Published January 1, 1978
Sodium and Calcium Movements
in Dog Red Blood Cells
JOHN
C. P A R K E R
F r o m the D e p a r t m e n t of Medicine, University of N o r t h C a rol i na School of Medicine,
C h a p e l Hill, N o r t h C a r o l i n a 27514
INTRODUCTION
D o g r e d b l o o d cells a r e u n u s u a l a m o n g a n i m a l cells in t h a t t h e y l a c k a N a - K
p u m p a n d a N a - K A T P a s e . T h e i r c y t o p l a s m i c N a c o n c e n t r a t i o n is a b o u t e q u a l
to t h a t o f p l a s m a . T h e m e c h a n i s m b y w h i c h t h e y p r o t e c t t h e m s e l v e s f r o m
o s m o t i c s w e l l i n g a p p e a r s to be d e p e n d e n t o n t h e availability o f m e t a b o l i c
s u b s t r a t e a n d also u p o n e x t e r n a l Ca ( P a r k e r , 1977b).
O m a c h i et al. (1961) r e p o r t e d s t u d i e s o f 4SCa m o v e m e n t s in d o g r e d b l o o d
cells i n c u b a t e d in a r t i f i c i a l e l e c t r o l y t e s o l u t i o n s . T h e y f o u n d t h a t t h e r a d i o i s o t o p e d i s a p p e a r e d f r o m t h e m e d i u m in s u s p e n s i o n s w h e r e KCI was t h e p r i n c i p a l
extracellular solute, but not when NaCI media were used. They interpreted
t h e i r r e s u l t s in t e r m s o f c o m p e t i t i o n b e t w e e n N a a n d Ca f o r e n t r y i n t o t h e cell
( M u l l i n s , 1956) a n d r e l a t e d t h e i r w o r k to s i m i l a r o b s e r v a t i o n s b y N i e d e r g e r k e
(1959) in p e r f u s e d f r o g h e a r t s .
T h e first p a r t o f this p a p e r is a n e x t e n s i o n a n d q u a n t i t a t i o n o f t h e r e s u l t s o f
O m a c h i et al. I n a d d i t i o n , a n e f f e c t o f e x t e r n a l C a o n N a e f f l u x is d e s c r i b e d .
THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 71, 1978 • pa ge s 1-17
1
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ABSTRACT Determinants o f 45Ca influx, ~5Ca efflux, and 22Na efflux were
examined in dog red blood cells. 4SCa influx is strongly influenced by the Na
concentration on either side o f the membrane, being stimulated by intracellular
Na and inhibited by extracellular Na. A saturation curve is obtained when Ca
influx is plotted as a function o f medium Ca concentration. T h e m a x i m u m Ca
influx is a function o f pH (increasing with greater alkalinity) and cell volume
(increasing with cell swelling). Quinidine strongly inhibits Ca influx. Efflux of 45Ca
is stimulated by increasing concentrations of extracellular Na. 22Na efflux is
stimulated by either Ca o r Na in the m e d i u m , and the effects of the two ions are
mutually exclusive rather than additive. Quinidine inhibits Ca-activated ~2Na
efflux. T h e results are considered in terms of a model for Ca-Na exchange, and it
is concluded that the system shows many features of such a coupled ion transport
system. However, the stoichiometric ratio between Ca influx and C a - d e p e n d e n t
Na efflux is highly variable u n d e r different experimental conditions. Because the
Ca fluxes may reflect a combination o f A T P - d e p e n d e n t , outward t r a n s p o r t and
Na-linked passive movements, the true stoichiometry of an exchanger may not be
ascertainable in the absence o f a specific Ca p u m p inhibitor. T h e meaning of
these observations for C a - d e p e n d e n t volume regulation by dog red blood cells is
discussed.
Published January 1, 1978
2
T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y " V O L U M E
71
• 1978
T h e two p h e n o m e n a , N a - i n h i b i t e d Ca i n f l u x a n d C a - s t i m u l a t e d Na e f f l u x , a r e
t h e n d i s c u s s e d in t e r m s o f t h e i r possible r e l a t i o n s h i p s to each o t h e r . A b r i e f
a c c o u n t o f this w o r k has b e e n p u b l i s h e d in a b s t r a c t f o r m ( P a r k e r , 1977a).
MATERIALS
AND
METHODS
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Blood from mongrel dogs was drawn into heparinized syringes and used within 1 h of
venipuncture. After centrifugation the plasma and bully coat were discarded and the
red cells washed in preparation for flux measurements. Unless otherwise noted, all
wash and incubation media contained (mM): HEPES 10; KHCO3 0.5; and glucose 5.
Other constituents are given below and in the figure legends. T h e pH of all media
(unless otherwise noted) was adjusted to 7.40-7.42 at 37°C with Tris-OH. Cell suspension
pH was measured with a thermostatted pH microelectrode unit (Radiometer, Copenhagen). Radioisotope purchases included 45Ca (as CaCI2, 10 mCi/mg Ca) from New
England Nuclear, Boston, Mass., and 22Na (as NaC1, 1 Ci/mg Na) from Radiochemical
Centre, Amersham, England. 45Ca was counted in a liquid scintillation spectrometer
(Packard Instruments Co., Downer's Grove, Ill.) with a scintillation mixture described
previously (Parker, 1970). 45Ca counting efficiency u n d e r all conditions described was
60-62%. 22Na was counted in a gamma scintillation spectrometer (Packard).
