478
Sodium-Lithium Exchange in Sarcolemmal
Vesicles From Canine Superior
Mesenteric Artery
Andrew M. Kahn, Julius C. Allen, Edward J. Cragoe Jr., Ruth Zimmer, and Harnath Shelat
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Exchange of intracellular sodium for extracellular lithium readily occurs in vascular smooth muscle,
but the mechanism of this exchange is not known. These studies examined whether a sodium-lithium
countertransport system was present in the cell membrane of vascular smooth muscle. A sarcolemmalenriched vesicle preparation was obtained from canine superior mesenteric artery via a magnesium
aggregation and differential centrifugation technique. An outwardly directed gradient for lithium
stimulated a Na uptake by the vesicles, and an inwardly directed gradient for lithium stimulated "Na
efflux. These effects were not due to an alteration in membrane potential, and sodium uptake was not
stimulated by lithium in the absence of a gradient for lithium. The lithium gradient-stimulated
component of sodium uptake was not affected by a change in membrane potential and was insensitive
to ouabain. Both sodium-lithium exchange and sodium-proton exchange in sarcolemmal-enriched
vesicles were inhibited by two compounds that inhibit the sodium-lithium countertransport system
in red cells, phloretin and quinidine. Ethylisopropylamiloride also inhibited both sodium-lithium
exchange and sodium-proton exchange in the vesicles. In support of the possibility that sarcolemmal
sodium-lithium exchange and sodium-proton exchange are mediated by a single cation exchange
mechanism with affinity for sodium, lithium, and protons, we found that an inwardly directed sodium
or lithium gradient stimulated proton efflux, and that the stimulation of sodium efflux by external
lithium or protons was not additive. It is concluded from these studies that sarcolemmal vesicles from
canine superior mesenteric artery contain an electroneutral, phloretin, quinidine, and ethylisopropylamiloride inhibitable sodium-lithium exchange transport system. This system may be analogous to
the sodium-lithium countertransport system in reds cells that has been linked to the hypertensive
process in man. Sodium-lithium exchange and sodium-proton exchange in the vascular smooth muscle
cell membrane may be mediated by a single transport system. (Circulation Research 1988;62:478—485)
I
t has been recognized for over a decade that
incubation of vascular smooth muscle tissue in a
lithium-containing solution results in replacement
of intracellular sodium ions with lithium.1-2 The mechanism of this exchange, however, is not understood.
Although a Na-Li countertransport system is present in
the red cell membrane, 3 ' 4 it has not yet been determined
whether a specific transport system is present in the
sarcolemma, which has affinity for both sodium and
lithium, and mediates the exchange of one cation for
the other. The possible presence of such a transport
system in vascular smooth muscle could have important
implications because several laboratories have reported
that the activity of Na-Li countertransport is elevated
in red cells from patients with essential hypertension.3"7
We have developed a method for preparing a
membrane vesicle fraction enriched in sarcolemma
from the superior mesenteric artery of the dog.8 In the
From the Department of Medicine, University of Texas Medical
School, Houston (A.M.K., R.Z., H.S.), the Department of
Medicine, Baylor College of Medicine, Houston (J.C.A.), and
Merck Sharp & Dohme Research Laboratories, West Point, Pennsylvania (E.J.C.).
Supported by grants HL35866 and HL24585 from the National
Heart, Lung, and Blood Institute and by a grant from the American
Heart Association, Texas Affiliate.
Address for correspondence: Andrew M. Kahn, MD, University
of Texas Medical School, P.O. Box 20708, MSB 4138, Houston,
TX 77025.
Received February 5, 1987; accepted October 1, 1987.
present study, we show that a Na-Li exchange transport
system is present in these membranes and that this
system shares several properties with the red cell Na-Li
countertransport system. Finally, we present data that
indicate that sarcolemmal Na-Li exchange and sarcolemmal Na-H exchange may be mediated by the same
transport system.
Materials and Methods
Vesicle Preparation
Mongrel dogs of either sex were killed with intravenous pentobarbital, and the superior mesenteric
arteries were dissected free. A sarcolemmal-enriched
vesicle preparation was prepared by a magnesium
aggregation and differential centrifugation technique,
as previously described and outlined as follows.8
Adhering veins, fat, and nervous tissue were removed
in the cold, and the arteries were thoroughly minced
with scissors. The tissue was suspended in 10 ml/g wet
wt in (mM) 200 mannitol, 10 tris(hydroxymethyl)aminomethane (Tris), and 16 N-2-hydroxyethylpiperazine-AT-2-ethanesulfonic acid (HEPES), pH 7.5,
at 4° C and homogenized with a Polytron homogenizer.
