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Cytosolic
By In this in
Free
Lee
Calcium
Eugene
nuclear ionized technique, calcium provides calcium precludes The mean erythrocytes normal RBCs in the value
Levels
Orringer,
in
Louis
Sickle
Levy,
(1 .2
Red
Scott
Blood
Cells
E. London
ethane were then assayed This of arising the significant incorporation These elevation as data in separated for the presquantiwas are total
Elizabeth
we
R. Berkowitz,
developed to The measure NMR
A. Gabel,
and Robert
recently
mag- FBAPTA
bis-(2-amino-5-fluorophenoxy) acid). they with had normal least process into intracellular cell calcium. erythrocytes in vesicles. vesicles consistent spectroscopy Stratton. Inc. to These Cells medium FBAPTA. a portion which and
technique
cal-N,N,N,N-tetraacetic the extracellular which from ence ties not use cell for of wasions gave sickle values show a of of FBAPTA; the extracellular with endocytotic are that in use by incorporated these ionized of NMR & deoxygenation, first at
19F NMR
a fluorinated acid]
chelator a erythro-
incorporated erythrocytes.
[2-(2-amino-4-methyl-5-fluorophenoxy)methyl-8approach since the calcium 2 nmol/L value of did under stimulated for to the study of oxygenated Experiments of ionized
aminoquinoline-N,N.N,N-tetraacetic cytes
of ionized presence calcium in (SE). 21 not oxygen. 1 hour mean hemoglobin
observed calcium an
consistent
in sickle
fluorescent
18
mean
nmol/L
for as
(SE).
ionized
After
calcium
1
with
hour
in
of further
increase the
S
values increase To
investigate in the
deoxygenwere impermeant of
processes.
endocytosis,
erythrocytes
1987
Grune
presence
T
and
sickle
HERE
IS A fiveerythrocytes calcium
an
to tenfold as influx
effect
elevation
of total
calcium in porated FBAPTA, we observed normal RBCs, level in these vesicles, demonstrating in deoxygenated by the vesicles on deoxygenation.
occur when
MATERIALS AND
an
increase dynamic
in
the Ca2
calcium uptake
erythrocytes,
normal total
pathogenesis
are
ofsickling
deoxygenated)2 sequestered,
by activating
Elevation should
K specific
of erythrocyte contribute
channels
METHODS
if it is not
Samples homozygous
channels would
gelation
for a minimum would transfused heparinized-vacuum tubes enhance of saline solution containing were
and
(in
subsequently
mmolar): NaCI
diluted
0.8,
base.
KC1
The
5.4,
cells
CaC12
1.25,
then
Hepes
centrifuged
5.0,
adjusted
at I ,000
5,4
Lew
and analysis
the supernatant and buffy coat were removed. Ca, cells were loaded with the fluorinated in quinMF, by incubating them at a hematocrit
For
modified saline solution, with 50 izmol/L of the acetoxymethyl ester an (quinMF-AM) for 20 minutes at 37#{176}C. The cells were sickle of quinMF at I ,000 g for 7 minutes and, after resuspension in conditions or during deoxy- then centrifuged the modified saline, were incubated for an additional 60 minutes to of Bookchin and co-workers6 allow the cells to recover from the loading procedure. For the NMR a transient increase in calcistudies, the cells were resuspended at a 40% hematocrit in modified cells. An indirect, nonkinetic saline.
method used to calculate Ca1 levels in erythrocytes suggests A byproduct of loading erythrocytes with that under basal conditions Ca levels in sickle and in dehyde, which can inhibit the glycolytic normal erythrocytes do not differ significantly.7 Ca levels were not, however, measured during deoxygenation, a condition the
cytes,
quinMF-AM enzyme
is formalglyceraldehyde
that presence
little
stimulates although
is known
45Ca Lew
about
uptake and
the
in
the
National ofMolecular
of
Furthermore,
colleagues5
formation of
vesicles
and
about
contained
using a nuclear magnetic allows direct measurement of 18 2 nmol/L (SE) for sickle
values
in well-oxygenated
significantly from
RBCs.
obtained
This
with
HL2860/ (E.O.)from the National Institutes ofHealth, MD. Address reprint requests to Dr Elizabeth Murphy. ionized ofEnvironmental Health Sciences, Laboratory value does notInstitute Biophysics, P0 Box 12233, Research Triangle normal human lar is found of suggesting addition, incor27709.
No
increase erythrocytes
in
deoxygena-
in
were defrayed in part by page therefore be hereby marked 18 U.S.C. 1734 solely to
endocytosis.
by
I 987
Grune
previously
0006-4971/87/6905-0031$3.0O/0
Blood,
Vol
69.
