Download Difference Between Human Red Blood Cell Na

Clinical Studies
Difference Between Human Red Blood Cell
Na + -Li + Counter transport and Renal
Na + -H + Exchange
ANDREW M.
KAHN
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SUMMARY Several laboratories have reported that the activities of sodium-lithium countertransport are increased in red blood cells from patients with essential hypertension. Based on the many
similarities between this transport system and the renal sodium-proton exchanger, a hypothesis has
been put forth in the literature that increased red blood cell sodium-lithium counter-transport activity
may be a marker for increased sodium-proton exchange activity in the renal proximal tubule. The
present studies were designed to test the hypothesis that sodium-lithium countertransport in red blood
cells from humans or rabbits is mediated by the same transport mechanism that mediates sodiumproton exchange in the renal brush border from those species. Similar to what has been reported for
the rabbit, the present studies show that an amiloride-sensitive sodium-proton exchanger is present in
human renal brush border vesicles. However, Na+-Li+ countertransport in human and rabbit red
blood cells, assayed under several different conditions, was not inhibited by amiioride. In agreement
with what has been reported for humans, the present studies show that extracellular proton-stimulated sodium efflux is inhibited by amiioride in rabbit red blood cells. These data demonstrate a
difference (amiioride sensitivity) between the red blood cell sodium-lithium countertransporter and
the renal brush border sodium-proton exchanger in humans and rabbits. These experiments detract
from the hypothesis that increased red blood cell sodium-lithium countertransport activity in patients
with essential hypertension is a marker for increased sodium-proton exchange activity In the renal
brush border. (Hypertension 9: 7-12, 1987)
KEY WORDS
• amiioride
• brush border vesicles • rabbit red blood cells
M
ANY studies have examined whether an abnormality in red blood cell (RBC) sodium
transport is linked to essential hypertension.1"6 An underlying hypothesis in these studies is
that an abnormality in a specific RBC sodium transport
system may be a marker for a similar abnormality in
the same transport system located in another tissue.
For instance, elevated activity of a specific sodium
influx pathway in RBCs from patients with essential
hypertension could be a marker for increased sodium
influx by that same pathway across the luminal membrane of certain renal tubular cells or the sarcolemma
of vascular smooth muscle. Such a transport defect in
From the Department of Medicine, University of Texas Medical
School, Houston, Texas.
Supported by a grant-in-aid from the American Heart Association, Texas Affiliate.
Address for reprints: Andrew M. Kahn, M.D., 4.138 MSMB,
University of Texas Medical School, P.O. Box 20708, Houston,
TX 77025.
Received January 6, 1986; accepted July 23, 1986.
the cell membrane of these cell types could be related
to the development or maintenance of hypertension.
The cell membrane of RBCs from humans and a
variety of mammals mediates Na + -Li + countertransport.7"9 This is an electroneutral transport system that
can operate in a sodium-for-lithium or sodium-forsodium exchange mode.7"9 Several laboratories have
reported that the activities of this transport system are
elevated in RBCs from patients with essential hypertension.2' 5> 6 Since lithium is only present in very low
concentrations in humans, this RBC transport system,
or its equivalent in other cell types, would be expected
to mediate Na + -Na + exchange.9 An increased activity
of Na + -Na + exchange would not alter net sodium
transport in any tissue. Aronson10 and Funder et al."
have independently suggested that RBC Na + -Li +
countertransport may be an operative mode of a sodium-proton exchange transport system. This hypothesis
is based on the many similarities between RBC Na + Li + countertransport and renal brush border Na + -H +
exchange. Both transport systems are quinidine-inhibi-
8
HYPERTENSION
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table,7-12 electroneutral monovalent cation exchangers 89 ' 13 with affinities for sodium and lithium,7"9' l3~13
greater affinity for lithium than sodium,8' M and no
affinity for choline, cesium, rubidium, or potassium.7' l3~15 Increased activity of Na+-Li+ countertransport in RBCs from hypertensive patients has been
proposed as a marker for increased activity of renal
brush border Na + -H + exchange.1'l0'16 Such an abnormality in the brush border membrane could be responsible for increased proximal tubular sodium reabsorption, which could contribute to the hypertensive
state.17' " This is an attractive proposal that would be
greatly strengthened if the aforesaid transport activities
from both tissues were indeed mediated by the same
system.
