signaling via c-Kit might be necessary for differenti-
ation of the progenitor cells into ICC, but not for their
890 Physiol Rev VOL 94 JULY 2014 www.prv.org
survival. Thus gain-of-function mutations in c-Kit in the
progenitor cells could yield continuous output of malignant
GIST cells that is not controlled by blocking the tyrosine
kinase activity of c-Kit (16). It was suggested that Kitlow
Cd44 Cd34 ICC stem cells are the source of GIST cells.
These are c-Kit independent stem cells that are not suscep-
tible to therapeutic approaches designed to inhibit c-Kit.
IV. INTERSTITIAL CELLS IN THE URINARY
TRACT
Smooth muscle tissues of the urinary tract generate sponta-
neous phasic contractions and tone. The intrinsic electrical
and contractile activities appear to be generated by special-
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ized pacemaker cells. Electrical and contractile activities are
also regulated by neurotransmitters, and interstitial cells
may be involved in transduction of neural inputs. Electrical
activity generated by specialized cells may propagate to
smooth muscle cells, control membrane excitability, and
generate phasic contractile activity. Two types of special-
ized cells have been reported: c-Kit and c-Kit cells. Re-
cently, PDGFR cells have been shown to be at least part
of the c-Kit interstitial cell population. In the following
sections, the distributions and physiological and patho-
physiological roles suggested for interstitial cells of the up-
per and lower urinary tract are discussed.
A. Renal Pelvis and Ureter
Pacemaker activity is generated in the most proximal re-
gions of pyeloureteric tissues, and this initiates peristaltic
contractions that spread toward the distal ureter. The cells
and ionic mechanisms responsible for pacemaker activity in
the upper urinary tract have received considerable study.
Two populations of specialized cells were observed in the
renal pelvis, and their ability to generate pacemaker activity
has been investigated (232). Short, spindle-shaped cells, re-
ferred to as atypical smooth muscle cells (ASMC), are lo-
cated in the proximal region of ureteropelvic junction.
These cells generate spontaneous electrical rhythmicity
(123, 124, 201). Another type of cell, referred to as ICC-like
cells (ICC-LC), has also been described. c-Kit immunoposi-
tive cells have been reported in the upper urinary tract (74,
231, 260, 261, 286, 356), but c-Kit immmunoreactivity is
not consistent in all species (e.g., ICC-LC in guinea pig
pyelouretic system were not labeled by c-Kit antibodies;
Ref. 201). ICC-LC are mainly distributed in more distal
regions of the ureteropelvic junction. ICC-LC may be in-
volved in generation of pyelrouteric pacemaker activity
(201, 232). Both ASMC (13pF) and ICC-LC (17 pF) are
smaller than SMC (20 pF) based on cell capacitance mea-
surements (181).
ASMC and ICC-LC cells display high (10 40/min) and low
(13/min) frequency of Ca2 transients, respectively (228,
 INTERSTITIAL CELLS IN SMOOTH MUSCLES
229). Based on the frequency of Ca2 transients and spon-
taneous transient depolarizations (STD), the dominant
pacemaker cells may be ASMC. Spontaneous Ca2 tran-
sients and STDs were inhibited in ASMC when extracellular
Ca2 was reduced or by application of cyclopiazonic acid
(CPA) and 2-APB (230). Ryanodine (up to 100 M) slightly
reduced Ca2 transients but failed to block STDs in ASMC
(229, 230). Reduced extracellular Na also decreased STDs
in ASMC, and these events were not affected by Cl chan-
nel blocking drugs or replacement of extracellular Cl
(230). The data suggested that pacemaker activity in ASMC
is due to openings of cation-selective channels stimulated by
release of Ca2 from IP3 receptor-operated stores.
A recent report, using eYFP-SMA transgenic mice,
showed that ASMC, isolated from smooth muscle cells by
the absence of an A-type K current and STOCs, displayed
two types of inward conductances that yielded STICs and
large inward currents (LICs) (181). LICs were blocked by
replacement of the external Na with TEA (181). Thus
LICs might arise from activation of nonselective cation
channels (NSCC), and these events may be responsible for
STDs in intact muscles. The importance of STICs, the in-
volvement of a Ca2-activated Cl condutance (CaCC),
and how STICs might link to the generation of LICs are not
clear at present.
ICC-LC have also been shown to generate STICs and LICs
(232, 233). The conductances responsible for STICs and
LICs were not affected by Cl channel blockers. The cur-
rents reversed close to 0 mV, suggesting the currents were
due to NSCC conductances (232). Current clamp (I 0)
experiments suggested that NSCC are responsible for STDs
in ICC-LC (233), and spontaneous generation of currents
may be due to Ca2 release via IP3 or ryanodine receptors.