No
5 (May),
1987:
pp
1469-1474
1469
1470
MURPHY
ET
AL
3-phosphate
dehydrogenase
as
consequence
of
an
increased
NADH/NAD
ameliorate the
ratio.
effect
This saline
lactate
can
lead
in turn
Na
to ATP
depletion.
5 mmol/L,
to <0.1 To such at
was ofcell NAD Ca
This
leads levels
to the error
further in the
ofcomplexation,
of formaldehyde,
pyruvate,
to a large inclusion
added
through
to the
the
modified
enzyme
solution.
dehydrogenase
Na presence
pyruvate
so that
generates
the cells loading only
erroneous
of a contribution
maintain
normal
previously with (<20%) by 9F calcium constant by Smith measuring a
AlP
similar
levels
showed that, calcium ofCa,.
despite
in the chelator
the
of
of
formaldehyde.
caused
presence (FBAPTA)
pyruvate,
fall in AlP.9
extracellular chelator We present at much higher we used a new fluorinated a slight has a lower dissociation
RBCs
with extracellular Ca2 these reasons, in this study chelator, quinMF, which for
shows
calcium (KD
63
Cytosolic
free
a
Ca2
(Ca)
was
measured
fluorinated dissociation
newly described has a much lower chelators is therefore originally more were
fluorinated
spectrum of quinMF-loaded, oxygenated Based on data from eight similar experifor extracellular quinMF) from seven cytosolic
a level
erythrocytes
suspended
a 20-mm
in suspension cells change were
N M R tube
and gassed with the with
fitted
either
with
an air-driven
oxygenation
stirrer
and inside This
to keep
in different 18 2
free
not
Ca2
significantly
(Ca)
values
different using
averaged
from to a
deoxygenation.9
The quinMF
100%
Once
N2, resulting
the gives cell, two
in no pH
the peaks
of 21
2 nmol/L
obtained
quinMF
erythrocytes.
Spectrum
B shows
the
one
arising
from
quinMF equation: or their
uncomplexed
(Ca-quinMF). Ca =
quinMF
Ca?
30 minutes of deoxygenation. in the Iinewidth of the based on The the increase paramagnetism in the
which be genated
is
K[Ca-quinMF]/
[quinMFj.
Under proportional determined of 63 nmol/L by to the assessed peaks of equal
9F resonances,
by
the
to the cutting was comparing area
concentrations
area weighing.
of Ca-quinMF
respective The level of were made for the of the loading quinMF performed previously
can reflect either an actual the presence of extracellular secondary to cell lysis. The by the fact that the
pretation
resonance
was
QuinMF
area known
concentration
6-tluorotryptophan.
studies
(Fremont,
Nicolet which shimmed width and at was
CA)
probe. tuned on half H2O,
NI-360
Observation to 339.7 and of
spectrometer
was MHz we -0. time 1 ppm. were routinely
using
through
a 20-mm
The
broad-band
decoupler sample
Ca-inMF
9F studies.
height acquisition
a 1 27-ms
to correct or quinMF).
therefore, to the ofcell measuring and
uncomplexed
The
this
percentage
with the
of cell
(Hb) total cell
lysis
Hb
hemoglobin
concentration
comparing
Hb
nm)2
concentration
Materials.
measured ester
Probes purchased from
spectrophotometrically of quinMF
(Junction ester from of Molecular
at
540 as
the other
20 15 10
was
City, quinMF.
synthesized
OR) The Probes. effected potassium All
-5
-tO
-15
-20
PPM
Fig 1. 19F spectra of fluorinated calcium chelator 2-(2-amino4-methyl-5-fluorophenoxy)methyl-8-aminoquinoline-N,N.N,N-teWe were initially interested in determining whether Ca traacetic acid (quinMF)-loaded sickle erythrocytes. (A) Spectrum levels increased in sickle erythrocytes particularly during shows oxygenated erythrocytes (5.000 acquisitions, 1 0-minute deoxygenation. FBAPTA has been used previously95 to accumulation). Values for control Ca level were obtained from spectra that had been corrected for extracellular quinMF. measure Ca in erythrocytes. The accuracy of the Ca similar suspension was then deoxygenated with 1 00% N2 for 30 determination in erythrocytes is limited, however, due to theThe minutes and spectrum shown in (B) was accumulated for 10 mismatch between the basal level of Ca and the binding minutes under 100% N2 (5,000 acquisitions). (C) Spectrum shows constant of FBAPTA for Ca2; thus, even for a I mmol/L a 10-minute accumulation of the same deoxygenated cells to which 10 mmol/L of EGTA was added. loading of chelator, the signal of Ca-FBAPTA corresponds
RESULTS
NMR
MEASUREMENTS
OF
CYTOSOLIC
FREE
CALCIUM
1471
sponding
to
Ca-quinMF
in
Fig
lB
appears
as
relatively
sharp potentially
intracellular
resonance reflecting
Ca-quinM
superimposed contributions
F, respectively.
on
To
a from EGTA
broader extracellular
distinguish
resonance, and
furexcess EGTA
ther cell
between
these
two
possibilities,
to an with
extracellular
ation
does
not basal
increase
Car.
