Katsuaki Endou, Kunihiko Iizuka, Akihiro Yoshii, Hideo Tsukagoshi, Tamotsu

Ishizuka, Kunio Dobashi, Tsugio Nakazawa and Masatomo Mori

Am J Physiol Lung Cell Mol Physiol 287:641-648, 2004. First published Apr 30, 2004;
doi:10.1152/ajplung.00287.2003

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CALL FOR PAPERS Rho GTPases in Lung Physiology and Disease
8-Bromo-cAMP decreases the Ca2 sensitivity of airway smooth muscle
contraction through a mechanism distinct from inhibition of Rho-kinase
Katsuaki Endou,1 Kunihiko Iizuka,1 Akihiro Yoshii,1 Hideo Tsukagoshi,1 Tamotsu Ishizuka,1
Kunio Dobashi,1 Tsugio Nakazawa,2 and Masatomo Mori1
1
Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine,
Maebashi; and 2Gunma University Faculty of Health Sciences, Gunma 371-8511, Japan
Submitted 21 August 2003; accepted in final form 26 April 2004
Endou, Katsuaki, Kunihiko Iizuka, Akihiro Yoshii, Hideo
Tsukagoshi, Tamotsu Ishizuka, Kunio Dobashi, Tsugio Naka-
zawa, and Masatomo Mori. 8-Bromo-cAMP decreases the Ca2
sensitivity of airway smooth muscle contraction through a mechanism
distinct from inhibition of Rho-kinase. Am J Physiol Lung Cell Mol
Physiol 287: L641L648, 2004. First published April 30, 2004;
10.1152/ajplung.00287.2003.To clarify whether cyclic AMP
(cAMP)/cAMP-dependent protein kinase (PKA) activation and Rho-
kinase inhibition share a common mechanism to decrease the Ca2
sensitivity of airway smooth muscle contraction, we examined the
effects of 8-bromoadenosine 3,5-cyclic monophosphate (8-
BrcAMP), a stable cAMP analog, and ()-(R)-trans-4-(1-aminoeth-
yl)-N-(4-pyridyl) cyclohexane carboxamide dihydrochloride, mono-
hydrate (Y-27632), a Rho-kinase inhibitor, on carbachol (CCh)-,
guanosine 5-O-(3-thiotriphosphate) (GTPS)-, 4-phorbol 12,13-
dibutyrate (PDBu)-, and leukotriene D4 (LTD4)-induced Ca2 sensi-
tization in -toxin-permeabilized rabbit tracheal and human bronchial
smooth muscle. In rabbit trachea, CCh-induced smooth muscle con-
traction was inhibited by 8-BrcAMP and Y-27632 to a similar extent.
However, GTPS-induced smooth muscle contraction was resistant to
8-BrcAMP. In the presence of a saturating concentration of Y-27632,
PDBu-induced smooth muscle contraction was completely reversed
by 8-BrcAMP. Conversely, PDBu-induced smooth muscle contrac-
tion was resistant to Y-27632. In the presence of a saturating concen-
tration of 8-BrcAMP, GTPS-induced Ca2 sensitization was also
reversed by Y-27632. The 8-BrcAMP had no effect on the ATP-
triggered contraction of tracheal smooth muscle that had been treated
with calyculin A in rigor solutions. The 8-BrcAMP and Y-27632
additively accelerated the relaxation rate of PDBu- and GTPS-
treated smooth muscle under myosin light chain kinase-inhibited
conditions. In human bronchus, LTD4-induced smooth muscle con-
traction was inhibited by both 8-BrcAMP and Y-27632. We conclude
that cAMP/PKA-induced Ca2 desensitization contains at least two
mechanisms: 1) inhibition of the muscarinic receptor signaling up-
stream from Rho activation and 2) cAMP/PKAs preferential reversal
of PKC-mediated Ca2 sensitization in airway smooth muscle.
calcium sensitization; cAMP; leukotriene D4
2
ALTHOUGH INTRACELLULAR calcium concentration Ca ([Ca2]i)
is the primary regulator of smooth muscle contraction, Ca2
sensitivity of the contractile apparatus can change in response
to agonists. An increase and a decrease in muscle tension at a
constant Ca2 concentration are correspondingly referred to as
Ca2 sensitization and desensitization of smooth muscle con-
Address for reprint requests and other correspondence: K. Dobashi, Dept. of
Medicine and Molecular Science, Gunma Univ. Graduate School of Medicine,
Maebashi, Gunma 371-8511, Japan (E-mail: dobashik@med.gunma-u.ac.jp).
http://www.ajplung.org 1040-0605/04 $5.00 Copyright 2004 the American Physiological Society
Am J Physiol Lung Cell Mol Physiol 287: L641L648, 2004.
First published April 30, 2004; 10.1152/ajplung.00287.2003.
traction. These are believed to be the results of changes in the
ratio of kinase and phosphatase activities toward the 20-kDa
light chain of myosin (MLC20) (22, 25). We have demonstrated
Downloaded from ajplung.physiology.org on November 17, 2008
that receptor-dependent, G protein-mediated Ca2 sensitiza-
tion occurs in canine, rabbit, and human airway smooth mus-
cles (28) and that a small G protein, RhoA, and its target
protein, Rho-kinase, play a key role in G protein-mediated
Ca2 sensitization of smooth muscle contraction (26), espe-
cially in the sustained phase. Rho/Rho-kinase signaling in-
creases phosphorylation of MLC20 through the inhibition of
myosin light chain phosphatase (MLCP)-associated mecha-
nisms, but it does not directly phosphorylate the MLC20 of
airway smooth muscle in situ (13).
Adenosine 3,5-cyclic monophosphate (cAMP)-elevating
agents such as -adrenergic agonists and phosphodiesterase
(PDE) inhibitors are most widely used clinically to relax
airway smooth muscle. Elevated cAMP not only decreases net
[Ca2]i by enhancing Ca2 extrusion to the extracellular space
and Ca2 sequestration to the [Ca2]i store but also decreases
the Ca2 sensitivity of the contractile mechanism (16). We
have demonstrated that ()-(R)-trans-4-(1-aminoethyl)-N-(4-