These studies were designed to test the hypothesis
that Na + -Li + countertransport in RBCs is mediated by
the same transport system that mediates Na + -H + exchange in the renal brush border membrane. The results demonstrate a difference between these two transport systems.
Materials and Methods
Human Brush Border Vesicles
Kidney tissue was obtained from fresh human kidneys immediately after surgical removal from patients
Withrenalcancer. Brush border vesicles were prepared
by a magnesium aggregation and differential centrifugation technique that was similar to the method used
by this laboratory to prepare rat renal brush border
vesicles." Normal portions of human renal cortex were
minced with a razor blade and homogenized with 10
strokes in a glass-teflon homogenizer in (in mM) 200
mannitol, 50 Tris, 80 HEPES, pH 7.5 (4 ml/g tissue).
Then, MgSO4 was added to the cortical homogenate to
a final concentration of 10 mM, and the homogenate
was incubated on ice for 20 minutes. The homogenate
was centrifuged at 4340 g for 10 minutes, and the
supernatant was centrifuged at 27,000 g for 25 minutes. The resultant pellet was resuspended in homogenizing solution with 10 mM MgSO4 and centrifuged
at 5900 g for 10 minutes, and the supernatant was
centrifuged at 27,000 g for 25 minutes. The resultant
pellet was resuspended in homogenizing solution with
10 mM MgSO4 and centrifuged at 7710 g for 10 minutes. The resultant supernatant was centrifuged at
27,000 g for 25 minutes, and the pellet was resuspended in homogenizing solution with 10 mM MgSO4 to a
final protein concentration of 10 to 15 mg/ml. Protein
was measured by the method of Lowry.20 The activities of the brush border membrane marker alkaline
phosphatase and the basolateral membrane marker
Na+,K+-ATPase were determined for the cortical homogenate andfinalvesicles, as previously described."
Alkaline phosphatase activity in the vesicles was enriched ninefold relative to the cortical homogenate,
while Na+,K+-ATPase activity was enriched by only
25%. Hence, the human renal vesiclesrepresentedprimarily brush border membranes.
The vesicles were preincubated with the desired solutions for 90 minutes at 22 °C before the sodium trans-
VOL 9, No 1, JANUARY
1987
port experiments. The timed uptake of 1 mM ^Na was
measured in triplicate by a rapid Millipore (Bedford,
MA, USA) filtration and washing technique, as previously described.21 The composition of the preincubation and incubation media are given in the legend to
Figure 1. This experiment was performed with at least
three separate vesicle preparations.
RBC Cation Transport
To determine RBC cation transport, fresh blood was
drawn into a heparinized syringe from the antecubital
vein of a human volunteer or from the central ear artery
of an adult male New Zealand White rabbit. The whole
blood was centrifuged at 5000 g for 5 minutes at 4°C,
and the plasma and buffy coat were removed. The
efflux of lithium or sodium from human or rabbit
RBCs was measured by methods modified from
Canessa et al.2 and are described in entirety as follows.
One volume of packed RBCs was incubated in a shaking water bath for 3 hours at 37 °C withfivevolumes of
(in mM) 10 glucose, 10 Tris morpholinepropanesulfonic acid (MOPS), pH 7.4, and 150 LiCl or 150 NaCl
plus 0.1 ouabain. The cells were centrifuged at 5000 g
for 5 minutes at 4°C, and the packed cells were resuspended in ice-cold washing solution that contained (in
mM) 75 MgCl2, 85 sucrose, 10 glucose, and 10 Tris
MOPS, pH 7.4. This suspension was centrifuged at
5000 g for 5 minutes at 4°C, andfivesuccessive identical washing and centrifuging steps were performed to
remove extracellular lithium or sodium. The hematocrit of the washed suspension was measured. The efflux of lithium or sodium from the preloaded washed
RBCs was initiated by incubating cells at 37 °C with
different external media. At time zero and at specified
intervals thereafter, three 1-ml aliquots from each suspension were pipetted into individual ice-cold plastic
centrifuge tubes. These tubes were immediately centrifuged at 5000 g for 3 minutes at 4°C, and the supernatants removed and stored in plastic tubes. The lithium
or sodium concentrations of the supernatants were
measured with a Corning 450flamephotometer (Corning Scientific Instruments, Medfield, MA, USA). The
mean concentration of cation in the initial supernatants
was subtracted from the mean concentrations in the
subsequent supernatants, and the efflux of lithium or
sodium into the different external media was calculated and expressed as millimoles per liter of RBCs.