For the 45Ca influx studies cells were washed four times in 20 times their volume of
the solution in which they were to be incubated and then resuspended at a cell:medium
ratio of 1:10 and placed at 37°C in a waterbath-shaker. After 5 min 4SCa was introduced
into the flask (to give a radioactivity concentration of about 50,000 dpm/ml suspension),
and at intervals thereafter samples were removed from the incubation flask and pipetted
into four times their volume of ice-cold "stopping solution" containing (mM): LiCI 150;
HEPES 5; EGTA 5; pH adjusted to 7.6 at room temperature with Tris-OH. The
resulting cold suspensions were centrifuged at 0°C, 28,000 g for 10 min in special lucite
tubes with a well at the bottom to localize the pellet. Each supernate was carefully
removed and the cell pellet transferred to a tared container where it was promptly
weighed (usual sample weight 0.25-0.40 g) and then solubilized in 5.0 ml distilled water
containing a drop of detergent (Actaionox; Scientific Products, McGaw Park, Ill.). T o
4.0 ml of the solubilized cell pellet was added 1.0 ml of 70% perchloric acid, and after
thorough mixing the sample was centrifuged. 1 ml of clear supernate was then added to
9 ml of scintillation fluid to give a homogeneous, clear solution for counting. Ca influx
was computed from the slope of the time course of 45Ca entry and the specific activity of
45Ca in the medium, as detailed in the legend to Fig. 2. For the experiments in Fig. 6 the
cells were pretreated before the Ca influx incubation so as to alter their internal ion
composition. This was done by first washing fresh cells four times with 20 times their
volume of Na-free medium (mM): LiCI 120; HEPES 5; KHCOa 0.5; pH 7.4 (37°C) with
Tris-OH. The cells were then divided into aliquots and resuspended in 10 times their
volume of solutions which contained ATP, an agent which rapidly and reversibly alters
Na-K permeability (Parker and Snow, 1972). The composition of these media was (mM):
(NaCI + KCI) = 150 (range: Na 0, K 150 to Na 150, K 0); HEPES 10; glucose 5; ATP 1;
pH 7.5 (room temperature). T h e suspensions were incubated at 37°C for 45 min, after
which the cells were washed free of ATP in the LiC1 wash medium detailed above and
then transferred to the influx media described in the legend of Fig. 6.45Ca was added,
and the procedure for 45Ca influx was followed as noted above.
Efflux studies (Fig. 8) were done by first loading the cells with 4SCa as in Fig. 1, then
washing the cells, and adding them to nonradioactive media. At suitable time points the
cell suspensions were processed exactly as for the influx studies, and the cell radioactivity
was measured.
Published January 1, 1978
PARKER Sodium and Calcium Movements in Red Blood Cells
3
\
R E S U L T S
T h e ' d a t a a r e p r e s e n t e d u n d e r t h r e e h e a d i n g s : Ca i n f l u x (Figs. 1-7); Ca e f f l u x
(Fig. 8); a n d N a e f f l u x (Figs. 9-15):
Ca I n f l u x
Fig. 1 s h o w s t h e t i m e c o u r s e o f *~Ca m o v e m e n t u n d e r c o n d i t i o n s w h i c h f a v o r
t h e e n t r y o f this i s o t o p e : t h e cells a r e s w o l l e n in h y p o t o n i c , N a - f r e e m e d i a at
p h y s i o l o g i c p H with a r e l a t i v e l y low c o n c e n t r a t i o n (40 /zM) o f t o t a l Ca. A f t e r
o.4fLiCI/,/~
o
Z
~,
0.2
~
0
EGTA.
0
-"~
C E O-4Fc,,o,,ooc,
0
TIME(rnin)
I0
20
30
FIGURE 1. T i m e course o f 4SCa entry into cells suspended in Na-free (less than l
mM Na), hypotonic media wi~h LiCI ( u p p e r panel) or choline C1 (lower panel) as
the principal extracellular solute. T h e studies were done in the absence (closed
circles, solid lines) or presence (open circles, dashed lines) of EGTA. Media
composition (mM): Li or choline CI 105; HEPES 10; KHCO3 0.5; glucose 5; CaCI2
0.040; plus or minus E G T A 5; pH 7.42 (37°C). Medium radioactivity was 50,000
d p m / m l (upper panel) and 63,000 dpm/ml (lower panel).
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T h e p r o c e d u r e for Z*Na efflux was as follows. Freshly drawn cells were washed three
times in 20 vol of the buffered, hypotonic LiC1 wash medium described in the
p r e p a r a t i o n of cells for the study in Fig. 6. T h e cells were then washed once in 10 times
their volume of "loading solution." In all cases except the study in Fig. 9 A, where KC1
was replaced by 140 mM NaC1, the "loading solution" contained (mM): KCI 140; HEPES
10; glucose 5; A T P 1; pH 7.5 at room t e m p e r a t u r e with Tris-OH. T h e cells were
r e s u s p e n d e d in twice their volume o f "loading solution," 2*Na was a d d e d (5 t~Ci per 10
ml suspension), and alter 30 min at 37°C the suspension was centrifuged. T h e cells were
freed of external A T P and *2Na by four washes in ice-cold, hypotonic LiC1 m e d i u m and
then pipetted into 100 times their volume o f efflux solutions (detailed composition in
the figure legends) p r e w a r m e d to 37°C. Samples of this suspension were p o u r e d off.at
suitable time intervals and centrifuged at 0°C. T h e radioactivity of the supernate was
then c o m p a r e d with the radioactivity of whole suspension in which the cells were lysed
by the addition of a d r o p of Acationox detergent. T h e results were expressed as noted
in the legend to Fig. 9.
Procedures for measuring the ion and water content of cells have been previously
described (Parker et al., 1975). T h e choline used was recrystallized from ethanol and
kept in a desiccator.
Published January 1, 1978
4
T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y • V O L U M E 7 1 • 1 9 7 8
t h e 1st r a i n , 45Ca e n t e r s t h e cell at a c o n s t a n t r a t e f o r at least 30 m i n a n d rises to
c o n c e n t r a t i o n s w h i c h e x c e e d t h a t o f t h e m e d i u m . Li a n d c h o l i n e a r e e q u a l l y
g o o d r e p l a c e m e n t s f o r e x t r a c e l l u l a r N a , a n d a l t h o u g h O m a c h i et al. (1961)
s h o w e d t h a t K c o u l d b e u s e d as well, we f o u n d t h a t this c a t i o n was lytic o v e r
l o n g i n c u b a t i o n t i m e s ( D a v s o n , 1942). I n t h e p r e s e n c e o f 5 m M E G T A v i r t u a l l y
n o 45Ca e n t e r s t h e cells, a n o b s e r v a t i o n w h i c h l e d us to use this c h e l a t o r as a
c o m p o n e n t o f t h e " s t o p p i n g s o l u t i o n " d e s c r i b e d in M a t e r i a l s a n d M e t h o d s .
Fig. 2 s h o w s a p l o t o f C a i n f l u x as a f u n c t i o n o f m e d i u m Ca c o n c e n t r a t i o n . A
s a t u r a t i o n c u r v e is o b t a i n e d with a h a l f - m a x i m u m v a l u e at a b o u t 0.1 m M C a .