In another study, interstitial cells immunonegative for
smooth muscle actin (most likely ICC-LC) displayed
Xe991-sensitive, Kv7 currents, spontaneous transient out-
ward currents due to activation of large conductance Ca2-
activated K channels, and niflumic acid-sensitive STICs
(possibly CaCC) (181). These findings were supported by
immunostaining for ANO1 and KV7.5 immunostaining.
How ASMC and ICC-LC interact with smooth muscle cells
to generate peristaltic contractions in the renal pelvis and
pyelouretic junction has not been fully clarified.
B. Bladder
Much has been written about interstitial cells in the urinary
bladder since vimentin interstitial cells in the detrusor
muscle were shown to display cGMP-like immunoreactivity
(353). Two general classes of interstitial cells have been
described on the basis of their histological distributions:
suburothelial and detrusor interstitial cells. Suburothelial
interstitial cells lie beneath the urothelium and are cGMP-
immunopositive (121). Vimentin immunoreactivity has
Physiol Rev VOL 94 JULY 2014 www.prv.org
been used typically to identify interstitial cells in the blad-
der. Vimentin cells have been reported in the suburothe-
lium of guinea pig (76, 129, 215, 302) and human bladders
(189, 268, 363), and there are reports of c-Kit cells in the
suburothelium of the human (189, 287, 288, 344) and pig
bladder (260). Some studies have noted that not all vimen-
tin cells are c-Kit (75, 256). Thus at least two classes of
interstitial cells may exist in the bladder. It should also be
noted that mast cells are also c-Kit and can be found in the
suburothelium and in detrusor muscles. However, mast
cells are typically rounded and of a less fusiform shape than
the c-Kit cells described in bladder tissues (189, 287).
Muscarinic receptor type 3 (M3) is expressed specifically by
suburethral interstitial cells, but the significance of these
receptors in interstitial cells is unknown (129, 130).
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Some investigators have referred to the interstitial cells of
the urothelium as myofibroblasts. These cells express vi-
mentin and connexin 43 and are in proximity to unmyeli-
nated nerves, suggesting possible neural interactions (107).
Vimentin myofibroblasts exhibit STICs that are mediated
by activation of a CaCC. ATP, acting via P2Y6 receptors,
increased intracellular Ca2 and activated CaCC (107). De-
veloping strains of animals expressing reporters or finding
immunoreactive proteins and antibodies directed toward
extracellular epitopes will be important advances for un-
equivocal identification of bladder myofibroblasts in future
studies.
Immunohistochemical evidence for c-Kit cells in the blad-
der detrusor muscles has been reported in human (189, 287,
288, 344), pig (260), dog (12), guinea pig (72, 75, 256, 268,
399), rat (79), and mouse (255). Transmission electron mi-
croscopy (TEM) identified a prominent network of detrusor
interstitial cells (CD34) in human detrusor (304). These
cells are described as bipolar with slender dendritic process,
abundant mitochondria, rough endoplasmic recticulum,
Golgi apparatus, and intermediate filaments. The cells were
found at the peripheral edges of muscle bundles and occa-
sionally could be found within muscle bundles. They were
considered to be similar to the ICC of the GI tract. How-
ever, these cells were immunonegative for CD117 (c-Kit).
The number of interstitial cells has been reported to increase
in overactive bladder generated in animal models of partial
obstruction or in patients with overactive bladder (34, 193,
216). Bladder contractions are induced by activation of
cholinergic M3 receptors (8, 55, 335), and smooth muscle
cells make close contacts with and are apparently directly
innervated by motor neurons (114, 115). Interstitial cells
are also found in close proximity to cholinergic nerves (63,
189, 256), so these cells might have neuromodulatory func-
tions in the bladder (128, 186). It should be noted that
interstitial cells immnopositive for c-Kit are not observed
consistently in the bladder. Some investigators have found
few cells immunopositive for c-Kit in the detrusor and subu-
rothelium, and the few cells that are c-Kit appear to be
891
 SANDERS ET AL.

mast cells in mice (207, 225, 245) and humans (303). Thus
the presence and significance of c-Kit interstitial cells in
A D
the bladder is controversial at present.

1. Functional observations on cells identified as

interstitial cells

Far less is known about the functional role of c-Kit inter-

stitial cells in detrusor muscles than in GI muscles. Studies
of these cells have relied on identification by morphological
characteristics, which are not definitive in dispersed cell
populations. Nevertheless, branched cells, thought to be B E
interstitial cells, were compared with smooth muscle cells.