MgSO4 was added to a final concentration of 10 mM,
and the homogenate was incubated on ice for 30
minutes. The suspension was centrifuged at l,035g for
4 minutes, and the resultant supernatant was filtered
through four layers of gauze and centrifuged at 48,000g
for 30 minutes. The resultant pellet was resuspended
Kahn et al
in 0.5 ml of homogenizing medium with 10 mM
MgSO4, vortexed in the presence of a glass rod, and
incubated on ice for an additional 30 minutes. This
suspension was brought up to 20 ml by the addition of
homogenizing medium with 10 mM MgSO4 and
centrifuged at l,035g for 4 minutes. The resultant
supernatant was centrifuged at 48,000g for 30 minutes
to yield the final pellet, which was enriched fourfold
in sarcolemmal membranes relative to a microsomal
fraction.8 This pellet was resuspended by vortexing in
the presence of a glass rod, recentrifuged twice in the
original homogenizing medium without MgSO4, and
resuspended to a protein concentration of about 3-5
mg/ml. Protein was measured by the method of Lowry
using bovine serum albumin standards.
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Sodium Transport
The transport of sodium by the vesicle preparation
was studied using ^Na and a rapid Millipore filtration
technique (Millipore, Bedford, Massachusetts). In
general, the transport of 2 Na was measured under
conditions in which the external and intravesicular ion
concentrations had been preset to certain values according to the goals of each particular experiment.
Aliquots of membranes (30-50 (xg protein) that had
been preincubated in the desired solutions at 22° C for
90 minutes were incubated with 1 mM ^Na at 22° C,
as described in the figure legends. Uptake was terminated at the desired time points by rapidly diluting the
incubating membranes with 3.5 ml of cold (0-4° C)
"stop solution" that contained 112 mM MgSO4, 1 mM
Tris, and 1.6 mM HEPES, pH 7.5. The membranes
were separated from external media by Millipore
filtration (0.65 \im, DAWP filter) and washed three
times with 3.5-ml aliquots of cold stop solution. The
filters were immersed in scintillation cocktail and
counted. Sodium uptake by the membranes was determined by subtracting a filter blank, which was
obtained in the absence of membranes. Sodium efflux
studies were performed by preincubating vesicles with
1 mM 2 Na for 90 minutes and diluting them 50-fold
in sodium-free media at 22° C. At 0- and 30-second
time points, the sodium content of the vesicles was
determined by the same cold stop, Millipore filtration,
and washing technique just described. Sodium efflux
was calculated as the difference in the sodium content
between the 0- and 30-second time points. Valinomycin was added to vesicles from an ethanolic stock to
achieve a valinomycin concentration of 50 ^g/ml and
an ethanol concentration of 1% (vol/vol). All presented
data represent the mean ± SEM of the mean of triplicate
determinations.
Proton Transport
The transport of protons by the final membrane
fraction was measured by monitoring the fluorescence
quenching of acridine orange. Acridine orange is a
fluorescent weak base that rapidly enters the intravesicular space and is trapped in its protonated form if the
intravesicular pH is lower than external pH. This results
in immediate quenching of acridine orange fluores-
Na-Li Exchange in Vascular Smooth Muscle
479
cence. As the inside acidic pH gradient dissipates,
trapped acridine orange leaves the vesicles, thereby
reducing fluorescence quenching. The fluorescence
signal increases with time until a steady-state level is
achieved, at which point protons are in electrochemical
equilibrium across the membrane.9 Vesicles were
preincubated with 181 mM mannitol, 40 mM MES, 2
mM Tris, and 3.2 mM HEPES, pH 5.0. Vesicles
(15-25 n,g protein) were rapidly mixed with 1 ml of
external buffer containing 6 u,M acridine orange plus
100 mM LiCl, NaCl, or W-methyl-D-glucamine (NMG)
Cl plus 16 mM HEPES-Tris, pH 7.5. Fluorescence was
recorded over time by activating at 493 nm and
recording the emission at 530 nm with a Perkin-Elmer
(Norwalk, Connecticut) 650-10S fluorescent spectrophotometer attached to a chart recorder, as previously described.8 The fluorescence data were fitted by
computer analysis to the general equation AF = c x e~ b ,
where AF is the difference between final fluorescence
(at 20 minutes) and fluorescence at time, t, after mixing
(3-20 seconds), and c and k are constants. In each
experiment, the data fitted the above equation with a
regression coefficient of 0.99 or greater. The value for
k was taken to represent the first-order rate constant for
proton gradient dissipation. Statistical analysis of these
data was performed using Student's t test.