The
levels
of Ca
excess Ca
difference
a sample of
of
cells
was
taken Microscopic
and
glutaraldehyde.
of 500 erythrocytes showed that >80% were in A conformation. Using 31P NMR, we also condeoxygenation of sickled cells caused no decrease
(data not shown). Identical deoxygenation
ATP
experiments
erythrocytes. cytes averaged
were
After
performed
deoxygenation, I 7 4
with
Ca
quinMF-loaded
of normal erythro-
(n = 3), oxygenated
a value
colleagues5
bis-(2-amino-5-fluorophe(FBAPTA) by sickle erytherythrocytes incubated with cytosis, FBAPTA, although impermeant FBAPTA (10 mmol/L) plus sufficient extracellular in the sickle erythrocytes. Results are shown in Fig 2. The calcium to give a ratio of CaFBAPTA/FBAPTA of 6/4. The suspeninitial spectrum, (Fig 2A), was taken with 10 mmol/L of sion was deoxygenated for 1 hour and then washed twice in saline. The cells were resuspended in modified saline FBAPTA and 6.0 mmol/L of CaCl2 in the incubating modified containing 1 .25 mmol/L of CaCI2 and the spectrum (B) was medium, so that the ratio of CaFBAPTA/FBAPTA was accumulated. Excess EGTA (20 mmol/L) was then added and 1 .5/ 1 .0. The high ratio of signal to noise reflects the high another spectrum (C) was accumulated. All spectra were 5.000 concentration of FBAPTA and the large extracellular volacquisitions. CaB, CaFBAPTA; B. FBAPTA.
To test this hypothesis, with the acetoxymethyl with the ionized form the membrane.
cells (that had not been 20 15 10 5 0 ester of FBAPTA) were of FBAPTA, which cannot Fig 2. Endocytosis of 1,2 acid If deoxygenation stimulates endo- noxy)ethaneN,N.N.N-tetraacetic rocytes. Spectrum (A) shows sickle impermeant, should be detected
ume. The cells were washed twice in the resuspended mmol/L 2B) and was then FBAPTA in the of CaCI2 obtained. peaks
then deoxygenated modified saline modified (room were air). In saline The
I hour at room
at 37#{176}C, air, and FBAPTA was intracellular in the form ofCa-FBAPTA. of the resonance intensity of the FBAPTA containing I .25ratio shown in Fig 2B to that in spectrum spectrum (Fig spectrum CaFBAPTA the ratio of than was have that the ly. 0.4%
Similarly,
peak A,
of
both
CaFBAPTA
the ratio of
was
the
sequestered
CaFBAPTA peak
intracellular-
CaFBAPTA spectrum extracellularly, intracellular. tosis EGTA (spectrum of the the free as (20
resonance shown in Fig the opposed mmol/L) shown in Fig intensity position, to free To demonstrate
to FBAPTA 2A. Because FBAPTA further added EGTA the confirming from to extracellular
was larger excess Ca peak the the shifted Ca-FBAPTA that must presence
in Fig 2C to that were obtained after normal similar initially FBAPTA erythrocytes, to with those suspended
of spectrum A was 0.6%. three repetitions of this spectra described in media of CaCl2 3A). two washes, were for obtained Fig 2. The a containing to give the cells Subsequent
of endocy-
3, using
all mmol/L 10
resonance FBAPTA
of CaFBAPTA/FBAPTA ofdeoxygenation
a fraction
of theto a I -hour
1472
MURPHY
ET
AL
(.iH
with
mmol/L
of
CaCI2).
After
erythrocytes 9F
the
for spectrum
while
shows
spectra
ratio
ofextracellular
ent The
when cells
the were
.1 25 obtained
cells then
were
for
I hour. in saline in
washed
mmol/L under
shown correspond-
Ca-B
y
excess Ca
B
y
y
lids.
