Circulation Research
JUNE
1985
VOL. 56
NO. 6
An Official Journal of the American Heart Association
BRIEF REVIEWS
The Plasma Membrane Sodium-Hydrogen Exchanger
and Its Role in Physiological and Pathophysiological
Processes
Rex L. Mahnensmith and Peter S. Aronson
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From the Departments of Medicine and Physiology, Yale University School of Medicine, New Haven, Connecticut
SUMMARY. The plasma membranes of most if not all vertebrate cells contain a transport system
that mediates the transmembrane exchange of sodium for hydrogen. The kinetic properties of
this transport system include a 1:1 stoichiometry, affinity for lithium and ammonium ion in
addition to sodium and hydrogen, the ability to function in multiple 1:1 exchange modes involving
these four cations, sensitivity to inhibition by amiloride and its analogues, and allosteric regulation
by intracellular protons. The plasma membrane sodium-hydrogen exchanger plays a physiological
role in the regulation of intracellular pH, the control of cell growth and proliferation, stimulusresponse coupling in white cells and platelets, the metabolic response to hormones such as insulin
and glucocorticoids, the regulation of cell volume, and the transepithelial absorption and secretion
of sodium, hydrogen, bicarbonate and chloride ions, and organic anions. Preliminary evidence
raises the possibility that the sodium-hydrogen exchanger may play a pathophysiological role in
such diverse conditions as renal acid-base disorders, essential hypertension, cancer, andtissueor
organ hypertrophy. Thus, future research on cellular acid-base homeostasis in general, and on
plasma membrane sodium-hydrogen exchange in particular, will enhance our understanding of
a great variety of physiological and pathophysiological processes. (Circ Res 57: 773-788, 1985)
FOR many years it was held that acid-base equivalents merely distributed passively across the plasma
membrane of cells according to a Donnan equilibrium. Neutralization of acid produced from intermediary metabolism was presumed to rely first upon
cytoplasmic and membrane buffer action, and, second, upon the diffusive movement of protons (H+)
out of the cell down an electrochemical gradient.
With the advent of refined and reliable electrode
techniques for actually measuring transmembrane
voltage differences and intracellular H + activity,
however, it has become clear that intracellular pH
(pHi) is maintained at a much higher value than
predicted if protons were in Donnan equilibrium
across the plasma membrane (Roos and Boron,
1981). Measurements from numerous vertebrate and
invertebrate cell types have given pHt values ranging
from 6.8-7.3, whereas if H+ were in electrochemical
equilibrium across the membrane, pH would average 6.3-6.5, because the typical cell has a large
interior-negative membrane potential. Thus, there is
continual passive influx of H+ into the cell, and/or
passive efflux of hydroxyl (OH ) or bicarbonate
(HCO3~) ions out of the cell (Fig. 1). It is clear that
maintenance of pHi must depend upon an energyrequiring active H+ extrusion (or OH~ accumulation)
system.
One such mechanism for active H+ extrusion that
has been documented in the plasma membrane of a
wide variety of cell types is a carrier-mediated Na+H+ exchange (antiport, countertransport) system
that catalyzes the coupled transmembrane exchange
of sodium (Na+) for H+. In the intact cell, the plasma
membrane Na + -H + exchanger mediates the uphill
extrusion of protons directly coupled to the downhill
flux of Na+ into the cell (Fig. 1). This antiport process
is not directly dependent upon any exergenic chemical reaction, such as ATP hydrolysis; rather, energy
for the uphill extrusion of H+ is obtained exclusively
from the steep inwardly directed Na + gradient,
which is maintained by the primary active extrusion
of Na+ via the separate plasma membrane Na+,K+ATPase system. Thus, Na + -H + antiport is an example of secondary active transport.
Circulation Research/Vo/. 56, No. 6, June 1985
774
FIGURE 1. Role of the Na*-H+ exchanger in cellular acid-base
homeostasis.
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This transport system has been identified in virtually every type of cell that has been examined to
determine its presence, including ova, sperm, erythrocytes, lymphocytes, skeletal muscle, cardiac muscle and Purkinje fibers, neurons, capillary endothelia, fibroblasts, renal tubular cells, intestinal and gall
bladder epithelia, and a variety of cells in culture
(Aronson, 1985). The principal physiological role of
the Na + -H + exchanger is to regulate intracellular
pH. However, as a corollary of this role, the Na+H + exchanger has an important additional role as a
signal-transducer for various stimuli that influence
cell function by altering intracellular pH. Examples
include sperm activation of oocytes, the initiation of
cell growth and proliferation in response to serum
growth factors, and certain actions of insulin and
thrombin. Moreover, as a transporter of Na+, this
system plays a role in the regulation of cell volume.