Voltage Measurements
The effect of an outwardly directed potassium
gradient in the presence of the potassium ionophore,
valinomycin, was assessed in the sarcolemmal vesicle preparation by monitoring the fluorescence of
the voltage-sensitive probe 3,3'-dipropylthiadicarbocyanine [diS-C,-(5)], as previously described.10 Two
milliliters of buffer, at 22° C, containing 55 or 11 mM
KC1, plus 2 ^M diS-C,-(5), were added to a magnetically stirred polystyrene cuvette in a spectrofluorometer that had previously been zeroed with deionized
water. The excitation and emission wavelengths were
620 and 669 nm, respectively. The fluorescence signal
was set to 90 units with the sensitivity control, and 10
\x\ of vesicles (about 50 u,g protein), which had been
preloaded with 55 mM KC1, was rapidly added to the
cuvette. Fluorescence was continuously recorded with
a strip chart recorder.
K
Na (200 pCi/\Lg Na) was obtained from Amersham, Arlington Heights, Illinois; diS-C,-(5) iodide
from Molecular Probes, Junction City, Oregon; and
quinidine, phloretin, valinomycin, and acridine orange
from Sigma Chemical, St. Louis, Missouri. 5-(NEthyl-jV-isopropyl) amiloride was synthesized as previously described."
Results
Na-Li Exchange
The uptake of 1 mM ^Na was measured in vesicles
in the presence of an outwardly directed gradient for
either lithium or choline (25 mM,,, 5 mM,,,,). As shown
in Figure 1, the presence of intravesicular lithium
stimulated sodium uptake by 89% at a 30-second time
point. In this experiment, sodium uptake was measured
480
Circulation Research
Vol 62, No 3, March 1988
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Choline m
1
25 mM
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FIGURE 1. Effect of an outwardly directed lithium gradient on
sodium uptake in sarcolemmal-enriched vesicles. Vesicles were
preincubated with (in mM) 25 choline Cl or LJCL, 55 KCl, 40
mannitoL, 16 HEPES-Tris, pH 7.5, plus 50 \ig/ml valinomycin
for 90 minutes at 22° C. The 30-second uptake of 1 mM nNa was
assayed at 22° C in the presence of (in mM) 5 Li, 5 choline, 55
K,66Cl,68 mannitol, and 16 HEPES-Tris, pH 7.5. Values are
means ± SEM from five experiments.
in the presence of equal internal and external potassium
concentration and in the presence of the potassium
ionophore valinomycin to clamp membrane potential to
zero. Thus, the stimulation of sodium uptake by
internal lithium could not be explained by an inside
negative lithium diffusion potential.
A representative time course of sodium uptake into
lithium and choline preloaded vesicles is shown in
Figure 2. Sodium uptake by lithium preloaded vesicles
was higher at 10-, 20-, and 30-second time points, but
uptake at the 90-minute equilibrium time point was the
same for both sets of vesicles. Thus, the stimulation of
sodium uptake by internal lithium at early time points
cannot be explained by an increase in the intravesicular
volume or binding of sodium to the membranes.
Figure 3 demonstrates the dependence of sodium
uptake on internal lithium concentration. As shown,
the rate of sodium uptake progressively increased as
intravesicular lithium concentration was raised from 0
to 5 mM. The stimulation of sodium uptake was fully
saturated at 10 mM intravesicular lithium. The concentration of internal lithium that half-maximal ry
stimulated sodium uptake was about 3 mM.
To rule out the possibility that the sarcolemmal
Na-K pump was responsible for lithium gradientstimulated sodium uptake, the 30-second uptake of 1
mM "Na by lithium and choline preloaded vesicles
was measured as in Figure 1, in the presence and
absence of 1 mM ouabain, an inhibitor of the Na-K
pump. Ouabain did not affect sodium uptake by
lithium or choline preloaded vesicles. The lithium
gradient-stimulated component of sodium uptake in
the presence of 1 mM ouabain was 105 ± 7% of uptake
in the absence of ouabain (n = 3, p = NS). Thus, the
lithium gradient-stimulated uptake of sodium in these
studies was not due to operation of the Na-K pump.