pyridyl) cyclohexane carboxamide dihydrochloride, monohy-
drate (Y-27632), a Rho-kinase inhibitor, relaxed airway
smooth muscle of guinea pigs, both in vitro and in vivo. The
Y-27632 was less potent than salbutamol, a selective 2-
adrenergic agonist, but was more potent than theophylline, a
PDE inhibitor (11). Nakahara et al. (21) reported that 2-
agonists and Y-27632 additively relaxed the bovine trachea.
However, if cAMP/cAMP-dependent protein kinase (PKA)
activation and Rho/Rho-kinase inhibition share at least one
common mechanism for modulating Ca2 sensitivity, Rho-
kinase inhibitors may compete with cAMP/PKA-mediated re-
laxation in airway smooth muscle. Thus the mechanisms of
these two pathways must be elucidated to clarify clinical
applications.
To address this issue, we focused on the mechanisms of
cAMP/PKA- and Rho/Rho-kinase-mediated changes in the
Ca2 sensitivity of airway smooth muscle contraction. In
-toxin-permeabilized rabbit tracheal smooth muscle, we ex-
amined the effects of 8-bromoadenosine 3,5-cyclic mono-
phosphate (8-BrcAMP), a stable cAMP analog, and Y-27632
on carbachol (CCh)-, guanosine 5-O-(3-thiotriphosphate)
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked advertisement
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
L641
L642 8-BRCAMP-INDUCED CA2 DESENSITIZATION IN SMOOTH MUSCLE
(GTPS)-, and 4-phorbol 12,13-dibutyrate (PDBu)-induced
Ca2 sensitization. We wanted to determine the following:
whether 8-BrcAMP blocks CCh- or GTPS-induced Ca2
sensitization in a similar manner to Y-27632; whether
8-BrcAMP affects myosin light chain kinase (MLCK)-associ-
ated mechanisms or MLCP-associated mechanisms; whether
8-BrcAMP reverses PDBu-induced Ca2 sensitization in the
presence of a saturating concentration of Y-27632, because
PDBu-induced contraction is resistant to Y-27632 (6); whether
the inhibitory effect of Y-27632 on GTPS-induced Ca2
sensitization is affected by 8-BrcAMP; and whether the
Y-27632- and 8-BrcAMP-responsive mechanisms are involved
in leukotriene D4 (LTD4)-induced Ca2 sensitivity in human
bronchial smooth muscle.
MATERIALS AND METHODS
Tissue preparation and isometric force measurement. The tissue
preparation and force measurement have been reported elsewhere (9,
10, 12). We administered the anesthesia by placing the animals in an
anesthetic chamber until the animals became anesthetized and unre-
sponsive to corneal stimulation. When the tracheal tissue had been
removed, the animals were killed by rapid exsanguination through the
carotid artery, in accordance with the recommendations of the Animal
Care and Experimentation Committee, Gunma University, Showa
Campus. The airways were first cut longitudinally at the center of the
cartilage opposite the smooth muscle. Small strips of tracheal smooth
muscle were then carefully separated from connective tissue, epithe-
lium, and cartilage with a razor blade under a binocular microscope.
Human bronchial smooth muscle was prepared from a macroscop-
ically normal section of lung tissue that was obtained during surgery
for lung cancer. Consent was obtained from each patient before
surgery. The surgically resected tissue was put in ice-cold Dulbeccos
modified Eagles medium, and small bronchi with an outer diameter
of 2 4 mm were carefully dissected as previously described (28).
Cartilage was removed to the greatest extent possible. Small strips of
rabbit tracheal smooth muscle (200 300 m wide, 40 50 m thick,
3 mm long) and human bronchial smooth muscle (150 200 m wide,
20 30 m thick, 3 mm long) were mounted on a bubble plate (400 ml
per bubble), and isometric force development was measured with a
force transducer (AE801; SensoNor, Horten, Norway). The developed
force was normalized to an initial pCa 5.0 (105 M) response in the
same strip (12, 28). Consent was obtained from each patient before
surgery. These protocols were approved by the Institutional Review
Board, Gunma University Faculty of Medicine, School of Medicine.
Solutions and permeabilization with -toxin. The method of per-
meabilization with -toxin has been described previously (12, 28).
The trachea was permeabilized with -toxin (16.4 g/ml) for 30 min
at 30C. We added a Ca2 ionophore, A-23187 (10 M), to the
trachea during -toxin permeabilization to block the sarcoplasmic
reticulum function. After permeabilization, all experiments except for
that depicted in Fig. 5 were performed at 24C (9, 10, 12).
The normal relaxing solution (G1) contained (in mM) 74.1 potas-
sium methanesulfonate, 2 Mg2, 4.5 ATP (Mg2 salt), 1 EGTA, 10
creatine phosphate, and 30 PIPES-KOH (pH 7.1 at 24C, ionic
strength 0.2). The same solution containing 10 mM EGTA rather than
1 mM EGTA and various amounts of calcium methanesulfonate was
used to achieve the desired concentration of free Ca2. According to
Zimmermann et al. (29), we prepared EGTA (10 mM)-buffered
ATP-free (rigor) Ca2-free solution (G10 rigor) and ATP-free high
Ca2 solution (pCa 4.5 rigor). These rigor solutions contained 50 M
P1,P5-di(adenosine-5) pentaphosphate, an inhibitor of kinase activity.
We obtained the desired concentration of free Ca2 by mixing the
G10 rigor and pCa 4.5 rigor solutions. All solutions contained ibu-