The branched cells expressed large-conductance Ca2-acti-
vated K channels (BK), TEA-sensitive delayed rectifier K

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channels (Kv), KCNQ channels, hyperpolarization-acti-
vated cyclic nucleotide-gated channels (HCN), L-type Ca2
channels, and T-type Ca2 channels (7, 79, 144, 186, 253,
254). A T-type Ca2 channel blocker inhibited spontaneous
phasic contractions in detrusor strips and decreased intra-
cellular Ca2 transients in detrusor interstitial cells, sug-
gesting that T-type Ca2 channels could be a Ca2 entry C F
source in generating spontaneous activity (79). Application
of carbachol increased Ca2 transients and phasic contrac-
tions through the M3-Gq/11-PLC pathway in the branched
cells of detrusor muscles, suggesting these cells may respond
to cholinergic neurotransmission in situ (186, 256, 382).
High concentrations of the c-Kit inhibitor imatinib abol-
ished action potential firing and significantly decreased in-
tracellular Ca2 transients in interstitial cells and muscle
contractions (79, 217). Thus it was suggested that a tyrosine
kinase inhibitor might be a useful target for treating symp-
FIGURE 10. Interstitial cells in bladder and oviduct. AC: PDGFR
toms of bladder overactivity (34, 218). However, imatinib cells in the detrusor muscle of cynomolgus monkey (Macaca fascicu-
has effects on Ca2 channels in smooth muscles, so the laris) bladder. A and B: cryostat cross sections through the detrusor
acute effects of this drug may not be through inhibition of wall. At least two populations of PDGFR cells were observed: cells
c-Kit (140). within (arrowheads) and surrounding detrusor muscle bundles (ar-
rows). PDGFR cells also appeared to bridge between adjacent mus-
cle bundles (*). C: flat-profile cryostat section through the detrusor.
There have been suggestions that bladder dysfunction in PDGFR cells located within (arrowheads) and between (arrows) mus-
diabetes and spinal cord injury could be related to changes cle bundles are present. DF: ICC and PDGFR cells within the mouse
in interstitial cell distribution (54, 187). However, it must and cynomolgus monkey oviduct. A and B: ICC within the myosalpinx of
be stated that a role of c-Kit cells in normal or diseased mouse (D) and monkey (E) form an anastomosing network of cells
bladder tissues is far from established. (arrows). These cells have been shown to provide electrical pacemaker
function for propulsive contractions in the oviduct (see text for details).
F: double labeling of PDGFR cells in a cross section of the isthmus
2. PDGFR cells in detrusor muscles region of the mouse oviduct. Tissue comes from Pdgfratm11(EGFP)Sor/J
mice that have constitutive expression of eGFP in nuclei driven off the
Pdgfra promoter. The tissues were double-labeled with antibodies
Recently, another type of interstitial cell was identified in against PDGFR. Thus the soma and cytoplasmic processes of
mouse, guinea pig, and human bladders (207, 268). These PDGFR cells are labeled with antibody (red), and nuclei of PDGFR
cells express PDGFR and therefore have been referred to cells are green. A dense population of these cells were observed within
as PDGFR cells, as in GI muscles (FIGURE 10, AC). the myosalpinx (arrowheads) and within the submucosa and endosal-
pinx (arrows) of the oviduct. The specific functions of these cells have
PDGFR cells are distributed throughout the bladder not been determined. Scale bars are as indicated in each panel. (Au-
wall, including the lamina propria and detrusor muscle. thors are grateful to Dr. Rose Ellen Dixon for images shown in D and E.)
These cells have a spindle-shaped or stellate morphology
and often possess multiple processes, possibly forming net-
works. PDGFR cells surround and are located between A mouse strain expressing eGFP in PDGFR cells (135)
smooth muscle bundles, and they are often in close proxim- has been used to identify PDGFR cells in cell dispersions
ity to intramural nerve fibers. and for purification of cells by FACS (207). PDGFR cells

892 Physiol Rev VOL 94 JULY 2014 www.prv.org

 INTERSTITIAL CELLS IN SMOOTH MUSCLES
are highly enriched in Pdgfra and Kcnn3 (SK3 channel)
transcripts in relation to the whole detrusor cell population.
SK3 protein was also observed in PDGFR cells by immu-
nohistochemistry but was not resolved in smooth muscle
cells. SK channel modulators, CyPPA and SKA-31, hyper-
polarized PDGFR cells and activated SK currents under
voltage clamp (239). Similar responses were not observed in
SMCs held at membrane potentials simulating physiologi-
cal potentials. The single-channel conductance of SK chan-
nels in PDGFR cells was 10 pS, and intracellular Ca2 frequency and longer duration than Ca2 transients in
activated the channels. PDGFR cells displayed STOCs at
potentials positive to 60 mV, and these events were inhib-
ited by apamin.