E x t r a c e l l u l a r N a i n h i b i t s Ca i n f l u x (Fig. 3). A b o v e 20 m M N a t h e r e is a l m o s t
8
•
•
o
O
~
6
"-J
I.L~4
~
• LITHIUM
MEDIUM
O C H O L I N E MEDIUM
o~
(D E
E 2
v
o
I
I
I
I
I
0.2
0.4
o.o
08
,.o
[CO]o (raM)
FIGURE 2. Ca influx as a function of extracellular Ca. LiC! (closed circles) or
choline C1 (open circles) was the principal extracellular solute. Media composition
(mM): LiCI or choline CI 105; HEPES 10; KHCOs 0.5; glucose 5; CaC12 as noted
on abscissa; pH 7.42 (37°C). Ca influx was c o m p u t e d by dividing the slope of the
45Ca influx curve (dpm/kg cells" h) by the specific activity of the medium Ca (dpm/
mmol Ca).
~
3i
z
E '
°o
I
Io
20
?o
FIGURE 3. Ca influx as a function o f external Na in hypotonic LiCl (closed
circles, solid line) or choline C1 (open circles) solutions. Media compositicn (raM):
sum o f LiCl or choline CI + NaCl = 110; HEPES 10; KHCO3 0.5; glucose 5; CaCl2
0.040; p i t 7.42 (37°C). All solutions were isosmotic with one another. Mean - SEM
for three LiCI studies; a single choline C1 study is shown.
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x_ ~
Published January 1, 1978
PARKE~t Sodium and Calcium Movements in Red Blood Cells
no measurable accumulation o f 45Ca in the cells. A strong d e t e r m i n a n t o f Ca
influx is the p H o f the cell suspension (Fig. 4). This finding is in g o o d
quantitative a g r e e m e n t with the curves published by O m a c h i et al. (1961). T h e
r i g h t - h a n d panel o f Fig. 4 suggests that inhibition o f Ca accumulation at low
p H reflects a decrease in the m a x i m u m rate r a t h e r than a c h a n g e in the affinity
o f the influx mechanism. Cell volume exerts a p r o f o u n d effect on Ca influx, as
shown in Fig. 5. T h e cells accumulate m o r e Ca as they are swollen. This result
~
~
F[co]-5o.,,M/"
I_ o /
|
I-
4
/
pH7.42
/
[co]ocmM
FIGURE 4. Ca influx as a function of pH at a single extracellular Ca level (left)
and as a function of medium Ca at two suspension pH values (right). Media
composition (raM): LiCl 105; HEPES 10; KHCOa 0.5; glucose 5; CaCl, as noted in
graphs; pH (37°C) adjusted over range indicated by titration with Tris-OH. Pooled
data from four experiments.
-[co];5o,,M/.¢
7 '°I
~5
-
z_-~
7o
% CELLWATER
3
0
CELL%w
TW.T
RwT
F~GUR~. 5. Ca influx as a function of cell water content at a single extracellular
Ca level (left) and as a function of medium Ca at three valuesfor cell water
(right). Media composition (mM): LiCI 105-200 mM; HEPES 10; KHCOa 0.5;
glucose 5; CaC12 as noted in graphs; pH 7.42 (37°C). Normal water content of
freshly drawn dog red blood cells in plasma (Parker, 1973a) indicated by arrow.
Pooled data from four experiments.
indicates that the p H effect shown in Fig. 4 c a n n o t be explained on the basis o f
pH-associated volume changes, since one would expect the cells to shrink with
increasing alkalinity. T h e result in Fig. 5 is not an ionic strength effect, since
addition of sucrose to media of constant LiCl concentration has the same
influence as a d d i n g m o r e salt (Parker et al., 1975). Fig. 5 shows a close
resemblance to a previously published g r a p h displaying K influx as a function
o f cell water (Parker a n d H o f f m a n , 1976). I n each case the major c h a n g e in
flux occurs as the cells rise above a n o r m a l water content.
Fig. 6 demonstrates that the entry o f Ca into d o g red blood cells is inhibited
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p.
Published January 1, 1978
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
• VOLUME
71 - 1978
by lowering their normally high Na concentration. T h e cation composition was
altered by preincubating the cells in media containing A T P as described in
Materials and Methods. Although K was used as a replacement for Na in the
studies shown, similar results were obtained with Li which, like K, exchanges
rapidly with Na in the presence o f external A T P (Brown and Obaid, 1976).
Quinidine strongly inhibits Ca influx (Fig. 7), and quinine is equally effective
(result not shown). T h e s e alkaloids were tried because o f reports that they
block Ca m o v e m e n t s in subcellular p r e p a r a t i o n s f r o m rabbit h e a r t ( H a r r o w
12 ~.E I O -
pH 7.6
ta8
4
z_
o 2
0
0
;
20
-
0"
40
-
60
80
~-IGURE
6. Ca influx as a function of intracellular Na concentration at three
values [or suspension pH. Cells with differing Na/K contents were prepared by
preincubation with solutions containing A T P as noted in Materials and Methods.
After removal of the A T P and resuspension in the flux medium, the cells had
70% +- 1% water content regardless of their Na/K composition. T h e medium in
which Ca influx was measured contained (raM): LiCI 120; HEPES 10; KHCO3 0.5;
glucose 5; CaC12 0.1; pH (37°C) adjusted to values indicated by titration with TrisO H . T h r e e separate experiments, one at each pH.
and Dhalla, 1976) and that they inhibit the influence o f Ca on K flux in h u m a n
red blood cells ( A r m a n d o - H a r d y et al., 1975).
Ca Efflux
Earlier work had led us to believe that Ca efflux might be stimulated by
extracellular Na (Parker et al., 1975). This idea was c o n f i r m e d by the studies in
Fig. 8.
Na efflux
T h e m e t h o d used here for loading cells with 22Na makes use o f the action o f
extracellular A T P , which causes a reversible increase in m o n o v a l e n t cation
permeability in dog red blood cells. D e p e n d i n g on the composition o f the
loading m e d i u m the cells can be made to contain any combination o f Na and K
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E
x
~
_J
Published January 1, 1978
PARKER
Sodium and Calcium Movements in Red Blood Cells
(see Fig. 6). In preliminary work with cells l o a d e d in h i g h - N a media so as to
preserve their n o r m a l ion content, we f o u n d the kinetics o f a2Na efflux u n d e r
some o f the conditions we wished to examine to be complicated (Fig. 9A).
W h e n cells were made low in Na at the time o f 22Na loading, however, the
subsequent efflux plots could be described with a single rate constant (Fig. 9 B,
C). Accordingly, all Na efflux movements with the exception o f those in Fig.
9 A were measured in cells with Na content r a n g i n g f r o m 24 to 34 mmol/liter
cells. Because o f variations in cell Na f r o m day to day the results are expressed
in terms o f rate constants r a t h e r than fluxes. Values for Na flux are presented
in the discussion (Table I I I ) where the stoichiometry between Ca and Na
movements is considered.