IGTA
C
V
#{149}zc.ss Cs
20
15
10
-IS
-20
PPC
Fig 3. Endocytosis of 1 ,2-bis(2-amino-5-fluorophenoxy)ethane N.N.NN-tetraacetic acid (FBAPTA) by normal erythrocytes. (A) Spectrum shows normal erythrocytes incubated with impermeant FBAPTA (10 mmol/L) plus sufficient extracellular calcium to give a ratio of CaFBAPTA/FBAPTA of 8/2. The RBCs were deoxygenated for 1 hour and then washed twice in modified saline. Spectra (B) and (C) were then accumulated with excess calcium and excess EGTA. respectively. All spectra were 5.000 acquisitions. Ca-B, CaFBAPTA; B. FBAPTA.
were CaCl2
FBAPTA
saline
containing
1 .25
resonance corresponding The addition of excess of the of CaFBAPTA the free that resonance FBAPTA all the FBAPTA sickle erythrocytes
of FBAPTA
in elimination with
with
the
V
-10 -15 -20 PPM
occur. The
cytotic
data
vesicles
presented
are
suggest
formed
that
that
on cell in
endoextracellular
incorporate
thereby was
is vesicles.
increasing to test
pumped For this study,
A final
expercalcium,
endocydeoxy-
designed transient
actively
possibility
and
deoxygenation
small
erythrocytes
genated FBAPTA
in
in
which to 1/15
the (15
ratio of mmol/L
CaFBAPTA/ of FBAPTA
Calcium uptake into endocytotic vesicles during deoxy(A) Spectrum shows erythrocytes incubated with 15 mmol/L of impermeant 1.2 bis-(2-amino-5-fluorophenoxy)ethane N,N,NN-tetraacetic acid (FBAPTA) plus calcium to give a ratio of CaFBAPTA/FBAPTA of 1 /1 5. Cells were deoxygenated for 1 hour. washed and resuspended in an oxygenated buffer containing excess extracellular calcium, and then spectrum B was accumulated. (C) Spectrum was accumulated after the cells were deoxygenated for a second time (20 minutes) in the presence of Ca2. Following addition of excess EGTA to the deoxygenated suspension, spectrum shown in (D) was accumulated. Cells were then washed and resuspended in excess Ca2, and spectrum shown in (E) was obtained.
NMR
MEASUREMENTS
OF
CYTOSOLIC
FREE
CALCIUM
1473
ing
to
uncomplexed of that vesicle. CaCI2 The in the in the varied was due
subsequent
the medium.
of
different
from
values
of
Ca
measured
in
normal
finding erythrocytes. endocy- but during caused ation minutes. we (20 that not saw although a could small be
of of
deoxygenation Ca ofCa are stimulus in sickle during signal-averaging for the presented the means
give
a slight
erythrocytes, deoxygenfor formation by which article on that in this FBAPTA vesicles and Ca2t either Similar oxygen10 of
4C,
elevations we
of CaFBAPTA/FBAPTA we deoxygenated peak; to donor. into FBAPTA added the this donor uptake we
In this
CaFBAPTA from to Ca
incorporation
leakage of
extracelluIn
Ca shown overestimated
medium, in Fig
spectrum tration
(in excess EGTA, extracellular FBAPTA would plexed), the ratio of CaFBAPTA/FBAPTA increased. was confirmed in the spectrum in Fig 4E, which intravesicular
washing and
ratio
resuspension
of
uncom- ated sickle erythrocytes or deoxygenated RBCs from normal This control subjects, suggesting that only deoxygenation of sickle shows the erythrocytes stimulates endocytosis. Incorporation of FBAPTA/CaFBAPTA following FBAPTA in sickle cells cannot be due to trapping of of the cells in modified saline. FBAPTA between the cells, since: (a) endocytosis could not be demonstrated in normal cells, and the trapped extracellular space is the oxygenated
calcium chelators provide
it has been shown same in comparisons or deoxygenated washed addition that Such twice,
using
fluorinated
and
sickle
of ionized portion of FBAPTA originally lular Ca2 or EGTA. to measure Data presented of two 9F vesicles
tion was
of extracelintracellular accumulawe we
thymocytes,
can had
actively
support accumulate
deoxygenating
demonstrated
by
erythrocytes
under
resonance
and
normal
the
erythro-
range from
in these stimulates
in the
extracellular
entirely in the CaFBAPTA tion of the Ca-FBAPTA tion KD of the more calculated than peak small a large Because calcium has a erythrocytes, 15 to 30 are tenfold level
of Can. basal
Because
present Ca2 uptake into these intracellular vesicles, consistent with will be transient a increase in Cai. to an overestimaThese studies indicate that despite the high total calcium to an overestimathat has consistently been observed in sickle RBCs the FBAPTA has ionized a calcium in the cytosol is not different from that of
FBAPTA therefore, produce peak. nated QuinMF normal from FBAPTA the
Ca levels, the Canormal cells. Deoxygenation of sickle RBCs does increase one-tenth of the FBAPTA peak; calcium permeability and may result in a slight transient of fluorinated chelator leakage increase in Ca1, but this excess calcium is rapidly pumped of increase in the Ca-FBAPTA into and sequestered by intracellular, endocytotic vesicles difficulties, we developed new fluori- and is pumped out of the cytoplasm by CaATPase. In this with tighter calcium binding. manner, such vesicles may have an important role in protect63 nmol/L calculated The Ca and, when level of values due loaded Ca into ing the ranges result using studies in capacity sickle from leave of may erythrocyte an open the exist leading increase the vesicles from in possibility, for in intracellular to ion and the adverse however, calcium water calcium loss effects calcium. that accumulation, and from despite known These the brief activate a the sickle to intracellular
nmol/L. probably
errors
sickle
erythrocytes
ACKNOWLEDGMENT
a considerable
These
of Ca
do
is sequestered
not provide
thank
Sorrell
Drs C. Tyler
and and critical Amy
Burt
reading Lewter
and
of for
John
the
C. Parker
manuscript.