To assess the effect of an inwardly directed lithium
gradient on sodium efflux, vesicles were preloaded with
1 mM "Na and diluted into external media containing
25 mM Li or choline. As shown in Figure 4, the
30-second efflux of "Na was stimulated 80% by external
lithium. Thus, an inwardly directed lithium gradient
stimulated the transport of sodium in the opposite
direction. The stimulation of sodium efflux by external
lithium could not be explained by an inside positive
lithium diffusion potential since this experiment was
performed in the presence of valinomycin and equal
internal and external potassium concentration.
To rule out the possibility that lithium merely
increased the permeability of the membrane to sodium,
the uptake of 1 mM "Na was measured in the presence
of 25 mM internal and external lithium or choline. As
shown in Figure 5, the 30-second uptake of sodium was
not affected by ambient lithium versus choline. Thus,
an oppositely directed gradient for lithium, but not
lithium per se, stimulated sodium transport. Taken
together, the preceding data indicate that the vesicles
used in these studies contain a Na-Li exchange transport system.
Effect of Membrane Voltage
To determine the effect of membrane voltage on
Na-Li exchange, the uptake of 1 mM "Na was
measured in vesicles preloaded with 25 mM Li or
choline, in the presence of valinomycin and an outwardly directed potassium gradient, or equal internal
and external potassium concentration. The outwardly
directed potassium gradient in the presence of valinomycin should hyperpolarize the vesicles, whereas
voltage should be clamped to zero by equal internal and
external potassium concentration. The relative membrane voltage of the vesicles in the presence and
absence of the outwardly directed potassium gradient
was assessed by monitoring the fluorescence of the
voltage-sensitive dye diS-C,-(5), as shown in Figure 6.
When vesicles preloaded with 55 mM KCl were mixed
with external solution containing 55 mM KCl and 2 ^M
diS-C3-(5) ( K ^ K ^ ) , the increased turbidity of the
solution caused the fluorescence to fall to a value that
1.0
2
0.9
0.4
o
0.3
c
<D
ra
Q.
25 Choline in
10
20
Seconds
30
90
min
FIGURE 2. Time course of sodium uptake by sarcolemmalenriched vesicles preloaded with or without lithium. Vesicles
were preincubated and the uptake of^Na at the indicated times
was assayed, as described in Figure 1 legend. Values are means
of triplicate determinations from a representative experiment.
Kahn et al
05
w
0.4
S
0-3
f
"I
o 0.1
E
0.0
10
15
U,n(mM)
20
25
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FIGURE 3. Dependence of sodium uptake on internal lithium
concentration in sarcolemmal-enriched vesicles. Vesicles were
preincubated for 90 minutes at 22° C with (in mM) 150 mannitol
and 16HEPES-Tris, pH 7.5, plus 0, 2.5, 5, 10, or25LiCl, and
enough choline Cl such that total salt concentration equaled 25
mM. The uptake of 1 mM "Na was assayed for 30 seconds at
22° C in the presence of (in mM) 5 UCl, 5 choline Cl, 178
mannitol, and 16 HEPES-Tris, pH 7.5. Values are means of
triplicate determinations from a representative experiment.
remained constant over time. When an identical aliquot
of the vesicle suspension was mixed with external
solution containing 11 mM KC1 and 2 (xM diS-C, — (5)
(K in >K M ), fluorescence of the solution fell to a lower
value than when K^ = K^,, and then slowly rose with
time. These findings indicate that the outwardly
directed potassium gradient induced an inside negative
membrane potential that caused the cationic dye,
diS-Cj-(5), to enter the vesicles, resulting in fluorescence quenching. As the potassium gradient dissipated
with time, the vesicles became less negative inside,
diS-C,-(5) effluxed from the vesicles, and fluorescence
gradually increased. It would appear, then, that the
vesicles were indeed rendered more electronegative
inside in the presence of an outwardly directed potassium gradient. Under each voltage condition, the
uptake of sodium by lithium preloaded vesicles minus
uptake by choline preloaded vesicles was taken as the
activity of Na-Li exchange. Although total sodium
uptake by both lithium and choline preloaded vesicles
was stimulated by the inside negative membrane
potential, Na-Li exchange activity was not affected by
the change in voltage (Figure 7). These data suggest
that Na-Li exchange in vascular smooth muscle is
electroneutral, as is the case for Na-Li exchange in the
red cell membrane. 412
Na-Li Exchange in Vascular Smooth Muscle
stimulated component of sodium uptake, as previously
described.8 These measurements were made in the
presence and absence of 1 mM phloretin or quinidine.