Fig. 9 B demonstrates that extracellular Ca stimulates the exit o f 2~Na f r o m
cells s u s p e n d e d in a hypotonic, Na-free m e d i u m . Mg ion had no effect. Fig.
1.0,
~o
oo
0.6
X
--J
Z
o
~
O.4
0.2
0
0
0.I
QUINIDINE (rnM)
•
0.2
FIGURE 7. Dose-response curve for quinidine inhibition of Ca influx. Ordinate,
ratio of Ca influx in the presence of various concentrations of quinidine to quinidine-free controls (a value of 0 means 100% inhibition by quinidine). Media composition (raM): LiCI 105; HEPES 10; KHCOz 0.5; glucose 5; CaC12 0.1; pH 7.42
(37°(]). Mean -+ SEM for three studies.
9 C shows that the release o f 22Na is likewise increased when Na is substituted
for choline in the m e d i u m . T h e effect o f Na was greater at a concentration o f
18 mM than at 110 mM.
Fig. 10A shows that the stimulation o f 2ZNa efflux by Ca is maximal at Ca
concentrations as low as 0.1 mM ( E D T A 0.01 mM was present in the medium).
In Fig. 10 B are displayed the results o f studies using 0.01 mM E D T A media to
which smaller concentrations o f Ca were a d d e d so as to give levels of ionized
Ca f r o m 10-7 to 10 -4 mM (Portzehl et al., 1964). T h e great sensitivity of Na
efflux to extracellular Ca (half-saturation at 10-6-10 -5 M Ca) led us to include
E D T A in all media so as to obtain a reliable value for the "zero Ca" point,
inasmuch as we f o u n d u p to 10 /~M contamination o f o u r nominally Ca-free
media with Ca. As will be pointed out, this consideration applies also to studies
o f Na-stimulated 22Na efflux.
Fig. 11 shows the c o n c e n t r a t i o n d e p e n d e n c e o f 2~Na efflux on extracellular
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o,,
Published January 1, 1978
8
THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 71 • 1978
Na. O p t i m u m stimulation occurs at a m e d i u m Na concentration o f 15-30 mM.
T h e c o m b i n e d effects o f Ca and Na on 22Na release are shown in Fig. 12. At
zero Na the stimulatory effect o f Ca is readily seen, but as Na is increased, Ca
ceases to affect 2~Na efflux. Conversely, Na stimulation o f 22Na efflux is readily
seen at zero Ca (with E D T A added), but when Ca is present n o stimulatory
effect o f Na can be observed. T h u s the effects o f Ca and Na are o f about the
same m a g n i t u d e , and each eclipses the other. It is likely that o u r failure in the
past to show Na stimulation o f Na efflux in dog red cells (unpublished
observations) was due to m i c r o m o l a r contamination o f the media by Ca.
IO0 q
Na] (mM)
30 MIN
~d 80
-
g.
'¢ z 6 0
f...-z(..9
Wz
o
-\
O,
"'o.
i
o
I00
I
30
60
TIME (rain)
0
I
I
50
I00
FIGURE 8. 45Ca efflux as a function of time (left panel) and medium Na (right
panel). Freshly drawn dog red blood cells were loaded with 45Ca by incubation for
30 min at 37°C under the conditions described in Fig. 1, i.e. in a medium
containing (mM): LiCI 105; HEPES 10; KHCO3 0.5; glucose 5; CaClz 0.040; pH
7.42 (37°C). The medium contained 4SCa 50,000 dpm/ml. The cells were then
washed at 4°C with the same medium (minus 4~Ca) so as to free them from
external radioactivity. At the beginning of the efflux period the cells were
resuspended at 37°C in media containing (mM): sum of LiC1 + NaCI = 110;
HEPES 10; KHCOa 0.5; glucose 5; pH 7.42 (37°C). At intervals thereafter cells
were centrifuged out of the suspension and prepared for counting as noted in
Materials and Methods. The cell radioactivity is expressed as a percentage of that
present at the beginning of the efflux period. Left panel shows efflux at media Na
of less than 1 mM (closed circles, solid lines) and 100 mM (open circles, dashed
lines). Right panel shows values for 30 min samples with various Na concentrations
in the medium as indicated on the abscissa. Two separate experiments.
Fig. 13 shows the p H d e p e n d e n c e o f 2~Na efflux in the presence a n d absence
o f external Ca and Na. T h e p H curve for Ca- or Na-stimulated Na efflux is
similar to that for Ca influx (Fig. 4) in that both show inhibition u n d e r acid
conditions; but the slope o f the curve for Na efflux is not as great as that for Ca
influx.
Fig. 14 shows that quinidine has a biphasic effect on 22Na efflux, causing
inhibition at levels o f 0-0.5 mM and stimulation at higher concentrations.
Above 2 mM the d r u g is hemolytic. Addition o f Ca or Na to the incubation
m e d i u m raises the base-line ~=Na efflux, as already shown, a n d the inhibitory
effect o f quinidine is greater with either o f these two ions in the solution (Fig.
14, Table I).
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uJ~
a_~
20
Ld
Published January 1, 1978
PARKER Sodium and Calcium Movements in Red Blood Cells
9
Fig. 15 shows that the effect o f shrinking the cells in h y p e r t o n i c m e d i a is to
raise the Na efflux rate constant, an observation which has been made
repeatedly on d o g red blood cells (Davson, 1942; Parker a n d H o f f m a n , 1976).
It is notable that neither the e x p o s u r e to A T P d u r i n g the isotope load n o r the
0
.
A
It0]0
O.L
It0]0
(raM)
.
~ox
\~
-T 0.2
I r N ~ 78mmol
tL QJc~
0.3 1
0
L
60
\o
\\ O.5
~,
~
N I ZSmmol~'~.b
°]c
i
I
120 0
60
TIME (min)
c
[.°]o
-
(0 M)
i
120
0.1 ~
-r o.2
.....
r.
!8%
~ 30retool
I LN°Jc ~
0.3
i
0
60
J
120
T I M E (min)
F]OVRE 9. A, B, e~Na efflux time course in a low (<0.5 mM)-Na medium, with
high Na (panel A) and low Na (panel B) cells. The effects of adding CaCle 0.5 mM
to the media are shown. The quantity f on the ordinate represents the ratio of
medium counts to counts in a hemolysate of the suspension. In Materials and
Methods the procedure for loading cells with 2~Na and simultaneously altering
their Na/K content is given. The basic efflux media for both studies was (mM):
choline C1 110; HEPES 10; KHCO3 0.5; free EDTA 0.1; pH 7.42 (37°C), plus CaCI2
as noted in the graphs. C, 2~Na efflux time course in a zero Ca medium with lowNa cells. The effects of substituting NaCI for choline CI in the medium are shown.