profen (2 M) to avoid cyclooxygenase activity that may attenuate
airway smooth muscle tone.
AJP-Lung Cell Mol Physiol VOL
Effect of 8-BrcAMP in ATP triggered Ca2 sensitization. To clarify
whether 8-BrcAMP-induced Ca2 desensitization is due to inhibition
of MLCK-associated mechanisms, we abolished the MLCP activity of
tracheal smooth muscle with calyculin A (300 nM) in rigor solutions
and then observed ATP (4.5 mM, Mg2 salt)-triggered contraction.
We knew that the rate of force would rise but the final amplitude
would not, reflecting MLCK-associated mechanisms (13, 17, 19).
After obtaining the maximum contraction at pCa 5.0, we relaxed the
tracheal smooth muscle in G10 solution. To activate PKA, we incu-
bated the tracheal smooth muscle with 8-BrcAMP (100 M) in G10
(containing ATP) for 10 min. In the continued presence of 8-BrcAMP,
the tracheal smooth muscle was quickly transferred to a G10 rigor
solution containing calyculin A (300 nM) for 55 min to inactivate
MLCP without contraction. After incubation of the tracheal smooth
muscle in a pCa 5.0 rigor solution containing 8-BrcAMP and calycu-
lin A for 5 min, ATP-triggered contraction was initiated. If 8-BrcAMP
inhibits MLCK-dependent contraction, the rate of ATP-triggered
contraction would be slowed by 8-BrcAMP. Time-matched, ATP-
triggered experiments were carried out in the absence of 8-BrcAMP,
as controls.
Downloaded from ajplung.physiology.org on November 17, 2008
Comparison of relaxation rates between PKA activation and Rho
inhibition of -toxin-permeabilized tracheal smooth muscle. To com-
pare the effects of PKA activation and Rho-kinase inhibition on
MLCP-dependent contraction and relaxation in situ, we measured the
relaxation rate of fully contracted tracheal smooth muscle in the
presence of 8-BrcAMP (100 M), Y-27632 (3 M), or both at 10C.
The low temperature conditions were required for comparing relax-
ation rates, because the relaxation rate at 24C was too fast to evaluate
the reagent effects on relaxation (19, 20). The tracheal smooth muscle
was fully contracted by high Ca2 with PDBu or GTPS and then
relaxed by combined treatment with Ca2 removal and 1-(5-chlo-
ronaphthalene-1-sulfonyl) homopiperadine-HCl (ML-9), an MLCK
inhibitor. We measured the half-time of the relaxation rate (time
required to reach 50% of maximum relaxation induced by a given
inhibitor) in the absence or presence of 8-BrcAMP, Y-27632, or both
(Fig. 5).
Inhibitory effect of 8-BrcAMP and Y-27632 on LTD4-induced Ca2
sensitization in -toxin-permeabilized human bronchial smooth mus-
cle. When submaximum contraction induced by pCa 6.8 plus GTP (3
M) was stable, LTD4 (1 M) was added to the -toxin-permeabil-
ized human bronchial smooth muscle. At the peak of additional
contractions, Y-27632 (3 M), 8-BrcAMP (100 M), or both were
added to the strip.
Reagents. Staphylococcus aureus -toxin was obtained from RBI
(Natick, MA); P1,P5-di(adenosine-5) pentaphosphate was from
Sigma (St. Louis, MO). The Y-27632 was a gift from Mitsubishi
Pharma (Osaka, Japan). The Y-27632 was dissolved in distilled water
to create a 10 mM stock solution, which was stored at 20C until
use. The GTPS was purchased from Boehringer Mannheim (India-
napolis, IN). The 8-BrcAMP, calyculin A, and PDBu were purchased
from Calbiochem (La Jolla, CA). The LTD4 was purchased from
Sigma. All other chemicals were of reagent grade.
Statistical analysis. Data were normalized to the pCa 5.0 response
measured before the reagent treatment of each strip and are shown as
means SE of the indicated numbers of experiments. Data were
compared by the Mann-Whitney U-test or Students t-test with the
Bonferroni correction for multiple comparisons. A P value of 0.05
was considered to be statistically significant.
RESULTS
Effects of 8-BrcAMP and Y-27632 on CCh- or GTPS-
induced Ca2 sensitization in -toxin-permeabilized tracheal
smooth muscle. After obtaining the maximum contraction at
pCa 5.0, we incubated the strip in G1 containing 8-BrcAMP,
Y-27632, or saline for 20 min. In the continuous presence of
the reagents, the tracheal smooth muscle was precontracted in
287 OCTOBER 2004 www.ajplung.org
 8-BRCAMP-INDUCED CA2 DESENSITIZATION IN SMOOTH MUSCLE L643
a pCa 6.5 solution containing GTP (3 M); CCh (100 M) was
then applied to the strip. In the experiments of GTPS-induced
Ca2 sensitization, the tracheal smooth muscle was precon-
tracted in a pCa 6.5 solution without GTP, followed by the
application of GTPS (10 M) to the tracheal smooth muscle.
As shown in Fig. 1A, CCh increased the contractile force from
the steady-state level at pCa 6.5 (15.8 2.2%) to 82.2 5.0%
(n 6). Pretreatment with 8-BrcAMP dose dependently in-
hibited the agonist-induced smooth muscle contraction. The
effect of 8-BrcAMP was saturated at 100 M, and the extent of
the maximum inhibition by 8-BrcAMP was comparable with
that by Y-27632 at 100 M. Similarly, GTPS caused rapid
contractions from 4.99 1.3 to 97.4 3.5% (n 8) at pCa
6.5, and the GTPS response was inhibited by Y-27632 (100
M). The inhibitory effect of 8-BrcAMP was only partial in
GTPS-induced smooth muscle contraction, and the resultant
contraction was 60.6 9.1% in the presence of 8-BrcAMP at
300 M (n 6, Fig. 1B). Thus the GTPS response was