As shown in Figure 8, 1 mM phloretin inhibited both
Na-Li exchange and Na-H exchange by about 50%. As
also shown in Figure 8,1 mM quinidine inhibited Na-Li
exchange and Na-H exchange by 60-70%.
The sensitivity of Na-Li exchange and Na-H exchange in the sarcolemmal vesicles to ethylisopropylamiloride was also tested. This drug inhibits Na-H
exchange in cultured vascular smooth muscle cells.15
As shown in Figure 9, both Na-Li exchange and Na-H
exchange were inhibited about 90% by 1 mM ethylisopropylamiloride and were inhibited about 50% by
0.1 mM ethylisopropylamiloride.
Proton Transport
The similarities between red cell Na-Li exchange and
renal brush border Na-H exchange have prompted
several investigators to propose that these transport
events are mediated by a single monovalent cation
exchanger with affinities for sodium, lithium, and
protons.1314-16 If such a relation exists between Na-Li
exchange and Na-H exchange in the sarcolemma of
vascular smooth muscle, then Li-H exchange should be
demonstrable in sarcolemmal vesicles. This was tested
in the following experiment. Vesicles were preloaded
with buffer at pH 5.0, and incubated in external
solutions at pH 7.5, containing 100 mM Li, Na, or
NMG, an ionic strength control. The rate of collapse
of the inside acidic pH gradient was monitored by
measuring the fluorescence quenching of the pHsensitive probe, acridine orange. As shown by a
representative experiment in Figure 10, external lith-
I
0.15
E 0.10
0.05
Inhibitor Studies
To explore further the possibility that the sarcolemmal and the red cell Na-Li exchangers are homologous systems, we tested whether phloretin and quinidine, two known inhibitors of red cell Na-Li exchange,3-4 would inhibit sarcolemmal Na-Li exchange.
In addition, we tested whether these agents would
inhibit sarcolemmal Na-H exchange, which has been
proposed to be homologous with red cell Na-Li
exchange.13-14 Na-Li exchange activity was measured
by determining the internal lithium-stimulated component of sodium uptake. Na-H exchange activity
was measured by determining the internal proton-
481
T
|
1i
25 mM
Chohne_
25 mM
FIGURE 4. Effect of an inwardly directed lithium gradient on
sodium effluxfrom sarcolemmal-enriched vesicles. Vesicles were
preincubated with (in mM) 1 uNa, 24 choline, 75 K 100 Cl, and
16 HEPES-Tris, pH 7.5, plus 50 \iglml valinomycin for 90
minutes at 22° C. The 30-second efflux of nNa was assayed at
22°Cin the presence of (in mM) 0.02 nNa, 25 choline ClorUCl,
75 KCl, and 16 HEPES-Tris, pH 7.5. Values are means ±SEM
from five experiments.
Circulation Research
°
Vol 62, No 3, March 1988
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it Uptake (% Cholino C ont rol
482
25 Choline*
25 Choline^
50
25 LiOT
25 Li^t
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FIGURE 5. Effect of ambient lithium on sodium uptake in
sarcolemmal-enriched vesicles. Vesicles were preincubated with
(in mM) 25 UCl or choline Cl, 55 KCl, 40 mannitol, and 16
HEPES- Tris, pH 7.5, plus 50 \ig/ml valinomycin for 90 minutes
at 22° C. The 30-second uptake of 1 mM uNa was assayed at
22° Cfrom an identical external solution. The uptake of sodium
by lithium preloaded vesicles was expressed as a percent of
uptake by choline preloaded vesicles that averaged 0.17
nmollmg protein. Values are means ± SEM from four experiments.
ium or sodium stimulated the efflux of protons compared to external NMG. The first-order rate constant for
proton gradient dissipation was 0.023 ± 0 . 0 0 3 ,
0.025 ±0.004, and 0.013 ±0.002 sec" 1 for outside
sodium, lithium, and NMG, respectively (n = 4;
p<0.05 for Na0 or Li0 versus NMGO). These data
confirm prior findings that demonstrated that sarcolemmal vesicles mediate Na-H exchange.8 The data
also indicate that the vesicles mediate Li-H exchange.
©
(55KCIJ 55V KCl
a|
11 KCl
Val.