Doxycycline-induced suppression of SK3 expression in
bladder muscles was associated with a decrease in SK cur-
rent density in bladder smooth muscle cells and increased
contractions of detrusor muscle strips (148). Cystometric
evaluations found that reduced SK3 channel expression
caused a marked increase in nonvoiding contractions.
Taken together, these studies indicate that SK3 channels are
important regulators of bladder muscle excitability. How-
ever, the current density of SK channels in smooth muscle
cells is very low relative to PDGFR cells (239). Thus it is
possible that the effects of suppressing SK3 channel expres-
sion could be mediated by loss of this conductance in
PDGFR cells. As in the GI tract, robust expression of
SK3 channels in PDGFR cells may be an important
means of controlling bladder excitability during the filling
phase.
Recently, the SK conductance in PDGFR cells was shown
to be activated by purines acting on P2Y1 receptors (240).
P2Y receptors (mainly P2ry1) were highly expressed in
PDGFR cells. ATP also induced significant hyperpolar-
ization responses in PDGFR cells under current clamp.
MRS2365, a P2Y1 agonist, mimicked the effects of ATP,
and MRS2500, an antagonist, inhibited ATP-activated SK
currents. ATP responses were largely abolished in
PDGFR cells of P2ry1/ mice. These data demonstrate hanced Ca2 oscillations, and NCX inhibitors, KB-R7943
that purines activate SK currents and would tend to stabilize
excitability in bladder muscles, thus providing an explana-
tion for purinergic relaxation of detrusor muscles (37, 38,
46, 257).
C. Urethra
Urethra smooth muscles also display spontaneous electrical
activity. Branched cells from rabbit urethra have been iso-
lated and studied by voltage-clamp and Ca2 imaging (188,
338). The branched cells were identified as interstitial cells
on the basis of vimentin expression, absence of smooth
muscle myosin, and presence of a discontinuous basal lam-
ina, sparse rough endoplasmic reticulum, abundant mito-
chondria, and a well-developed smooth ER. Significant dif-
ferences were observed in the behaviors of branched cells
Physiol Rev VOL 94 JULY 2014 www.prv.org
and smooth muscle cells. The exact nature of the interstitial
cells in the urethra (i.e., whether mainly c-Kit or
PDGFR) is unclear (286, 282).
Urethral smooth muscles exhibit STDs and slow wave-like
events. The electrical events are due to activation of CaCC
in response to release of Ca2 from intracellular Ca2
stores (142). Ca2 transients occur in interstitial cells in
intact urethral muscles; however, these events were of lower
smooth muscle cells. Ca2 transients in interstitial cells
were inhibited by CPA, ryanodine, and 2-APB (141), as also
found in isolated interstitial cells (see below).
Isolated urethral interstitial cells generate spontaneous elec-
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trical activity, but smooth muscle cells from the urethra are
normally electrically quiescent. The spontaneous electrical
activity in interstitial cells appears to be due to intracellular
Ca2 oscillations that generate pacemaker currents. CaCC
were activated by Ca2 release from IP3 and ryanodine
receptors on Ca2 stores (67, 156, 339). Mitochondria also
regulate the activity of CaCC in interstitial cells, and elec-
tron transport chain blockers, rotenone and antimycin A,
and protonophore uncouplers, FCCP and CCCP, inhibited
intracellular Ca2 waves and abolished STICs in these cells.
The evidence for functional expression of CaCC in urethral
interstitial cells is strong from studies of isolated cells, but a
recent report suggested that Ano1 is expressed in smooth
muscle cells, but not in interstitial cells (325). Thus deter-
mining the molecular species responsible for CaCC in ure-
thral interstitial cells will be a goal for the future.
Urethral smooth muscle cells express L-type and T-type
Ca2 channels (41, 157), but expression of voltage-depen-
dent Ca2 channels in interstitial cells has not been deter-
mined. The Na-Ca2 exchanger type 3 (NCX3) has been
suggested as an important contributor of Ca2 influx in
urethral interstitial cells. Lowering extracellular Na en-
and SEA0400, decreased Ca2 oscillations and STICs. Thus
the Ca2 influx mode of NCX may drive or regulate spon-
taneous Ca2 release events and electrical rhythmicity (40).
How NCX3 is switched to reverse mode in interstitial cells
is not fully understood, but might occur by localized in-
creases in intracellular Na due to actions of other trans-
porters or cation channels.
Urethral interstitial cells may also have a role in transducing
or modulating neural inputs from autonomic motor neu-
rons. Adrenergic neurotransmission affects urethral tone
via postjunctional 1 adrenoceptors (39). Norepinephrine
(NE) activated STICs in interstitial cells, and this is blocked
by 1 antagonists, a phospholipase C inhibitor, IP3 receptor
antagonists, and CaCC blockers (341). NO is a prominent
inhibitory neurotransmitter in the urethra (9), and inhibi-
tory effects are mediated through activation of protein ki-
893
 SANDERS ET AL.
nase G (285). NO donors and membrane-permeable cGMP
analogs inhibited STICs in urethral interstitial cells by re-
ducing intracellular Ca2 waves (340). NO may also target
cyclic nucleotide-gated channels (CNG), which are ex-
pressed in urethral interstitial cells (381).