Downloaded from ajplung.physiology.org on November 17, 2008

relatively more resistant to 8-BrcAMP.
Lack of effect of 8-BrcAMP on ATP-triggered contraction of
calyculin A-treated tracheal smooth muscle. As shown in Fig.
2A, with or without 8-BrcAMP (100 M), ATP elicited rapid
contractions of tracheal smooth muscle that had been treated
with calyculin A in the rigor solutions. The final force devel-
opments were not different between the two groups (80
7.2% in the control, 80.9 5.3% in the 8-BrcAMP-treated
group, n 4), and the values of T1/2 (time required to reach
50% of maximum force induced by a given stimulant) in the
control and the 8-BrcAMP-treated strips were also comparable
Fig. 2. Lack of effect 8-BrcAMP on kinase activity toward 20-kDa myosin
light chain (MLC20). After obtaining the maximum contraction at pCa 5.0, we
relaxed the tracheal smooth muscle in G10 solution. To activate PKA, we
incubated the tracheal smooth muscle with 8-BrcAMP (100 M) in G10
(containing ATP) for 10 min. In the continued presence of 8-BrcAMP, tracheal
smooth muscle was quickly transferred to a G10 ATP-free (rigor) solution
containing calyculin A (300 nM) for 55 min to inactivate myosin light chain
phosphatase (MLCP) without contraction. After incubation of tracheal smooth
muscle in a pCa 6.5 rigor solution containing 8-BrcAMP and calyculin A for
5 min, ATP-triggered contraction was initiated (A). The final force develop-
ments were not different between the 2 groups, and the values of the time
required to reach 50% of maximum force induced by a given stimulant in the
control strips (E) and the 8-BrcAMP-treated strips (F) were also comparable
(n 4, B). Relative force was normalized to the initial pCa 5.0 (105 M)
response in the same strip.