( J
20
- Tune
FIGURE 6. Effect of an outwardly directed potassium gradient
and valinomycin on membrane potential in sarcolemmalenriched vesicles. Vesicles were preincubated with (in mM) 25
UCl, 55 KCl, 40 mannitol, and 16 HEPES-Tris, pH 7.5, plus
50 \ig/ml valinomycin for 90 minutes at 22° C. Ten-microliter
vesicles (about 50 \ig protein) were mixed with 2 ml of buffer
at 22° C containing (in mM) 0.002 diS-C,-(5), 5 UCl, 5 choline
Cl, 1 NaCl, 68 mannitol, and 16 HEPES-Tris, pH 7.5, plus 55
KCl (right tracing) or 11 KCl, 44 choline Cl (left tracing).
Fluorescence was recorded over time (right to left) as described
in "Materials and Methods. " Tracings are from a representative
experiment.
Inside
Neutral
Inside
Electronegative
FIGURE 7. Effect of membrane voltage on Na-U exchange in
sarcolemmal-enriched vesicles. Vesicles were preincubated with
(in mM) 25 UCl or choline Cl, 55 KCl, 40 mannitol, and 16
HEPES- Tris, pH 7.5, plus 50 \yglml valinomycin for 90 minutes
at 22° C. Inside neutral: 30-second uptake of 1 mM nNa was
assayed at 22° C in the presence of (in mM) 5 U, 5 choline, 55
K 66 CI.J68 mannitol, and 16 HEPES-Tris, pH 7.5. Inside
electronegative: 30-second uptake ofl mM nNa was assayed at
22° C in the presence of (in mM) 5 U, 49 choline, 11K,66CI,
68 mannitol, and 16 HEPES-Tris 7.5. For each voltage
condition, sodium uptake by lithium preloaded vesicles minus
sodium uptake by choline preloaded vesicles was taken as the
activity of Na-U exchange. Na-U exchange by inside electronegative vesicles was expressed as a percent of Na-U exchange
by inside neutral vesicles. Values are means ± SEM from four
experiments.
Concurrent Na-Li and Na-H Exchange
To test further the possibility that sarcolemmal Na-Li
exchange and Na-H exchange are mediated by the same
transport system, we determined whether the stimulation of sodium efflux by external lithium and external
protons was additive. Vesicles were preloaded with 1
mM ^Na at pHj,, 7.5 and diluted into external solutions
containing 25 mM Li or NMG at pH 7.5 or 6.5. As
shown in Figure 11, at pH0 7.5, external lithium
stimulated sodium efflux by 80%. In the absence of
lithium, lowering external pH from 7.5 to 6.5 stimulated sodium efflux by 73%. This latter effect is
consistent with the operation of the Na-H exchanger.
However, when both lithium and increased acidity (pH0
6.5) were present in the external solution, the stimulation of sodium efflux was not additive. In fact,
sodium efflux under these conditions was not higher
than when external lithium or external acidity alone
was present in the outside solution. These data are
consistent with the possibility that Na-Li exchange and
Na-H exchange are mediated by the same system. If
such were the case, saturation of the external cation
binding site of the exchanger by 25 mM Li, or
separately by protons (pH0 6.5), would preclude the
further stimulation of sodium efflux by the simultaneous presence of 25 mM Li and protons (pHo 6.5).
Discussion
Incubation of intact vascular smooth muscle tissue
in a lithium-containing solution leads to lithium uptake
accompanied by sodium efflux. Exchange of lithium
for sodium occurs, at least in part, across the vascular
Kahn et al
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smooth muscle cell membrane.1-2 This exchange process in intact tissue has several features characteristic of
a mediated membrane transport system. For example,
it has been demonstrated that ouabain-resistant sodium
efflux from the cellular compartment of rabbit portal
anterior mesenteric vein into an external lithium
solution was markedly inhibited by lowering temperature from 37° C to 2° C. In addition, removal of
calcium from the external media greatly inhibited
ouabain-resistant sodium efflux.2 Nevertheless, the
mechanism of Na-Li exchange in intact vascular
smooth muscle cell has not been determined. Several
different possibilities could potentially underlie this
exchange phenomenon. Extracellular lithium could
enter the vascular smooth muscle cell via a conductive
pathway, such as diffusion or a channel, and electrically
induce the efflux of sodium through a conductive
pathway for that ion. It is also possible that lithium,
once inside the cell, could displace sodium from
intracellular binding sites, resulting in higher intracellular free sodium concentration and greater sodium
flux out of the cell. A third possibility is that, as in the
red cell, a discrete membrane transport system with
affinity for sodium and lithium is present in the
sarcolemma, which exchanges one cation with the
other.