V. INTERSTITIAL CELLS IN THE
REPRODUCTIVE TRACT
A. Uterus
The uterus has extraordinary requirements to accomplish
the tasks of implantation and embryogenesis. The hypertro-
phy that accommodates the growth of an embryo and main-
tenance of a low level of smooth muscle excitability facili-
tate uterine function during pregnancy. Several studies have
reported the presence of ICC or ICC-like interstitial cells in
the human uterus (60, 89, 170, 171, 290, 345), but the role
of these cells in uterine contractile activity and maintenance
of pregnancy is poorly understood.
A few studies have attempted to study the role of c-Kit
ICC-like cells in the uterus by testing the effects of imatinib
mesylate (5, 169). This drug caused concentration-depen-
dent (100 M) inhibition of spontaneous uterine contrac-
tions and contractions initiated by elevated external K. It
is possible that the effects of imatinib were mediated by a
tyrosine kinase independent signaling pathway since the
concentrations required to affect contractions were much
greater than concentrations required to inhibit c-Kit (69).
When acute and sustained application of imatinib was stud-
ied in GI muscles, it was found that chronic application for
several days was required to affect c-Kit populations of
cells and inhibit electrical rhythmicity (23, 139). ICC net-
works were lost from the small intestines after at least 3
days of imatinib treatment (35 M), and this was similar
to the time course of the effects of neutralizing c-Kit anti-
bodies on ICC and pacemaker activity. In studies of gastric
antrum, imatinib (10 M) was shown to reduce spontane-
ous contractions, intracellular Ca2 transients, and basal
[Ca2]i levels without affecting pacemaker activity (139).
Thus imatinib may generally suppress spontaneous con-
tractions of smooth muscles by blocking L-type Ca2 chan-
nels.
Studies of cells isolated from uterine muscles and identified
as interstitial cells are limited. Cells distinct from myocytes,
based on morphology and ultrastructure, were obtained
from pregnant human and rat uterus (89, 427). The ICC-
like cells were multipolar with spiderlike projections and
enlarged nuclear regions, but c-Kit immunoreactivity was
not resolved (89). The cells were noncontractile and con-
tained no myofilaments, but numerous mitochondria, cave-
olae, and intermediate filaments were observed and the cells
were immunopositive for vimentin (89). The cells had rest-
ing membrane potentials of 58 mV and displayed no in-
894 Physiol Rev VOL 94 JULY 2014 www.prv.org
ward currents or action potentials in contrast to myocytes.
Outward currents were elicited by depolarization of the
ICC-like cells. The numbers of ICC-like cells increase in
pregnancy, and the suggestion was made that the cells might
help to stabilize uterine excitability during the later stages of
pregnancy (89, 427).
B. Oviduct
Oviducts, or fallopian tubes in humans, are smooth muscle-
lined tubular organs that transport ova from ovary to uterus
(284). Several stages of the reproductive process occur
within the oviduct and are subject to physiological regula-
tion by a variety of processes. The four main functions of
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the oviduct include 1) transport of the ovum from the ovary
to the site of fertilization. After delivery to the oviduct,
oocytes are propelled down the tube, assisted by peristaltic
contractions and oviductal fluid moved by ciliated epithelial
cells, into the ampulla where fertilization takes place (134).
2) The oviduct aids in transport of spermatozoa from the
site of deposition to the site of fertilization within the am-
pulla. Delivery of sperm to the site of fertilization is also
thought to involve muscular movements of the oviduct (3).
3) The oviduct provides a suitable environment for the egg.
Secretions along the oviduct provide a suitable environment
and maintain the viability of the egg during the descent
through the oviduct (71). 4) Transportation of the fertilized
ovum (embryo) to the uterus where implantation and fur-
ther development occurs. After fertilization, the developing
morula descends through the isthmus of the oviduct rela-
tively rapidly, followed by successful implantation within
the endometrium of the uterus (70).