(63.1 3.6 and 61.9 7.6 s, respectively; n 4). Thus

Fig. 1. Inhibitory effect of 8-bromoadenosine 3,5-cyclic monophosphate
(8-BrcAMP) or Y-27632 on carbachol (CCh)- or guanosine 5-O-(3-thio-
triphosphate) (GTPS)-induced Ca2 sensitization. After a stable pCa 5.0
response was obtained, the -toxin-permeabilized tracheal smooth muscle was
incubated in G1 containing saline, 8-BrcAMP (0.1300 M), or Y-27632 (100
M) for 20 min. In the continuous presence of reagents, the tracheal smooth
muscle was precontracted in a pCa 6.5 solution containing GTP (3 M); then
CCh (100 M) was applied to the strips (n 3 6, A). In the experiments of
GTPS-induced Ca2 sensitization, tracheal smooth muscle was precontracted
in a pCa 6.5 solution, followed by the application of GTPS (10 M) to the
trachea (n 3 8, B). Relative force was normalized to saline.
AJP-Lung Cell Mol Physiol VOL
8-BrcAMP did not affect the MLCK-associated mechanisms.
Involvement of a distinct mechanism between 8-BrcAMP-
and Y-27632-induced decreases in Ca2 sensitivity. To find a
qualitative difference between 8-BrcAMP- and Y-27632-in-
duced changes in Ca2 sensitivity, we attempted inhibition of
PDBu-induced Ca2 sensitization by 8-BrcAMP in the pres-
ence of a saturating concentration of Y-27632. As shown in
Fig. 3A, after obtaining the maximum contraction at pCa 5.0,
we treated the tracheal smooth muscle with Y-27632 (30 M)
for 20 min, and then PDBu (10 M)-induced Ca2 sensitiza-
tion was evoked at pCa 6.5. We verified that the concentration
of Y-27632 was saturated, because an increase in the concen-
tration of Y-27632 from 30 to 100 M had no effect on force.
In the saturated concentration of Y-27632, 8-BrcAMP dose
dependently reversed contraction of the tracheal smooth mus-
cle. Time-matched experiments were carried out in the absence
of Y-27632 as controls. As shown in Fig. 3B, although
Y-27632 showed a tendency to decrease PDBu-induced con-
tractions, the contractions before the application of 8-BrcAMP
287 OCTOBER 2004 www.ajplung.org
L644 8-BRCAMP-INDUCED CA2 DESENSITIZATION IN SMOOTH MUSCLE

smooth muscle following PDBu- or GTPS-induced Ca2

sensitization. After obtaining the maximum contraction at pCa
5.0 at 10C, we precontracted the strips in a pCa 6.5 solution
containing PDBu (10 M) or GTPS (100 M). The strips
were then treated with a pCa 4.5 solution containing either
PDBu or GTPS and the inhibitors 8-BrcAMP (100 M) or
Y-27632 (3 M) or both as indicated in Fig. 5. In the contin-
uous presence of all reagents, the strips were moved into a G10
solution containing ML-9 (100 M). The 8-BrcAMP acceler-
ated the relaxation rate of PDBu-treated strips but not that of
GTPS-treated strips. In contrast, Y-27632 was effective in
GTPS-treated strips, but not in PDBu-treated strips (Table 1).
Inhibitory effects of 8-BrcAMP and Y-27632 on LTD4-
induced Ca2 sensitization in -toxin-permeabilized human
bronchial smooth muscle. To estimate the relative contribu-
tions of PKA activation and Rho-kinase inhibition to LTD4-
mediated Ca2 sensitization, we treated strips of permeabilized
human bronchial smooth muscle with 8-BrcAMP (100 M),

Downloaded from ajplung.physiology.org on November 17, 2008

Y-27632 (3 M), or both. Sensitization to Ca2 was evoked
with a saturated solution of 1 M LTD4. The pCa 5.0-induced
contractions before Ca2 sensitization were 1.81 0.17 mN in
human bronchial smooth muscle (n 8). As shown in Fig. 6,
in the presence of GTP (3 M), LTD4 (1 M) induced an

Fig. 3. Inhibition of 4-phorbol 12,13-dibutyrate (PDBu)-induced Ca2 sen-

sitization by 8-BrcAMP in the presence of Y-27632. After obtaining the
maximum contraction at pCa 5.0, we treated tracheal smooth muscle with
Y-27632 (30 M) or saline for 20 min, and then PDBu (10 M)-induced Ca2
sensitization was evoked at pCa 6.5. After we verified that the concentration of
Y-27632 was saturated, 8-BrcAMP dose dependently reversed contraction of
the trachea (A), although Y-27632 tended to decrease PDBu-induced contrac-
tions. The contractions before 8-BrcAMP application were not statistically
significant (P 0.147, Mann-Whitney U-test), and the IC50 values with or
without Y-27632 were also comparable; the amplitude of the PDBu response
and the IC50 values for 8-BrcAMP in the Y-27632-treated group (F) were
53.1 7.1% and 5.24 0.8 M (n 7); those in the control group (E) were
70.0 8.1% and 8.21 1.0 M (n 11), respectively (B). Relative force was
normalized to the initial pCa 5.0 (105 M) response in the same strip.