In the present studies, we have demonstrated that an
outwardly directed lithium gradient stimulated the
uptake of sodium in a sarcolemmal-enriched vesicle
preparation from canine superior mesenteric artery. In
addition, an inwardly directed lithium gradient stim-
lmM Phloretin
o
lmM
T
= 50
1
1—
n
Na/Li
N»/H
Quinidine
Na/Li
Na-Li Exchange in Vascular Smooth Muscle
483
Na/H
Na/Li
too
80
|
60
X)
a?
20
1.0
0.1
1.0
Ethylisopropylamiloride (mM)
FIGURE 9. Effect of ethylisopropylamiloride on Na-Li exchange and Na-H exchange in sarcolemmal-enriched vesicles.
The 30-second activities of Na-Li exchange and Na-H exchange
were determined as described in the legend of Figure 8, and the
percent inhibition by 1.0 and 0.1 mM ethylisopropylamiloride
calculated. Results are means ± SEM from four experiments.
ulated sodium efflux. These effects were not the result
of an alteration in membrane voltage due to a lithium
diffusion potential. The effect of lithium on sodium
uptake was not due to an increased sodium permeability due to the mere presence of lithium, since
sodium uptake was stimulated by an oppositely directed lithium gradient but not by ambient lithium per
se. The stimulation of sodium transport by an oppositely directed lithium gradient could not be accounted
for by operation of the Na-K pump since lithium
gradient-stimulated sodium uptake was not affected by
1 mM ouabain. Although these data do not rule out the
possibility that lithium gradient-stimulated sodium
transport is actually due to Li-H exchange coupled to
Na-H exchange, taken together the data strongly
pH i n = 7.5
Li O ut
Na/H
FIGURE 8. Effect of phloretin and quinidine on Na-Li exchange
and Na-H exchange in sarcolemmal-enriched vesicles. Na-Li
exchange was measured by determining the 30-second uptake of
1 mM nNa by lithium preloaded vesicles minus the uptake by
choline preloaded vesicles, as described in the legend of Figure
1, in presence or absence of 1 mM phloretin or quinidine. Na-H
exchange was measured by preincubating vesicles for 90
minutes at 22° C with (in mM) 181 mannitol, 17 Tris, and 28
HEPES, pH 7.5, or 181 mannitol, 40 MES, 2 Tris, and 3.2
HEPES, pH 5.0. The 30-second uptake of 1 mM "Na was
assayed in the presence of (in mM) 1 Cl, 104 mannitol, 8 MES,
47 Tris, and 65 HEPES, pH 7.5, in the presence or absence of
1 mM phloretin or quinidine. The uptake of sodium by vesicles
preincubated at pH 5.0 minus the uptake by vesicles preincubated at pH 7.5 was taken as the activity of Na-H exchange.
Na-Li exchange and Na-H exchange in the presence of phloretin
or quinidine are expressed as percent inhibition relative to these
transport rates in the absence of inhibitor. Results are
means ± SEM from four experiments.
pH i n = 5.0
Time
pH o u t
/pHinN
2 min
FIGURE 10. Effect of external lithium or sodium on the
dissipation of an inside-acidic pH gradient in sarcolemmalenriched vesicles. Vesicles were preincubated with (in mM) 181
mannitol, 40 MES, 2 Tris, and 3.2 HEPES, pH 5.0, for 90
minutes at 22° C. Five microliters of vesicle suspension (30 \ig
protein) were rapidly mixed with 1 ml of external buffer
containing 6 \iM acridine orange, plus (in mM) 100 LiCl or
NaCl or NMG Cl, plus 16 HEPES-Tris, pH 7.5. Fluorescence
was measured starting 3 seconds after mixing, by activating at
493 nm and recording the emission at 530 nm with a chart
recorder (right to left). Data shown are from a representative
experiment.