Video imaging and spatiotemporal mapping showed that
cumulus oocyte complex (COC) movements are due to the
contractile activity of the myosalpinx, and ciliary beating
stirs the fluid environment that provides lubrication and
viability to the egg and sperm (1, 192). Thus the source of
peristaltic contractions in oviduct is important to under-
standing the physiology of this organ. Morphological stud-
ies using light and electron microscopy identified ICC-like
cells in the fallopian tube, and it was postulated the cells
might provide pacemaker activity (290, 291, 346). These
studies described elongated c-Kit cells with dendritic pro-
cesses located within the circular muscle layer and also be-
tween the circular and longitudinal muscle layers in all seg-
ments of the oviduct. The elongated c-Kit cells were dis-
tinct from the rounded and unbranched mast cells also
detected within the muscularis. c-Kit cells were also de-
tected beneath the epithelium in a layer that was 10 15 m
thick. The ICC-like cells represented 9 and 7% of total
cell population in the lamina propria and muscularis, re-
spectively (291). Although no direct functional evidence
was presented, based on physiological studies in the GI
tract, it was proposed that the ICC-like cells might provide
pacemaker activity and/or mediate neurotransmission.
 INTERSTITIAL CELLS IN SMOOTH MUSCLES
ICC have also been described in oviducts of mouse and hen
(85, 117). In mice and monkeys, ICC, termed ICC-OVI, are
found within the myosalpinx and organized into an exten-
sive network throughout the oviduct (Figure 10, D and E).
Functional studies demonstrated the importance of ICC-
OVI in oviduct motility. Selective removal of ICC-OVI us-
ing a c-Kit neutralizing antibody resulted in loss of ICC,
electrical slow waves, and propulsive contractions of the
oviduct (85). It is not known whether removal of ICC-OVI
leads to loss of egg propulsion and infertility as these studies
were performed in neonatal animals where ICC-OVI phe-
notype is susceptible to neutralizing antibody (23).
PDGFR cells are also widely distributed in the oviduct
(FIGURE 10F).
Mice infected with Chlamydia muridarum develop dilated
oviducts, pyosalpinx, and loss of spontaneous contractile
activity (85). Morphological inspection showed disruption
of ICC-OVI networks, and electrophysiological recordings
showed loss of pacemaker activity without change in basal
smooth muscle membrane potential. Although spontaneous
activity was absent in infected mice, membrane depolariza-
tions, similar in amplitude and duration to slow waves,
could be evoked by electrical field stimulation, suggesting
the oviducts remained excitable. Chlamydia-induced infec-
tion of oviducts was associated with increased expression of
inducible nitric oxide synthase (Nos2) and prostaglandin
synthase 2 (Ptgs2) in stellate-shaped, macrophage-like cells
that lie in close proximity to ICC-OVI in the oviduct. A
correlation was shown to exist between damage to ICC-
OVI and loss of pacemaker activity and increased Nos2
expression. It was suggested that loss of ICC-OVI could be
a major contributing factor to the development of tubal
factor infertility.
C. Prostate
Movement of prostatic contents from the peripheral acini
into the prostatic ducts is driven by myogenic activity in the
stromal wall (94). Interstitial cells, immunopositive for c-
Kit, were identified in guinea pig prostate stroma, and these
cells were in close contact with smooth muscle cells and
varicose axon bundles (94). Examination of tissues with
electron microscopy showed typical smooth muscle cells
and interstitial cells with more electron-dense cytoplasm,
ovoid nuclei, an abundance of mitochondria, rough ER,
some caveolae, and an incomplete basal lamina. Thick fila-
ments were not observed in these cells. These are general
ultrastructural features of the ICC of the GI tract. Intersti-
tial cells have also been reported in human prostate tissues
(347). A slow wave-like electrical event drives phasic con-
tractions of prostate smooth muscles, and these events may
be driven by the c-Kit interstitial cells (270).
Prostate interstitial cells generate spontaneous Ca2 tran-
sients that were abolished by nicardipine, suggesting that
Physiol Rev VOL 94 JULY 2014 www.prv.org
Ca2 influx through L-type Ca2 channels is a crucial fac-
tor for intracellular Ca2 oscillations. However, an early
study reported that the STDs were not affected by nifedi-
pine (94) in the same species. CPA, caffeine, or reduced
extracellular Ca2 also abolished spontaneous Ca2 tran-
sients, suggesting that intracellular Ca2 stores are involved
in the generation of Ca2 transients in interstitial cells
(226).
Interstitial cells in the prostate are associated with sympa-
thetic neurons, and the interstitial cells express 1-adreno-
ceptors and the gap junction protein connexin 43 as dem-
onstrated by immunohistochemistry (394). Norepinephrine
evoked inward currents in isolated cells identified as inter-
stitial cells, and these responses were blocked by phentol-
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amine and prazosin, suggesting the responses to norepi-
nephrine were mediated by 1-adrenoceptors. These au-
thors suggested that interstitial cells may be targets for
adrenergic regulation of contractions in prostatic ducts.
Interestingly, GISTs have shown a relationship with pros-
tatic GIST, either primarily or as a result of metastasis (6,
77, 83). Thus expression of CD117 (c-Kit) might be useful
for the diagnosis of GIST-like tumors in the prostate.