were not statistically significant (P 0.147, Mann-Whitney

test), and the IC50 values with or without Y-27632 were
comparable; the amplitude of the PDBu response and IC50
values for 8-BrcAMP in the Y-27632-treated group were
53.1 7.1% and 5.24 0.8 M (n 7), whereas those in the
control group were 70.0 8.1% and 8.21 1.0 M (n 11),
respectively.
Next, we verified inhibition of GTPS-induced Ca2 sensi-
tization by Y-27632 in the presence of 8-BrcAMP (Fig. 4). The
amplitude of the GTPS response and IC50 values for Y-27632
in the 8-BrcAMP-treated group were 81.4 7.0% and 3.06
0.5 M (n 6), whereas those in the control group were Fig. 4. Inhibition of GTPS-induced Ca2 sensitization by Y-27632 in the
104.9 7.0% and 2.24 0.7 M (n 5), respectively. presence of 8-BrcAMP. After obtaining the maximum contraction at pCa 5.0,
we treated the trachea with 8-BrcAMP (30 M) for 20 min, and then GTPS
Time course of relaxation of -toxin-permeabilized trachea (10 M)-induced Ca2 sensitization was evoked at pCa 6.5. After we verified
in the presence of 8-BrcAMP, Y-27632, or both. We measured that the concentration of 8-BrcAMP was saturated, 8-BrcAMP dose depen-
the relaxation rate of fully contracted tracheal smooth muscle dently reversed contraction of the tracheal smooth muscle (A). The amplitude
in the presence of 8-BrcAMP, Y-27632, or both at 10C to of the GTPS response and the IC50 values for Y-27632 in the 8-BrcAMP-
treated group (F) were 81.4 7.0% and 3.06 0.5 M (n 6); those in the
estimate the effect of MLCP-associated mechanisms. Figure 5 control group (E) were 104.9 7.0% and 2.24 0.7 M (n 5), respectively
shows relaxation by ML-9/G10 after treatment with Y-27632, (B). Relative force was normalized to the initial pCa 5.0 (105 M) response in
8-BrcAMP, or both in -toxin-permeabilized rabbit tracheal the same strip.

AJP-Lung Cell Mol Physiol VOL 287 OCTOBER 2004 www.ajplung.org

 8-BRCAMP-INDUCED CA2 DESENSITIZATION IN SMOOTH MUSCLE L645

Fig. 5. Relaxation by Y-27632, 8-BrcAMP, or

both in -toxin-permeabilized smooth muscle
with GTPS- or PDBu-induced Ca2 sensiti-
zation. After maximum contraction was
achieved with pCa 5.0 at 24C, the tracheal
smooth muscle was contracted with pCa 5.0 at
10C. When the submaximal contraction in-
duced by pCa 6.5 became stable, PDBu (10
M) or GTPS (100 M) was added to the
strip, and then the tracheal smooth muscle was
treated with PDBu (10 M) or GTPS (100
M) to induce Ca2 sensitization at pCa 4.5.
After the contraction reached a peak,
8-BrcAMP (100 M), Y-27632 (3 M), or
both were added to the strips, and the strips
were incubated for 20 min. Then the strips
were transferred in G10 containing PDBu or

Downloaded from ajplung.physiology.org on November 17, 2008

GTPS, reagents, and ML-9 (100 M). The
kinetics of tracheal smooth muscle relaxation
on PDBu-induced contraction (B) and GTPS-
induced contraction (C) with 8-BrcAMP (E),
Y-27632 (), or both (). *, , # and P
0.05 vs. control (F, n 4 6). Relative force
was normalized to the initial pCa 5.0 (105 M)
response in the same strip.

additional contraction at a fixed free Ca2 concentration of pCa DISCUSSION

6.8 in -toxin-permeabilized human bronchial smooth muscle.
-Adrenergic agonists can be understood as a cascade in-
The peak force achieved was 70.4 4.7% (n 8) of the initial
volving activation of adenyl cyclase, elevation of cytoplasmic
contraction at pCa 5.0. The LTD4 response was reversed by
cAMP levels, and PKA activation leading to phosphorylation
8-BrcAMP to 43.6 3.7%, by Y-27632 to 33.7 6.0%, and
of target proteins. However, the precise mechanisms are still
by their combination to 9.14 2.3% (n 4 8, Table 1).
unknown. In Fig. 2, the contractile rate (T1/2) was comparable
between 8-BrcAMP-treated strips and control strips. The T1/2
of the strips treated with a phosphatase inhibitor (such as
Table 1. Effects of 8-BrcAMP, Y-27632, or both in -toxin- microcystin-LR or calyculin A) is dependent on the MLCK-
permeabilized rabbit tracheal smooth muscle with PDBu-,
GTPS-, or LTD4-induced Ca2 sensitization
Maximum
Contraction
at pCa 5.0,
Half-Time of Relaxation, s %