The ordinate notation and 22Na loading procedure are as noted above (A, B).
Efflux media contained (mM): sum of choline CI + NaC1 = 110; HEPES 10;
KHCO3 0.5; glucose 5; free EDTA 0.1, pH 7.42 (37°C). Na concentrations in cells
and medium are as noted in the graph.
low Na concentration o f the cells following the loading phase inhibited the
effect o f cell shrinkage on Na efflux. O f i m p o r t a n c e is the observation that the
i n c r e m e n t in the rate constant for ~2Na efflux with 0.5 mM Ca present is about
the same in swollen as in s h r u n k e n cells.
In Figs. 5 and 15, m o v e m e n t s o f Ca a n d Na in cells o f different volumes are
c o m p a r e d . A , p r o p e r interpretation o f such data should take account o f the fact
that when cells are s h r u n k e n or swollen there is a c h a n g e in n u m b e r o f cells
Downloaded from on June 18, 2017
0
"~
B
(m~v~)
Published January 1, 1978
10
THE
0.10 - ~ - - -
JOURNAL
.O- . . . . .
OF
GENERAL
PHYSIOLOGY'VOLUME
7]"
1978
O
P
)
T 0.O8
r/
X
~F'-
d
006
~. o
004
oJ
oJ
L~
I-<
0.02
or"
A
°o
B
,I
l
0.5
I
1.0
I
I
I
-oo -7 -6 -5 -4
O. lO
•
•
0
0
J
0.05
I
0.00 ~-
0
Choline CI
Li CI
L
L l
20
i
40
L
6O 0
20
I
t
40
I ,
}
60
FIGURE 11. Rate constant tor 22Na efflux as a function of extracellular Na in
predominantly LiC1 (left panel) or choline CI (right panel) media with equimolar
substitutions of NaC1 in the concentrations indicated. Media composition (raM):
sum of LiCl or choline CI + NaCl = 110; HEPES 10; KHCO3 0.5; free EDTA 0.1;
pH 7.42 (37°C). Cell Na = 24-34 mmol/kg cells. Left, two separate studies plotted
in open and closed circles. Right, mean - SEM for three studies.
( a n d t h e r e f o r e o f total c e l l - s u r f a c e a r e a , if o n e a s s u m e s t h a t s u r f a c e a r e a p e r
cell is c o n s t a n t ) p e r v o l u m e o f cells. A way o f e x p r e s s i n g the d a t a , r e c o g n i z i n g
this, is to c o m p u t e " a p p a r e n t p e r m e a b i l i t i e s " as is d o n e in T a b l e I I . T h e s e
c a l c u l a t i o n s show in t h e case o f t h e Ca fluxes t h a t the effect o f cell swelling o n
Downloaded from on June 18, 2017
FIGURE 10. Rate constant for 22Na efflux as a function of extracellular Ca in
predominantly LiCI (closed circles, solid lines) or choline C1 (open circles, dashed
lines) solutions. Left panel (A) shows a range of Ca from 0 to 1.0 raM; right panel
(B) shows levels of ionized Ca from 0 to 10.4 M in log units, as calculated from Ca
and EDTA concentrations by the method of Portzehl et al. (1964). Media composition (raM): LiC1 or choline C1 110; HEPES 10; KHCO3 0.5; glucose 5; free EDTA
0.010; pH 7.40 (37°C), plus CaCI~ as noted. Cell Na = 24-34 mmol/kg cells. Four
separate studies.
Published January 1, 1978
11
PARKEe Sodium and Calcium Movemen~ in Red Blood Cells
0.10
LI_~-oO
CO]o
0.05
--¢-- 0
--o- 0.5 mM
ea ta9
<t
0.00
I
0
I
I
20
I
40
i
I
I
I
600
[O]o(mM/
I
I
20
40
1
I
60
Rate constant for Z2Na efflux as a function of extracellular Na in a
predominantly choline CI medium in the presence (open circles, dashed lines) and
absence (closed circles, solid lines) of CaCI2 0.5 raM. Media composition (mM):
choline CI + NaCI --- 110; HEPES 10; KHCO3 0.5; free EDTA 0.1; pH 7.42 (37°C)
plus or minus CaC12 as indicated in the graphs. Cell Na = 24-34 mmol/kg cells.
Two separate studies are shown.
FIGURE 12.
d8
0.05
W
l.cr
0
6.8
I
I
7.0
i
I
7.2
I
I
I
7.4
pH
FIGURE 13. Rate constant for 22Na efflux as a function of suspension pH in a 110
mM choline medium with no added Ca or Na (closed circles), with Ca 0.5 mM
(open circles), or with equimolar substitution of NaC1 18 mM for choline (crosses).
Media composition (mM): control (closed circles)-choline CI 110, HEPES 10,
KHCO3 0.5, glucose 5, free EDTA 0.1; Ca (open circles)-choline CI l l 0 , HEPES
10, KHCO3 0.5, glucose 5, free EDTA 0.1, CaC12 0.5; Na (crosses)--choline CI 92,
NaCl 18, HEPES 10, KHCOa 0.5, free EDTA 0.1. pH was adjusted io indicated
values (37°C) with Tris-OH. Celt Na = 24-34 mmol/kg cells. T h r e e separate
studies are shown, one for each medium.
a p p a r e n t Ca p e r m e a b i l i t y is actually g r e a t e r t h a n s u g g e s t e d by t h e d i m e n s i o n s
u s e d i n Fig. 5. T h e rise i n 22Na e f f l u x r a t e c o n s t a n t with cell s h r i n k a g e (Fig. 15)
reflects a n i n c r e a s e i n a p p a r e n t p e r m e a b i l i t y , a l t h o u g h t h e m a g n i t u d e o f t h e
effect is s o m e w h a t less t h a n t h e r a t e c o n s t a n t s p e r se w o u l d i n d i c a t e . T h e s e
Downloaded from on June 18, 2017
x/
0.I0
Published January 1, 1978
12
THE JOURNAL
OF GENERAL PHYSIOLOGY
• VOLUME
71 • 1978
c o m p u t a t i o n s also b r i n g o u t t h e p o i n t t h a t N a p e r m e a b i l i t y does n o t rise
a p p r e c i a b l y u n t i l t h e cell v o l u m e d r o p s b e l o w a n o r m a l v a l u e .