484
Circulation Research
Vol 62, No 3, March 1988
T T T
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25 Li 0
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FIGURE 11. £#ecf of external lithium and protons on sodium
efflux from sarcolemmal-enriched vesicles. Vesicles were preincubated with (in mM) 1 "Na, 24 NMG, 75 K, 100 Cl, and 16
HEPES- Tris, pH 7.5, plus 50 \iglml valinomycin for 90 minutes
at 22° C. The 30-second efflux of nNa was assayed at 22° C in
the presence of (in mM) 0.02 aNa, 75 KCl, plus 25 NMG Cl,
16 HEPES-Tris, pH 7.5, or 25 UCL, 16 HEPES-Tris, pH 7.5,
or25NMG Cl, 16MES-Tris, pH6.5, or25 UCl, 16MES-Tris,
pH 6.5. Results are expressed as a percent of sodium efflux in
the presence of 25 mM NMG CL, pH 7.5. Values are means ±
SEM of four experiments.
suggest the presence of a Na-Li exchange transport
system in the sarcolemma.
The Na-Li exchange mechanism examined in the
present studies has several features in common with the
red blood cell Na-Li countertransport system. The
present studies have demonstrated that Na-Li exchange
in sarcolemmal-enriched vesicles was insensitive to a
change in membrane voltage and was inhibitable by
quinidine and phloretin. These features are also characteristics of the red cell Na-Li countertransporter. 3412
Several laboratories have demonstrated that the
activities of Na-Li countertransport are elevated in red
cells from patients with essential hypertension.5"7 The
relation between this transport system and blood
pressure has been obscure; however, increased red cell
transport activity could be a marker for increased
transport activity of a similar system in vascular
smooth muscle. Indeed, Friedman has demonstrated
increased ouabain-resistant sodium efflux into a lithium solution from intact tail arteries of the spontaneously hypertensive rat versus the Wistar-Kyoto
control rat.17
Since lithium is not normally present in the body, the
physiological role of Na-Li exchange in the red cell or
vascular smooth muscle cell is obscure. Aronson16 and
Funder et al13 have independently proposed the hypothesis that red cell Na-Li countertransport could be
an operative mode of a Na-H exchanger. This proposal
is based on the many similarities between the red cell
Na-Li countertransporter and the renal brush border
Na-H exchanger. Both systems are quinidine
inhibitable,3-18 electroneutral monovalent cation
exchangers412-19 with affinities for sodium and lithium,
greater affinity for lithium than sodium,12-20 and no
affinity for cesium, rubidium, or potassium.31*-21 It is
possible that vascular smooth muscle Na-Li exchange
and Na-H exchange are mediated by the same transport
system.
In the present study, we show that Na-Li exchange
and Na-H exchange in the sarcolemmal-enriched vesicle preparation were similarly inhibited by two compounds that inhibit Na-Li countertransport in red cells,
phloretin and quinid ine .3-4 We show that ethyl isopropylamiloride, which inhibits Na-H exchange in cultured
vascular smooth muscle cells,15 similarly inhibited
Na-Li exchange and Na-H exchange in the vesicle
preparation. We also show that an inwardly directed
lithium gradient stimulated proton efflux and that
sodium efflux rates, which were stimulated by an
oppositely directed gradient for lithium or protons,
were not additive. These data are consistent with the
single transport system hypothesis. It is of interest to
note that Wasserman et al2 found that calcium removal
inhibited the exchange of intracellular sodium for
external lithium in intact vascular smooth muscle.
These data are also consistent with a common identity
of the sarcolemmal Na-Li and Na-H exchangers since
intracellular calcium has been shown to stimulate the
Na-H exchanger in a variety of cell types including
cultured vascular smooth muscle cells.22-23 It must be
pointed out, however, that it is not yet known with
certainty whether the Na-Li exchanger and Na-H
exchanger in vascular smooth muscle are indeed the
same transport system.
The physiological functions of vascular smooth
muscle Na-Li exchange, and the range of transport
events mediated by this system in vivo, are not currently
known and require further study. Additional investigation is needed to determine whether a link exists
between this transport system in vascular smooth
muscle and the hypertensive process.
Acknowledgment
The authors acknowledge the excellent secretarial
assistance of Ms. Nhung Le.
References
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KEY WORDS • vascular smooth muscle • sodium-proton
exchange • phloretin • quinidine • acridine orange •
ethylisopropylamiloride • 3,3'-dipropylthiadicarbocyanine iodide
Sodium-lithium exchange in sarcolemmal vesicles from canine superior mesenteric artery.
A M Kahn, J C Allen, E J Cragoe, Jr, R Zimmer and H Shelat
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
Circ Res. 1988;62:478-485
doi: 10.1161/01.RES.62.3.478
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