The effect of Na,, on lithium efflux was measured by
incubating lithium-loaded RBCs with 150, 25, or 0
mM NaCl and 0.1 mM ouabain, pH 7.4. The effect of
Li0 on sodium efflux was measured by incubating sodium-loaded RBCs with 5, 1, 0.5, or 0 mM LiCl and 0.1
mM ouabain, pH 7.4. In all cases, choline chloride
was added where necessary to maintain extracellular
salt concentration at 150 mM. The effect of extracellular acidity on Na efflux was measured by incubating
Na-loaded RBC with (in mM) 150 choline chloride,
0.1 ouabain, 10 Tris MOPS, pH 7.4, or 10 Tris morpholineethanesulfonic acid, pH 6.0. In addition, 4,4'diisothiocyano-2,2'-disulfonic stilbene (DIDS) was
present in the external media (0.125 mM) to help
maintain the imposed pH gradients. All efflux expert-
RED BLOOD CELL Na+-Li+ AND RENAL Na+-H+ COUNTERTRANSPORT/tfa/in
ments were performed in the presence and absence of 1
mM amiloride. The exact composition of the external
media is given in the legends to Figures 2 through 5.
Each RBC transport experiment was performed on at
least three separate occasions.
Materials
The n N a (200 AiCi/>g Na) was obtained from
Amersham (Arlington Heights, IL, USA) and DIDS
from Sigma Chemical (St. Louis, MO, USA). Amiloride HCl was a gift from Merck Sharp Dohme (West
Point, PA, USA). All other chemicals were reagent
grade.
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Results
Brush Border Na+-H+ Exchange
In the presence of an outwardly directed proton gradient (pHta = 5.7, p H ^ = 7.4), the 10-second uptake
of 1 mM H Na in human brush border vesicles was
stimulated sevenfold as compared with the absence of
a pH gradient (pH^ = p H ^ = 7.5). As shown in Figure
1, sodium uptake in the presence of an outwardly directed proton gradient was stimulated up to and including the 1-minute time point. At the 90-minute equilibrium time point, sodium uptake in control and
acid-loaded vesicles was the same. When 1 mM amiloride was added to the incubation medium in the presence of an outwardly directed proton gradient, the
stimulation of sodium uptake was inhibited. These
data indicate that human renal brush border vesicles
mediate Na + -H + exchange and that amiloride can inhibit the Na + -H + exchanger in these vesicles.
RBC Na+-Li+ Countertransport
The efflux of lithium from human RBCs into different external media was measured and plotted in Figure
2. The rates of lithium efflux into external media containing sodium were greater than the efflux rate into
the sodium-free medium. The increment in lithium
efflux rate caused by the presence of external sodium
represents the activity of Na + -Li + countertransport.
The rates of lithium efflux into media containing 150,
25, or 0 mM NaCl were not affected by 1 mM amiloride. Thus, the activity of Na + -Li + countertransport in
human RBCs in the presence of 150 or 25 mM Nao was
not inhibited by 1 mM amiloride.
Rabbit RBCs were also preloaded with lithium,
washed, and incubated with the same external media as
the human RBCs. As shown in Figure 3, 1 mM amiloride did not inhibit rabbit RBC Na + -Li + countertransport in the presence of 150 or 25 mM external sodium.
RBC Li+-Na+ Countertransport
The Na efflux from rabbit RBCs was measured in
the presence and absence of 1 mM amiloride and various concentrations of external lithium. The sodium
efflux rate into lithium media minus the efflux rate into
lithium-free media was taken as the activity of Li + Na + countertransport. As shown in Figure 4, the presence of 1 mM amiloride in the external media did not
inhibit the activity of Li + -Na + countertransport in the
presence of 5, 1, or 0.5 mM external lithium.