DISCUSSION
T h e f o r e g o i n g e x p e r i m e n t s w e r e d e s i g n e d to e x a m i n e C a a n d N a m o v e m e n t s
in d o g r e d b l o o d cells as f u n c t i o n s o f several variables: m e d i u m p H ; cell
I,°c° //
020[-- l"c°'m°LI
x
v
u..~:
1.1_ I--
O. 15
x
A
No
o,o
///
0.05
(]1::
~
i
o.~
~.0
QUINIDINE
i
f
L5
(mM)
2.0
FIGURE 14. Rate constant ['or 22Na efflux as a function of quinidine concentration. Media composition (mM)- control (closed circles)-choline CI 110, HEPES 10,
KHCO3 0.5, glucose 5, free EDTA 0.1; Ca (open circles)-choline Ci 110, HEPES
10, KHCO3 0.5, glucose 5, free EDTA 0.1, CaCI2 0.5; Na (crosses)-choline C1 92,
NaC1 18, HEPES 10, KHCO3 0.5, glucose 5, free EDTA 0.1. All media pH 7.42
(37°C). Cell Na = 24-34 mmol/kg cells. A single study is shown. For further data
see Table 1.
TABLE
1
I N H I B I T I O N OF 22Na EFFLUX BY Q U I N I D I N E
Media conditions (raM)
Ca 0,
Na 0
Na efflux rate constant (h -a) in absence
of quinidine
0.040_+0.003
Maximum inhibition by quinidine (%)
16.3_+4.0
Ca 0.5,
Na 0
0.097--+0.002
47.2-4-4.9
Ca 0,
Na 18
0.101_+ 0.006
35.5_+5.5
Media composition (mM): Ca0, Na 0-LiCI or choline CI 110, HEPES 10, KHCO3 0.5, glucose 5,
free EDTA 0.1; Ca 0.5, Na 0-LiCI or choline C1 110, HEPES 10, KHCOs 0.5, glucose 5, free
EDTA 0.1, CaCI2 0.5; Ca 0, Na 18-LiCI or choline CI 92, NaCI 18, HEPES 10, KHCO3 0.5, glucose
5, free EDTA 0.1. All were at pH 7.40-7.42 (37°C). The quinidine concentration is that at which
maximum inhibition was seen and ranged from 0.2 to 0.5 mM (see Fig. 14). Mean -+ SEM for five
studies with each medium.
v o l u m e ; cell a n d m e d i u m Na; m e d i u m Ca; a n d q u i n i d i n e . T h e h y p o t h e s i s
u n d e r c o n s i d e r a t i o n is w h e t h e r t h e r e is a n y l i n k a g e b e t w e e n N a a n d Ca
t r a n s p o r t . T h e c o n c l u s i o n will be t h a t since N a - i n h i b i t a b l e Ca i n f l u x r e s p o n d s
to s o m e o f the i m p o s e d c o n d i t i o n s d i f f e r e n t l y f r o m C a - s t i m u l a t e d N a e f f l u x ,
the r e l a t i o n s h i p b e t w e e n the two fluxes is n o t s t r a i g h t f o r w a r d . B e f o r e d i s c u s s i n g
these m a t t e r s in detail, several f e a t u r e s o f these e x p e r i m e n t s s h o u l d be p o i n t e d
out.
Downloaded from on June 18, 2017
0.00~
o
Published January 1, 1978
13
P^RXER Sodium and Calcium Movements in Red Blood Cells
Except for the studies o f Na efflux into Na-containing media, the radioisotopes in these e x p e r i m e n t s are m o v i n g into c o m p a r t m e n t s w h e r e the initial
concentration o f their u n l a b e l e d chemical c o u n t e r p a r t s is very low (dog red cell
Ca is a b o u t 0.020 mmol/liter cells; the nominally N a - f r e e m e d i a contain less
t h a n 0.1 m M Na). T h e m e a s u r e m e n t s do not r e p r e s e n t "steady-state" fluxes in
any sense. T h e tracer m o v e m e n t s are indicative o f net ion m o v e m e n t s (except
in the case o f the Na efflux studies m e n t i o n e d ) . T h e radioisotopes were used
because they are easier to m e a s u r e in the small concentrations dealt with in
these e x p e r i m e n t s . All the conditions which favor the m o v e m e n t s o f 45Ca a r e
the s a m e as those which in a previous r e p o r t were shown to stimulate chemical
0.5
o
~j:: 0.4
v
x
Ii
Z{3O 0 . 2
N
oa
LLI
C2
F-r<r
Oo g
0.1
NORMAL •
CELL
WATER- ~
I
I
I
60
I
I
Iq
•
•
I
65
I
I
I
I~
I
I
I
70
CELL WATER
% WET WT
FIGURE 15. Rate constant for 22Na efflux as a function of cell water content in
the presence (clear symbols) and absence (solid symbols) of CaCI2 0.5 mM. Efflux
media consisted principally of choline CI (square symbols) or LiCI (round symbols).
Media composition (mM): LiCI or choline C1 110-210 raM; HEPES 10; KHCO8 0.5;
glucose 5; free EDTA 0.1, plus or minus CaC12 as noted, pH (37°C) was 7.42.
Normal water content of freshly drawn dog red blood cells in plasma (Parker,
1973a) indicated by arrow. Cell Na ranged from 34 mmol/kg cells at 71% cell
water to 44 mmol/kg cells at 58% cell water. Pooled data from four experiments.
Ca flux (Parker et al., 1975). Lack o f i n f o r m a t i o n a b o u t the physical state o f Ca
in cells, however, prevents a quantitative s t a t e m e n t a b o u t the electrochemical
potential g r a d i e n t f o r this ion. T h u s , the finding that ~ C a concentrations in
the cell m a y rise to levels which exceed that o f the m e d i u m (Fig. 1) is difficult
to i n t e r p r e t in t e r m s o f active versus passive t r a n s p o r t .