Reagent PDBu GTPS LTD4

Control 41137.4 37337.5 70.44.7

8-BrcAMP 27415.3* 38222.2 43.63.7
Y-27632 3528.95 2697.51 33.76.0
8-BrcAMP Y-27632 24715.7* 2531.52 9.142.3
Values are means SE. Half-times of relaxation of the strips pretreated
with 8-bromoadenosine 3,5-cyclic monophosphate (8-BrcAMP, 100 M)
alone, Y-27632 (3 M) alone, and both followed by 4-phorbol 12,13-dibuty-
rate (PDBu)-induced (10 M) or guanosine 5-O-(3-thiotriphosphate) Fig. 6. Inhibitory effect of 8-BrcAMP and Y-27632 on leukotriene D4 (LTD4)-
(GTPS)-induced (100 M) contraction. Percentage of maximum contraction induced Ca2 sensitization in human bronchial smooth muscle. In -toxin-
at pCa 5.0 showed that force development of 8-BrcAMP (100 M), Y-27632 permeabilized human bronchial smooth muscle, after the maximum contrac-
(3 M), and both added at the peak of leukotriene D4 (LTD4)-induced tion at pCa 5.0 and the submaximum contraction from pCa 6.8 were obtained,
contraction was normalized to the initial pCa 5.0 response. Data are given as LTD4 (1 M) was added to strips. When LTD4 caused contraction became
Bonferroni corrections for multiple comparisons: control (saline) vs. stable, 8-BrcAMP (100 M) or Y-27632 (3 M) or both were added to the
8-BrcAMP, Y-27632, or both. *, , and P 0.05, considered to be significant strips. Developed force was normalized to the initial pCa 5.0 response
by the Bonferroni methods (n 4 8). (n 4 8).

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L646 8-BRCAMP-INDUCED CA2 DESENSITIZATION IN SMOOTH MUSCLE

associated mechanisms in the presence of Ca2 (13, 16). These
results suggest that cAMP/PKA signaling preferentially affects
MLCP-associated mechanisms of Ca2 sensitization in rabbit
tracheal smooth muscle.
The relaxing rate was mainly dependent on MLCP under our
experimental conditions (19, 20). We considered that cellular
events other than MLCK/MLCP-induced mechanisms might
be involved in our experiment conditions. Previous studies
have suggested that MLCK/MLCP-associated mechanisms
play a key role in at least the initiation of contraction and early
phase of maintenance of force (19, 20). Many cellular events
other than MLCK/MLCP-induced mechanisms may be slower
than changes in the phosphorylation state of myosin and thus
rate limiting. However, precise time-course studies suggest that
initiation of contraction occurred within milliseconds and that
the early phase of maintenance of force occurred within several
seconds (25). Furthermore, we have demonstrated that MLCK
inhibition with wortmannin but not Rho-kinase inhibition with
signaling. Alternatively, this observation could have mechanis-
tic implications; for example, one of many interpretations
would be that 8-BrcAMP modulates activity of MLCP-associ-
ated mechanisms whereas Y-27632 modulates activation of
MLCP-associated mechanisms, because 8-BrcAMP acceler-
ates the rate of relaxation without changing the time of onset,
whereas Y-27632 prolongs the onset of relaxing rate without
changing its rate (Fig. 5, B and C).
Muscarinic receptor signaling for airway smooth muscle
contraction contains Ca2 sensitization mechanisms mediated
by Rho/Rho-kinase (14, 28). Although CCh-induced Ca2
sensitization was inhibited by both 8-BrcAMP and Y-27632 to
a similar extent, GTPS-induced Ca2 sensitization was resis-
tant to 8-BrcAMP. In permeabilized canine tracheal smooth
muscle, both PDBu and acetylcholine induce Ca2 sensitiza-
tion (3, 4). Rho/Rho-kinase-mediated signaling may be distinct
from the PKC system because the effects of saturating concen-
trations of GTPS and PDBu were additive (9). Eto et al. (5)

Downloaded from ajplung.physiology.org on November 17, 2008

Y-27632 slowed force developments under the same experi-
mental conditions (13). The final amplitudes were not different.
Therefore, other cellular events appear to precede the devel-
opment of maximum force by minutes.
The 8-BrcAMP accelerated the relaxation rate in PDBu-
treated strips but not in GTPS-treated strips (Fig. 5). In
contrast, Y-27632 accelerated the relaxation rate in GTPS-
treated strips but not in PDBu-treated strips. It is difficult to
assess cellular events that occur much faster than the force
changes. A simple delay mechanism may explain the longer
half-time of relaxation but not the shorter half-time of relax-
ation. This is why we decreased the temperature conditions in
the relaxation experiments. These results suggest that cAMP/
PKA signaling impairs the inhibition of MLCP-mediated re-
sponses by PKC signaling but not by the Rho/Rho-kinase
reported that PDBu-induced Ca2 sensitization was partially
inhibited by Y-27632 in -toxin-permeabilized rabbit femoral
artery. However, we previously reported only a minor contri-
bution of PKC to GTPS-induced Ca2 sensitization in rabbit
trachea. In our present study, PDBu-induced Ca2 sensitization
was not inhibited by Y-27632 (13), although the reason for the
discrepancy is unknown.
In the present study, we demonstrated leukotriene-induced
Ca2 sensitization in human bronchial smooth muscle. Leuko-
trienes, as well as CCh, increased Ca2 sensitivity in human
bronchial smooth muscle, and leukotriene-induced Ca2 sen-
sitivity is reversed by 8-BrcAMP and Y-27632 (Fig. 6). Leu-
kotrienes evoke a potent, sustained contraction of intact human
airway smooth muscle (2, 24). Leukotrienes transiently in-
creased Ca2 sensitivity in -toxin-permeabilized porcine tra-