Proton-Stimulated Na Efflux
7 4 0 / 5 7,
Rabbit RBCs were preloaded with sodium and
washed, and the efflux of sodium was measured in
sodium-free and lithium-free external solutions at various pH concentrations, in the presence and absence of
06
£
1
-.
o
m
|
05
150 Na0
0.4
o
I 0.3
125 chohne0
Minutes
150 cholineu
0.1
FIGURE 1. Effect of a pH gradient and amiloride on sodium
uptake in human renal brush border vesicles (*). Vesicles were
preincubatedfor 90 minutes at 22 °C in (in mM) 196 mannitol, 2
MgSO4, 50 Tris, 80 HEPES, pH 7.5, and the uptake of 1 mM
u
Na was assayed at 37"C in the presence of (in mM) 199
mannitol, 0.4 MgSO4, 50 Tris, 80 HEPES, pH 7.5. Vesicles
were preincubated for 90 minutes at 22 "C with (in mM) 260
mannitol, 2 MgSO4, 10 Tris, 16 HEPES, 40 morpholineethanesulfonic acid (MES), pH 5.7, and the uptake of 1 mM ^Na was
assayed at 37°C in the presence of (in mM) 212 mannitol, 0.4
MgSO4, 42 Tris, 67 HEPES, 8 MES, pH 7.4, with (A) or
without (*) 1 mM amiloride. Data presented are the results
from a representative experiment.
30
60
90
Minutes
FIGURE 2. Effect of amiloride on Na + -Li* countertransport
in human RBCs. Human RBCs were preloaded with lithium,
and the efflux of lithium at 37"C was assayed by incubating the
cells in media containing (in mM) 4 MgCl2, 4 sucrose, 10
glucose, 0.1 ouabain, 10 Tris morpholinepropanesulfonic acid,
pH 7.4, and 150 NaCl (O •), 25 NaCl and 125 choline chloride
(O, • ) , or 150 choline chloride (A, A), with (• • A.) or without
(o, a, A) 1 mM amiloride. Data presented are the results of a
representative experiment.
HYPERTENSION
10
VOL 9, No
1, JANUARY
1987
1 5
80
|
| Control
Y/
\ 1 mM Amilonde
60
•S
io
tf>,
150 Na.
§4.0
II
o —
I 0.5
20
5 mM L i .
1 mM L i .
0.5 mM LiD
Minutes
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FIGURE 3. Effect of amiloride on Na +-Li+ countertransport
in rabbit RBCs. Lithium transport in rabbit RBCs was measured
as described for human RBCs in the legend for Figure 2. Data
presented are the results of a representative experiment.
1 mM amiloride. As shown in Figure 5, the efflux of
sodium was stimulated when the external pH was lowered from 7.4 to 6.0. As also shown in Figure 5, the
stimulation of sodium efflux by external protons was
inhibited by 1 mM amiloride.
Discussion
In a number of studies, the activities of Na+-Li+
countertransport in RBCs from patients with essential
hypertension were elevated.2'3> 6 The relation between
thesefindingsand elevated arterial pressure is obscure.
It has been suggested that RBC Na+-Li+ countertransport may be mediated by the same transport system as
renal brush border Na + -H + exchange. 11016 If this
were the case, increased RBC Na+-Li+ countertransport activity could be a marker for increased Na + -H +
exchange activity in the brush border membrane of the
proximal tubule. Such an abnormality could cause increased proximal tubular reabsorption of salt and water, which could lead to a series of events resulting in
elevated blood pressure. "•" This attractive hypothesis
would be strengthened if RBC Na+-Li+ countertransport and renal brush border Na + -H + exchange were
indeed mediated by the same system. Much circumstantial evidence gives credence to this possibility. The
purpose of the present studies was to test the hypothesis that Na+-Li+ countertransport in RBCs from humans and rabbits is mediated by the same transport
system that mediates Na + -H + exchange in the renal
brush border membrane from those species.