VI. SUMMARY AND CONCLUSIONS
Smooth muscle tissues contain a variety of interstitial cells.
These cells provide important regulatory controls in normal
and pathophysiological responses of smooth muscles, and
we are just beginning to understand the full range of behav-
iors imposed by interstitial cells on the mechanical perfor-
mance of smooth muscles. In the GI tract, several basic
regulatory functions have been attributed to interstitial cells
and a mechanistic understanding of these cells is develop-
ing. Electrical coupling between smooth muscle cells and
interstitial cells is a key structural feature that plugs inter-
stitial cells into smooth muscle cells, forming the SIP syncy-
tium. ICC and PDGFR cells express unique conduc-
tances and generate spontaneous transient inward and out-
ward currents, respectively, that modulate the excitability
of the SIP syncytium. ICC are pacemaker cells, generating
electrical slow waves that conduct to smooth muscle cells
and provide the basis for the phasic contractions of muscle
strips and segmental and peristaltic contractions of GI or-
gans. ICC and PDGFR cells are innervated and contrib-
ute to the transduction of neural inputs from enteric motor
neurons, and they appear to be specialized, generating
responses to specific neurotransmitters. ICC have also been
shown to have a role in mechanotransduction; however, the
specific mechanosensitive responses of each cell type in the
SIP syncytium and the contributions of each cell to inte-
grated mechanosensitive responses of GI organs is not fully
understood at present. More research is needed to under-
stand the complete molecular apparatus required for devel-
opment and maintenance of pacemaker activity in ICC. A
895
 SANDERS ET AL.
complete description of the ion channels and transporters
responsible for generating and sustaining pacemaker activ-
ity may provide ideas for drugs that can normalize gastric
dysrhythmias or improve slow colonic transit. Therapeutic
benefits might also be realized by understanding the
postjunctional mechanisms involved in neuro- and mecha-
notransduction.
It is very difficult to deduce the role of specific cell types in
tissues as complicated as smooth muscles, particularly
when the cells are electrically coupled. Functional studies
on muscle strips and expression studies on whole muscle
extracts do not reveal the cells that express specific genes or
are responsible for specific functions. Having fluorescent
reporters expressed in SIP cells makes it possible to isolate
and purify each cellular component. Studies focused on the
cellular components are building data bases about the
unique molecular and functional features of each class of
cell in the SIP syncytium. The integrated output and re-
sponses of the SIP syncytium are fundamental to GI motil-
ity. We suggest that the classic term myogenic to describe
the subneural, subhormonal level of motor regulation in the
gut is limited and not inclusive of the important behaviors
and regulation provided by interstitial cells. Thus the term
SIPgenic is suggested as a replacement for myogenic, or
to be used as an intermediate term, to incorporate the inte-
grative functions and full range of activities and responses
of smooth muscle cells and interstitial cells of the SIP syn-
cytium.
Based on the anatomical localization and morphologies of
interstitial cells, there are subclasses of SIP cells. Therefore,
better vital cellular markers or reporters driven by promot-
ers of genes expressed in specific subclasses will be needed
for efficient molecular and functional subclassifications of
interstitial cells. Questions about why slow waves are prop-
agated actively by ICC-MY but not by ICC-IM of the fun-
dus, why interstitial cells develop in specific anatomical
niches, and why there are differential sensitivities to inacti-
vation of c-Kit will require a finer level of granularity in
future studies of SIP cells.
Tissue engineering efforts have considered growing smooth
muscle tissues and organs for reconstructive therapies.
Maintenance of the smooth muscle phenotype is a continu-
ing obstacle in these efforts, as smooth muscle cells rediffer-
entiate readily when isolated from the native extracellular
matrix. Another issue to consider in tissue engineering, and
indeed in studies of cultured smooth muscle cells, is the
impact of losing or abnormal growth or development of
interstitial cells in culture. Few studies of cultured smooth
muscles have evaluated the content or status of interstitial
cells and either loss- or gain-of-function or loss of growth
factor support might impact the behavior of smooth muscle
cells in culture or in cultured muscle tissues. Expression
studies that include cellular markers for the many cell phe-
896 Physiol Rev VOL 94 JULY 2014 www.prv.org
notypes at play in smooth muscle tissues should be included
in studies of cultured smooth muscles and tissue engineering
protocols to better understand the fates of cell types that are
important to the performance of native muscles.