for their
We also
helpful
thank assistance,
suggestions
on
whether of sickle
in Ca occurs during Robin Our studies show that and is not signifi- stirrer.
excellent
secretarial
Robert
Hall
for
designing
and
constructing
the
air-driven
well-oxygenated
erythrocytes
1474
MURPHY
ET AL
REFERENCES
10. Smith GA, Hesketh RI, Metcalfe JC, Feeney J, Morris P0: Intracellular calcium measurements by F NMR of fluorine labeled I973 chelators. Proc NatI Acad Sci 80:7178, 1983 2. Palek J: Calcium accumulation during sickling of hemoglobin 1 1. Levy LA, Murphy E, London RE: Synthesis and characterS red cells. Blood 42:988, 1973 ization of 9F NMR chelators for measurement of cytosol-free Ca. 3. Gardos G: The function ofcalcium in the potassium permeabilAm J Physiol (in press) ity of human erythrocytes. Biochem Biophys Acta 30:653, 1958 I 2. liffert I, Garcia-Sancho J, Lew VL: Irreversible ATP deple4. Bookchin RM, Lew VL: Red cell membrane abnormalities intion caused by low concentrations of formaldehyde and of calciumsickle anemia, in Brown EB (ed): Progress in Hematology, vol XIII. chelator esters in intact human red cells. Biochem Biophys Acta Orlando, FL, Grune & Stratton, 1983, pp 1-23 773:143, 1984 5. Lew VL, Hockaday A, Sepulveda MI, Somlyo AP, Somlyo 13. Metcalfe JC, Hesketh TR, Smith GA: Free cytosolic Ca2 #{192}y, Ortiz OE, Bookchin RM: Compartmentation of sickle-cell measurements with fluorine labelled indicators using 9F NMR. Cell calcium in endocytic inside-out vesicles. Nature 315:586, 1985 Calcium 6:183, 1985 6. Bookchin RM, Ortiz OE, Lew VL: Cell dehydration from 14. Fabry ME, San George RC: Effect of magnetic susceptibility sickling-induced calcium-sensitive K -channe1 activation. BlOOd on nuclear magnetic resonance signals arising from red cells: A warning. Biochemistry 22:41 19, 1983 66:56a, 1985 (abstr) 7. Rhoda MD, Giraud F, Craescu CT, Beuzard Y: Compartmen15. Gupta RK, Schanne FAX: 9F NMR measurements of intratation of Ca2 in sickle cells. Cell Calcium 6:397, 1986 cellular free calcium in human red cells. Fed Proc 45:549, 1986 8. Orringer EP, Mattern WD: Formaldehyde-induced hemolysis (abstr) 16. Williamson P. Rubin L, Schlegel R: Internal Ca2 containing during chronic hemodialysis. N EngI J Med 294:1416, 1976 in sickle erythrocytes. J Cell Biol 99:430a, 1986 (abstr) 9. Murphy E, Levy L, Berkowitz LR, Orringer EP, Gabel SA, vesicles London RE: NMR measurements of cytosolic free calcium in I 7. Berkowitz LR, Orringer EP: Passive sodium and potassium movements in sickled erythrocytes. Am J Physiol 249:C208, 1985 human red blood cells. Am J Physiol 25 1:C496, 1986
I . Eaton JW, Skelton ID, Swofford HS, Kolpin CE, Jacob HS:
Elevated
erythrocyte
calcium
in sickle
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246:105,