Data are presented that demonstrate that renal brush
border vesicles from the human kidney mediate amiloride-sensitive Na + -H + exchange. Previous studies
with renal brush border vesicles from the rabbit have
reached the same conclusion.1322 The present studies
could not demonstrate the amiloride sensitivity of
Na+-Li+ countertransport in RBCs from humans or
rabbits. In the rabbit RBCs, inhibition of countertransport by amiloride was not observed when the transport
FIGURE 4. Effect of amiloride on Li+-Na+ countertransport
in rabbit RBCs. Rabbit RBCs were preloaded with sodium, and
the efflux of sodium was assayed by incubating cells for 15
minutes at 37"C with external solutions containing (in mM) 4
MgCl2, 4 sucrose, 10 glucose, 0.1 ouabain, 10 Tris
morpholinepropanesulfonic acid, pH 7.4, and 5, 1, 0.5, or 0
IACI, with or without 1 mM amiloride. Choline chloride was
present such that external salt concentration was 150 mM. The
Li+-Na + countertransport activity was calculated as the rate of
sodium efflux into lithium media minus the rate into lithium-free
media. Data are the means ± SE of three experiments.
30
a.
20
^m 6 0 + amclonde
74
7.4 + amiloride
1.0
15
30
Minutes
45
60
FIGURE 5. Effects of extracellular acidity and amiloride on
sodium efflux in rabbit RBCs. The RBCs were preloaded with
sodium, and the efflux of sodium was measured by incubating
the cells in media containing (in mM) either 4 MgCl2, 4 sucrose, 10 glucose, 150 choline chloride, 0.1 ouabain, 0.125
4,4' -diisothiocyano-2,2' -disulfonic stilbene, and 10 Tris, morpholinepropanesulfonic acid, pH 7.4 (o, 9), or 10 Tris
morpholineethanesulfonic acid, pH 6.0 (O, *), with C, U) or
without (o, O) 1 mM amiloride. Data presented are the results of
a representative experiment.
system operated in a Lita for Na^ mode or a Nata for
Li^, mode. These findings suggest that RBC Na+-Li+
countertransport and renal brush border Na + -H + exchange are different transport systems. Nevertheless,
the same system may indeed be responsible for these
RED BLOOD CELL Na+-Li+ AND RENAL Na+-H+ COUNTERTRANSPORT/tfaAn
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two transport activities, but amiloride is not able to
effectively inhibit cation transport in the RBC membrane. This possibility seems unlikely since an amiloride-sensitive Na + -H + exchanger has been found in
the RBC membrane from humans, dogs, and amphiuma.23-2* Although Na + -H + exchange is not demonstrable under normal conditions in these cells, an
elevation in intracellular calcium24 or a decrease in cell
volume23-M clearly brings out this amiloride-sensitive
cation translocation pathway. The present studies
demonstrate that amiloride can also inhibit sodium
transport in the rabbit RBC membrane. As shown in
Figure 5, sodium efflux, which was stimulated by external protons, was abolished by 1 mM amiloride.
Amiloride is a competitive inhibitor of Na + -H + exchange in the renal brush border membrane.22 An inherent property of a competitive inhibitor is that a fixed
concentration of inhibitor will become less effective as
the substrate concentration is raised. It is important to
consider whether the failure of 1 mM amiloride to
inhibit RBC Na + -Li + countertransport in these studies
was due to such kinetic restraints, rather than to an
actual lack of sensitivity to amiloride. Indeed, amiloride is not an effective inhibitor of the Na + -H + exchanger in the presence of physiological concentrations of sodium.27 The \DK of amiloride for the human
RBC Na + -H + exchanger has been estimated to be
about 17 /iM. 24 If human RBC Na + -Li + countertransport and human RBC Na + -H + exchange are mediated
by the same process, then Michaelis-Menton inhibition kinetics predict that 1 mM amiloride should inhibit
Na + -Li + countertransport by about 97% when the external sodium concentration equals its Km value (25
mM). 8 Figure 2 shows that amiloride did not inhibit
human RBC Na + -Li + countertransport activity under
these conditions. Figure 5 shows that about 90% of
external proton-stimulated sodium efflux from rabbit
RBCs was inhibited by 1 mM amiloride. Thus, the KK
of amiloride for this process is less than 0.11 mM. If
rabbit RBC Na + -Li + countertransport and external
proton-stimulated sodium efflux are mediated by the
same process, then 1 mM amiloride should inhibit
Na + -Li f countertransport by at least 82% when the
external sodium concentration is less than its Km value
(50 mM). 9 Figure 3 shows that amiloride did not inhibit rabbit RBC Na + -Li + countertransport activity under
these conditions. Thus, the failure of amiloride to inhibit RBC Na + -Li + countertransport probably was not
due to kinetic restraints imposed by the external sodium and amiloride concentrations used in these studies.