A better understanding of the roles of interstitial cells in
non-GI muscles is necessary. Studies of interstitial cells in
many smooth muscle organs have stalled somewhat after
the initial morphological descriptions of these cells. It is
tempting to speculate that similar cells will be widespread in
smooth muscle tissues and have important functions in reg-
ulating smooth muscle excitability or in mediation of re-
sponses to neurotransmitters. However, the lack of pheno-
types in various smooth muscle tissues of animals with Kit
mutations suggests that c-Kit cells may not be present or
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important in some smooth muscle organs. For example, the
presence and functional significance of c-Kit interstitial
cells, other than mast cells, in bladder detrusor muscles, is
questionable. Use of imatinib mesylate is a poor method of
testing the function of interstitial cells because the concen-
trations needed for acute effects have nonspecific effects on
voltage-dependent Ca2 channels and/or the contractile
mechanism of smooth muscle cells. These effects are down-
stream from generation of pacemaker activity and/or trans-
duction of neural responses by interstitial cells. Effects of
tyrosine kinase inhibitors on interstitial cells are likely to
require long-term treatments to block the signaling func-
tions of c-Kit or PDGFR. New studies examining the pres-
ence of PDGFR cells should be performed in various
types of smooth muscles. Finding these cells in the bladder
has provided novel hypotheses about the role of interstitial
cells in this organ. Strains of mice with reporters expressed
in interstitial cells should be used in studies of non-GI mus-
cles. These experiments will give the opportunity to make
unequivocal identification of cells purported to be intersti-
tial cells and provide better cell preparations for determina-
tion of interstitial cell functions.
Considerable additional work is needed to understand the de-
velopment and plasticity of interstitial cells. Many studies have
made associations between failures of interstitial cells to de-
velop or loss of cells in adults and motor dysfunction, but the
factor(s) responsible for developmental failures or interstitial
cell loss have not been identified. One intriguing lead is that
many of the pathological conditions that result in loss of ICC
are associated with inflammatory responses. Thus it is possible
that these cells are victims of inflammatory mediators. At pres-
ent, there is little information about the response of interstitial
cells to paracrine substances, hormones, and/or cytokines. It is
possible that responses or loss of responses to one of these
factors during the development of pathological conditions
could negatively impact interstitial cells, causing cell death or
remodeling of the phenotype. Now that it is possible to purify
each cellular component of the SIP syncytium, studies can be
designed to understand cellular mechanisms and cellular re-
sponses to pathological challenges. Deep sequencing of tran-
 INTERSTITIAL CELLS IN SMOOTH MUSCLES
scriptomes of each type of SIP cell will yield important insights
into the molecular personalities of these cells and predict the
stimuli to which the cells will respond. Having cell-specific
iCRE strains will make it possible to test these hypotheses
without total reliance on pharmacology. Fate mapping studies
using molecular markers will make it possible to finally deter-
mine what happens to interstitial cells in response to patholog-
ical challenges. If remodeling of the interstitial cell phenotype
is responsible for the disappearance of interstitial cells in
pathophysiological states, then it may be possible to learn the
signaling that controls remodeling and loss of interstitial cell
functions and reestablishment of interstitial cells after patho-
logical conditions are resolved. Detailed knowledge of SIP cell
transcriptomes may help identify biomarkers that will provide
noninvasive tests to assess the health of specific types of SIP
cells in disease. As in most areas of biomedical research, mo-
lecular technologies have made rapid progress on the physiol-
ogy and pathophysiology of interstitial cells possible. New
dimensions of understanding of the significance of interstitial
cells in the behavior of smooth muscle organs will result from
these endeavors.
ACKNOWLEDGMENTS
We thank the many post-docs and students that have
worked in our labs on interstitial cells. We are eternally
grateful to Nancy Horowitz and Yulia Bayguinov for their
technical support on all projects related to interstitial cells.
We are especially grateful to Drs. Masaaki Kurahashi, Peter
Blair, and Rose Ellen Dixon for images used in FIGURES 1
AND 10; Dr. Grant Hennig for FIGURE 9; and Professor Wei
Yan for helpful discussions about gene inactivation studies.
We are also most grateful for the insightful comments and
collaborations with Professor David Hirst and Dr. Gerard
Sergeant regarding the pacemaker mechanism in ICC.
Address for reprint requests and other correspondence: K.
Sanders, Dept. of Physiology and Cell Biology, Univ. of
Nevada School of Medicine, Reno, NV 89557 (e-mail:
ksanders@medicine.nevada.edu).
GRANTS
We are extremely grateful to the National Institutes of
Health (via the IDeA Program and the National Institute of
Diabetes and Digestive and Kidney Diseases) for funding
from R37 DK40569 and R01 DK91336 (to K. M. Sanders),
R01 DK57236 and DK078736 (to S. M. Ward), R01
DK98388 (to S. D. Koh), as well as P01 DK41315 and P20
GM103513 (shared by K. M. Sanders, S. M. Ward, and
S. D. Koh).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared
by the authors.
Physiol Rev VOL 94 JULY 2014 www.prv.org
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