Many o f the p r e s e n t studies were d o n e in N a - f r e e media, a n d the possibility
t h e r e f o r e exists that s o m e o f the ion m o v e m e n t s were driven by h y p e r p o l a r i z a tion o f the m e m b r a n e resulting f r o m the large o u t w a r d Na c o n c e n t r a t i o n
gradient. We have e x a m i n e d this question in several ways, including the use o f
a fluorescent dye which m o n i t o r s m e m b r a n e potential ( H o f f m a n a n d Laris,
1974). Dog red blood cells s u s p e n d e d in hypotonic choline solutions do not
show m e a s u r a b l e h y p e r p o l a r i z a t i o n unless agents are included in the system
which cause the Na p e r m e a b i l i t y to be increased. Ca is not o n e o f those agents
Downloaded from on June 18, 2017
oo
0.3
Published January 1, 1978
14
THE JOURNAL
OF G E N E R A L P H Y S I O L O G Y
" VOLUME
71
• 1978
( P a r k e r e t a l . , 1977). W i t h Li as t h e p r i n c i p a l e x t r a c e l l u l a r s o l u t e n o h y p e r p o l a r i z a t i o n w o u l d b e e x p e c t e d , s i n c e t h e Li a n d N a p e r m e a b i l i t i e s i n d o g r e d b l o o d
c e l l s a r e v e r y c l o s e ( P a r k e r e t a l . , 1975).
The interactions between Ca and Na described in these experiments
defy a
simple interpretation.
E a r l i e r w o r k ( P a r k e r e t a l . , 1975) l e d u s t o b e l i e v e t h a t
d o g r e d b l o o d cells m i g h t h a v e a p a s s i v e t r a n s p o r t p a t h w a y , l i k e t h a t s e e n i n
TABLE
APPARENT
PERMEABILITIES
II
T O Ca A N D Na O F C E L L S A T V A R I O U S
VOLUMES*
Solvent ~ o l u m e
Apparent perme
ability
Cell w a t e r
Surface area
Rate constant
% wet wt
cm
s -1
cm/s
3.9x 10-4
22.2x 10-4
47.3x 10-4
12 x 10-9
69x 10-9
176x 10-9
65
66
70
4.3 x 10-5
4.5x 10 5
5.4x 10 5
Na efflux (Fig. 15)
58
60
64
66
70
3.2x
3.5x
4.1x
4.5x
5.4x
10 5
10-5
10.5
10-5
10-5
1.ix
0.7x
0.3x
0.2x
0.1 x
10-4
10-4
10 4
10.4
10-4
4.2x I0-9
2.9x 10-9
1.3x 10.9
1.1 x 10-9
0.9x 10-~
Cell solvent volume:surface area ratios were calculated from the information that under normal
conditions dog red blood cells have a volume of 63 x 10 t5 liter/cell, a density of 1.105 kg/liter cells,
a water content of 0.636 kg/kg cells, and a surface area of 117 x 10-8 cm2/cell. Rate constants were
calculated from Fig. 5 at an external Ca of 0.5 mM. Rate constants for Na are from Fig. 15 in the
absence of extracellular Ca. Apparent permeabilities were computed according to the following
tbrmulas (Sachs et al., 1975):
V k ( e-n - 1~
For Ca influx, P = .~ \ ~ e~e_n j ;
For Na efflux, P
A
where P (cm/s) is the apparent permeability, V (cm z) is the cell solvent volume, A (cm 2) is the cell
surface area (assumed to be constant), and k (s -x) is the rate constant. The quantity B equals zEF/
RT, where z is the valence, E is the membrane potential (assumed constant at - 1 0 mV), and P, R,
and T (310°K) have their usual meanings.
* U n d e r the conditions of Figs. 5 and 15.
e x c i t a b l e cells, w h i c h c o n d u c t s a c o u p l e d e x c h a n g e o f C a f o r N a , N a f o r N a , o r
C a f o r C a ( R e u t e r a n d S e i t z , 1968; B a k e r e t a l . , 1969; B l a u s t e i n , 1974). T h e
e v i d e n c e f o r s u c h a m e c h a n i s m c a n b e l i s t e d as f o l l o w s : ( a ) C a m o v e m e n t s i n
either direction across the membrane are accelerated when the Na concentration
is l o w o n t h e s i d e f r o m w h i c h C a m o v e s a n d / o r h i g h o n t h e s i d e t o w h i c h C a
m o v e s ( F i g s . 3, 6, 8); (b) N a e f f l u x is s t i m u l a t e d b y e x t e r n a l C a ( F i g s . 9, 10, 1 2 15); (c) i n h i b i t i o n o f C a e n t r y b y e x t e r n a l N a ( F i g . 3) o c c u r s a t a b o u t t h e s a m e
c o n c e n t r a t i o n o f N a (20 m M ) w h i c h m a x i m a l l y s t i m u l a t e s 22Na e f f l u x ( F i g . 11).
T h u s it a p p e a r s t h a t N a is c o m p e t i n g w i t h C a f o r a m e c h a n i s m w h i c h m e d i a t e s
Downloaded from on June 18, 2017
Ca influx (Fig. 5)
Published January 1, 1978
PARXER Sodium and Calcium Movements in Red Blood Cells
15
b o t h Ca e n t r y a n d Na exit; (d) the effects o f e x t e r n a l Ca a n d N a o n 22Na e f f l u x
are o f t h e s a m e m a g n i t u d e a n d a r e m u t u a l l y exclusive (Fig. 12), a g a i n s u g g e s t i n g
c o m p e t i t i o n b e t w e e n t h e two ion species for s o m e site o n t h e cell s u r f a c e ; (e)
q u i n i d i n e i n h i b i t s 4SCa i n f l u x (Fig. 7) a n d C a - s t i m u l a t e d 22Na e f f l u x (Fig. 14,
T a b l e I); (J) the p H d e p e n d e n c e c u r v e s for Ca i n f l u x a n d C a - d e p e n d e n t N a
e f f l u x a r e q u a l i t a t i v e l y s i m i l a r (Figs. 4, 13).
A g a i n s t a s i m p l e m o d e l for C a - N a e x c h a n g e are the d a t a s u m m a r i z e d in
T a b l e I I I , w h i c h c o m p a r e f l u x values for Ca a n d N a u n d e r v a r i o u s c o n d i t i o n s
o f cell v o l u m e a n d N a c o n t e n t . T h e r e is n o c o n s t a n t ratio b e t w e e n Ca i n f l u x
(which is c o m p l e t e l y N a - i n h i b i t a b l e , as s h o w n in Fig. 3) a n d C a - s t i m u l a t e d N a
TABLE
III
S T O I C H 1 O M E T R Y BETWEEN Ca INI~LUX AND Ca-DEPENDENT Na EFFLUX
Osmotically s h r u n k e n cells (cell water
60-62% wet wt)
Ca flux (mmol/kg cells, h)
Ca-dependent Na eftlux (retool/
kg cells, h)
Ratio:
Ca influx
Ca-dependent
Na efflux
H i g h Na ceils
160-170
Low N a cells
42-44
H i g h Na cells
70-80
Low Na cells
24-34
<0.2*
9.1§
<0.1"
2.5
7.8:i:
3.5
<0.2*
1.8
<0.03
<0.04
2.2
<0.02
All studies were done in Na-free media at pH 7.40-7.42 (37°C).