Fig. 7. Signaling pathway for Ca2 sensitization in smooth muscle. Activation of the muscarinic receptor (M3) initiates signaling
through the illustrated cascades that inhibit MLCP, increasing MLC20 phosphorylation and contraction. The Rho/Rho-kinase and
PKC/CPI-17 pathways inhibit the phosphorylation of MLCP and enhance the Ca2 sensitivity of myosin phosphorylation. GTPS
increases the activity of G-binding proteins, including the heterotrimetric G protein and small G proteins such as Rho/Rho-kinase.
PDBu increases the activity of PKC. GTPS-induced Ca2 sensitization is inhibited by Y-27632, but not by 8-BrcAMP. On the
other hand, PDBu-induced Ca2 sensitization was inhibited 8-BrcAMP, but not by Y-27632. Thus 8-BrcAMP has the potential to
affect the PKC/CPI-17 pathway and upstream of the Rho/Rho-kinase pathway.

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 8-BRCAMP-INDUCED CA2 DESENSITIZATION IN SMOOTH MUSCLE
cheal smooth muscle (23). However, it is not known whether
mechanisms of Ca2 sensitization in the sustained contraction
were evoked by leukotrienes. Leukotrienes increased contrac-
tile force by 25%, and the LTD4-induced Ca2 sensitization
was inhibited by 8-BrcAMP and Y-27632 together in an
additive manner. This is the first report stating that Ca2
sensitization is present in human bronchial smooth muscle
contraction induced by leukotrienes and that mechanisms of
both 8-BrcAMP sensitivity and Y-27632 sensitivity are in-
volved. The LTD4-induced contraction in human bronchial
smooth muscle is at least in part independent of increases in
Ca2 sensitivity through activation of PKC (1). Thus inhibition
of Rho/Rho-kinase signaling may become a second-line bron-
chodilator (in addition to 2-stimulants) for resolving limited
airflow in asthma. Further studies are required to determine the
relative contributions of Rho/Rho-kinase and PKC/CPI-17 in
human tissue (27).
In smooth muscle, receptor-dependent, trimetric G protein-
mediated Ca2 sensitization was inhibited by both 8-BrcAMP
and Y-27632 (Fig. 1B). Theoretically, GTPS may activate
both trimetric and monometric G proteins, resulting in direct
activation of Rho/Rho-kinase signaling and indirect activation
of trimetric G protein-mediated PKC signaling. However, prac-
tically, PKC plays a minor role in GTPS-induced Ca2
sensitization in rabbit tracheal smooth muscle (13). Hepler et
al. (8) showed that purified Gq had only a slight binding affinity
to GTPS. Additionally, AlF 4 is known to activate the trimet-
ric G protein. Contraction induced by AlF 4 but not that
induced by GTPS was insensitive to GDPS (15, 28). These
findings suggest that GTPS mainly activates small G proteins,
although the precise mechanism is still unknown.
The next question was why 8-BrcAMP effectively blocked
CCh- or LTD4-induced Ca2 sensitization, in which PKC
signaling might be involved. In fibroblasts, 8-BrcAMP blocks
Rho activation by phosphorylation of heterotrimetric G pro-
teins (7, 18). Thus the cAMP/PKA system may inhibit CCh-
and leukotriene-induced Ca2 sensitization through two steps.
First, the cAMP/PKA system may inhibit the muscarinic re-
ceptor signaling upstream of Rho activation. Second, the
cAMP/PKA system might be present downstream of the PKC
pathway (Fig. 7).
In conclusion, cAMP/PKA preferentially reverses PKC-
mediated Ca2 sensitization to Rho/Rho-kinase-mediated
Ca2 sensitization. Activation of the MLCP-associated system
is a mechanism of cAMP/PKA-induced Ca2 desensitization,
and this is distinct from Rho/Rho-kinase inhibition in airway
smooth muscle. Human bronchial smooth muscle has Ca2
sensitivity mechanisms that respond not only to contractile
agonists but also to cAMP/PKA elevating agents.
ACKNOWLEDGMENTS
The authors thank Y. Ohtani for preparing human lung tissue samples.
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