One must consider the possibility, however, that Na + H + exchange and Na + -Li + countertransport are indeed
mediated by the same system, but that the affinity for
amiloride is absent or greatly reduced when the system
operates in a sodium for lithium exchange mode. The
present studies have not ruled out this possibility.
Jennings et al.28 recently reported studies designed
to determine whether the rabbit RBC Na + -Li + countertransport system can also mediate Na + -H + exchange. These authors found that Na + -Na + exchange,
an operative mode of the Na + -Li + countertransport
system,9 occurred at a velocity over 10 mmol Na/L
11
RBC/hr in rabbit RBCs. Despite this brisk transport
rate, little stimulation of sodium efflux into sodiumfree media was observed when extracellular pH was
reduced from 7.5 to 6.9. These authors concluded that
rabbit RBC Na + -Na + (or Na + -Li + ) countertransport
does not function appreciably in a Na + -H + exchange
mode.28 The experimental conditions and results of the
present study are different from those of Jennings et
al.28 In the present study, amiloride-inhibitable sodium
efflux from rabbit RBCs was stimulated by reducing
extracellular pH from 7.4 to 6.0. The difference in the
ability of external protons to stimulate sodium efflux in
the present studies and those of Jennings et al.28 is not
known, but it may be related to the larger increase in
external proton concentration in the present study.
Despite the differences in experimental conditions
between the present study and that of Jennings et al. ,M
the conclusions are the same: the RBC Na + -Li +
countertransport system probably is not the Na + -H +
exchanger.
The hypothesis that elevated Na + -Li + countertransport activity in RBCs from patients with essential hypertension is a marker for elevated Na + -H + exchange
activity in the renal brush border membrane1'i0> 16
would be a more attractive possibility if the two transport activities were mediated by the same system. The
present studies detract from this hypothesis. Since
Na + -H + exchange in sarcolemmal vesicles from vascular smooth muscle is inhibitable by amiloride,29 the
present data also detract from the recent suggestion by
Funder et al." that elevated Na + -Li + countertransport
activity may be a marker for increased vascular smooth
muscle Na + -H + exchange activity." On the other
hand, a physicochemical disturbance may exist in cell
membranes throughout the bodies of patients with essential hypertension. Such a global membrane abnormality might increase the activities of both RBC Na + Li + countertransport and renal brush border Na + -H +
exchange, even if the two transport systems were distinct. If this were the case, RBC transport activity
would be a marker for renal brush border transport
activity. In support of this possibility, Weder30 recently
has reported that RBC Na + -Li + countertransport activity was increased and renal fractional lithium clearance
was decreased in patients with essential hypertension.
The finding that renal lithium clearance was decreased
suggests that proximal tubular sodium reabsorption by
the brush border Na + -H + exchanger was increased in
the hypertensive patients described by Weder.30
Further studies are necessary to confirm the relation
between RBC Na + -Li + countertransport and renal
brush border Na + -H + exchange. The present studies
demonstrate a difference between these two transport
systems. The relation between RBC Na + -Li + countertransport and the hypertensive process remains
obscure.
Acknowledgments
The author expresses gratitude to Drs. Michael Boileau and Stuart Flechner for the provision of human kidneys. The author acknowledges the excellent technical assistance of Brian Lee and the
secretarial assistance of Ms. Nhung Le.
12
HYPERTENSION
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Difference between human red blood cell Na+-Li+ countertransport and renal Na+-H+
exchange.
A M Kahn
Hypertension. 1987;9:7-12
doi: 10.1161/01.HYP.9.1.7
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