* Values obtained from experiments exactly like those in figs. 5 and 6, but with external Ca raised
to 0.5 raM.
:~ Value averaged from Figs. 2, 4, and 5 at 0.5 mM medium Ca. Na effluxes were calculated from
Figs. 9 and 15 as tollows: (Na efflux rate constant with 0.5 mM external Ca [h 1] _ Na efflux rate
constant with 0 mM external Ca [h 1]) x cell Na concentration (mmol/kg cells) = Ca-dependent Na
efilux (mmol/kg cells, h). In the case of the high-Na cells where Na efllux is not linear with time
(Fig. 9 A), the flux was calculated from the amount ot isotope released at 60 rain.
§ Value obtained from experiments performed exactly as in Fig. 9A (high-Na cells), but in a
hypertonic medium consisting of (raM): LiCI 200 HEPES 10; KHCOa 0.5; glucose 5; EDTA 0.1;
plus or minus CaCI2 0.5, pH 7.42 (37°C). This value is the mean of two results: 10.3 and 7.8 mmol/
kg cells- h.
e f f l u x . As cell v o l u m e is r e d u c e d Ca i n f l u x falls, while C a - d e p e n d e n t Na e f f l u x
is i n c r e a s e d . As cell N a is l o w e r e d , Ca i n f l u x falls m o r e s h a r p l y t h a n does Cad e p e n d e n t N a e f f l u x . T h u s , the s t o i c h i o m e t r y o f c o u p l i n g o f Ca to Na
m o v e m e n t s is a p p a r e n t l y v a r i a b l e o v e r a wide r a n g e a n d d e p e n d s o n the
c o n d i t i o n s o f the e x p e r i m e n t . A f u r t h e r d i f f i c u l t y with a s t r a i g h t f o r w a r d m o d e l
for C a - N a e x c h a n g e is t h a t the two fluxes r e s p o n d q u a n t i t a t i v e l y d i f f e r e n t l y to
e x t e r n a l Ca: C a - s t i m u l a t e d N a e f f l u x is 10-100-fold m o r e sensitive to e x t e r n a l
Ca t h a n is Ca i n f l u x (Figs. 2 a n d 10).
T h u s , while q u a l i t a t i v e e v i d e n c e for C a - N a e x c h a n g e exists in d o g r e d b l o o d
cells, c o n s i d e r a t i o n s o f s t o i c h i o m e t r y a n d the a c t i v a t i o n by e x t e r n a l Ca s u g g e s t
that the process m a y be c o m p l e x . S o m e r e s o l u t i o n o f the c o m p l e x i t y m i g h t be
possible if o n e k n e w m o r e a b o u t active Ca e x t r u s i o n i n d o g r e d b l o o d cells.
H u m a n r e d b l o o d cells h a v e a p o t e n t , A T P - d e p e n d e n t Ca p u m p ( S c h a t z m a n n
Downloaded from on June 18, 2017
Cell Na (mrnol/kg cells)
Osmotically swollen cells (cell water
69-71% wet wt)
Published January 1, 1978
16
THE
JOURNAL
OF
GENERAL
PHYSIOLOGY
• VOLUME
71 • 1978
The technical assistance of Paula S. Glosson is gratefully acknowledged. This work was supported
by United States Public Health Service grant AM 11357 from the National Institutes of Health.
Receivedfor publication 16June 1977.
REFERENCES
ARMANDO-HARDY, M., J. C. ELLORY, H. G, FERRE1RA,S. FLEMINGER,and V. L. LEW.
1975. Inhibition of the calcium-induced increase in potassium permeability of human
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across cell membranes. Rev. Physiol. Biochem. Pharmacol. 70:33-82.
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roles of Na and cellular metabolism. Physiologist. 19:140.
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activities of the rabbit heart mitochondria and sarcotubular vesicles. Biochem. Pharmacol. 25:897-902.
HOFFMAN, J. F., and P. C. LAreS. 1974. Determination of membrane potentials in
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and Vincenzi, 1969). It is likely that d o g red cells have a similar mechanism,
a l t h o u g h the effects o f Na on Ca efflux make it difficult to characterize (Parker
et al., 1975). T h e data presented here d o not allow one to tell whether a
condition which favors Ca entry (cell swelling, low external Na, high internal
Na, alkalinity) exerts its effect by raising the inward m o v e m e n t o f Ca or by
inhibiting the Ca p u m p . Similarly, when cells are placed in circumstances
which do not favor Ca accumulation (cell shrinkage, high external Na, low
internal Na, acid conditions, quinidine), the effect could be due to decreased
Ca entry or stimulation o f the Ca p u m p . T h u s , the d o g red blood cell may have
a tightly c o u p l e d Ca-Na e x c h a n g e r , but it might not be possible to study its true
stoichiometry without selectively inhibiting the Ca extrusion p u m p , and this
has not been possible to date.
T h e s e experiments were originally u n d e r t a k e n to clarify the mechanism by
which extracellular Ca activates the uphill, o u t w a r d t r a n s p o r t o f Na by d o g red
blood cells d u r i n g the course o f a d j u s t m e n t f r o m a swollen state toward a
n o r m a l volume (Parker, 1973a, b; Parker et al., 1975). T h e v o l u m e - r e g u l a t o r y
studies were all d o n e with cells s u s p e n d e d in high-Na, plasma-like media, a
circumstance which should suppress inward Ca flux (Fig. 3). Nevertheless,
some net influx o f Ca did occur d u r i n g the cells' adjustment o f Na and water
c o n t e n t (Parker et al., 1975), and it was on this basis that we postulated a Na
p u m p which derives its e n e r g y from the passive, inward m o v e m e n t o f Ca
t h r o u g h a Ca-Na e x c h a n g e r . T h e studies presented here d o c u m e n t n u m e r o u s
interactions between Ca a n d Na movements, but the role o f a Ca-Na e x c h a n g e r
in mediating the net salt a n d water m o v e m e n t s which occur in swollen d o g red
blood cells u n d e r physiological circumstances (Parker, 1973a) remains an o p e n
question.
Published January 1, 1978
PARKER Sodium and Calcium Movements in Red Blood Cells
17
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61:146-157.
PARI~ER, J. C. 1973b. Dog r e d blood cells: adjustment o f salt and water content in vitro.
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PARKER, J. C. 1977a. I n t e r d e p e n d e n c e of Ca and Na movements in dog r e d blood cells.
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