Finally, the Na + -H + exchanger has an important role
in mediating the net transport of acid-base equivalents and Na+ across various epithelia, such as the
renal proximal tubule and small intestine.
This review presents a current overview of the
physiological roles subserved by the plasma membrane Na + -H + exchanger in different animal cells.
The intrinsic properties of the antiporter will be
presented first, as these have bearing on the interpretation of the many investigations that pertain to
its various roles in diverse cells and tissues. Of
burgeoning interest is how this transport system
might be regulated and how its state of activity
might be important in certain pathophysiological
states.
Properties of the Na+-H+ Exchanger
The distinctive nature of this transport system,
like any other transport system, resides in its specific
properties with regard to stoichiometry, selectivity
for substrates, sensitivity to inhibitors, and capacity
for regulation. The first clear-cut demonstration of
directly coupled Na + -H + exchange was made in
microvillus membrane vesicles derived from the luminal membranes of rat intestinal mucosa and renal
proximal tubular cells (Murer et al., 1976). In this
experimental preparation, uphill 22Na influx can be
driven by an outward proton gradient, and outward
extrusion of H + can be driven by an inward Na+
gradient. In intact cellular preparations, the technique is essentially the same: Na+-dependent H +
extrusion can be assayed with intracellular H+-sensitive microelectrodes, with pH-sensitive fluorescent
dyes, or with nuclear magnetic resonance (NMR);
and H+-coupled Na+ movements can be assayed
isotopically or with intracellular Na+-sensitive electrodes (see Nuccitelli and Deamer, 1982). The transport properties derived from these various approaches show remarkably close agreement.
Stoichiometry
Several observations indicate that the stoichiometry of the Na + -H + antiport process is one-for-one.
In a number of studies with intact cell systems, the
net flux of Na+ in one direction and the net flux of
H+ in the opposite direction have been directly
quantified (Cala, 1980; Moolenaar et al., 1981b;
Boron and Boulpaep, 1983; Grinstein et al., 1984).
In each of these studies, the directly measured ratio
of these fluxes did not differ significantly from 1.0.
Second, in vesicle studies, maneuvers that alter the
transmembrane electrical potential difference have
no effect on Na+ influx via this transport pathway
(Murer et al., 1976; Kinsella and Aronson, 1980;
Cohn et al., 1982; Knickelbein et al., 1983), and in
intact cell systems, stimulating or inhibiting Na + -H +
antiport does not cause any measureable change in
the transmembrane electrical potential difference
(Aickin and Thomas, 1977; Cala, 1980; Deitmer and
Ellis, 1980; Vigne et al., 1982; Grinstein et al., 1983),
indicating that the operation of the Na + -H + exchanger is electrically silent and must have a Na+:H+
coupling ratio dose to 1.0. Third, when the transmembrane Na+ gradient is thermodynamically balanced by an H gradient of the same magnitude
(i.e., when Na^/Na^ = Ho7Hi+), the Na + -H + exchanger is at equilibrium and mediates no net flux
of Na+ and H + (Moody, 1981; Kinsella and Aronson,
1982), which also implies that the exchange is electroneutral and that the coupling ratio is 1.0. A
Na+:H+ coupling ratio of 1.0 is consistent with stoichiometries of 1:1, 2:2, 3:3, etc., but the interaction
of external Na+ and external H + with the Na + -H +
exchanger conforms to simple saturating MichaelisMenten kinetics, with a Hill coefficient of 1.0 (Burnham et al., 1982; Aronson et al., 1983; Ives et al.,
1983a; Gunther and Wright, 1983; Frelin et al., 1983;
Paris and Pouyssegur, 1983; Grinstein et al., 1984).
This is most consistent with the presence of only a
single binding site for external Na+ or H+, and
indicates that the stoichiometry of exchange is actually one Na+ for one H+.
Alternative Substrates and Modes
The Na + -H + exchanger can operate in more than
one mode with a narrow selection of monovalent
cations. In addition to mediating Na + -H + exchange
Mahnensmith and Aronson/Plasma Membrane Na+-H+ Exchanger
+
+
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in either direction, it can mediate Na -Na exchange, and can bind and transport Ii + under certain
conditions. Thus, exchange of external Na+ for internal H+, external H+ for internal Na+, external Na+
for internal Na+, external Ii + for internal H+, and
internal Li+ for external H+ have all been demonstrated (Murer et al., 1976; Kinsella and Aronson,
1980, 1981a; Deitmer and Ellis, 1980; Rindler and
Saier, 1981; Moolenaar et al., 1981b; Aronson et al.,
1982; Burnham et al., 1982; Paris and Pouyssegur,
1983; Gunther and Wright, 1983; Abercrombie and
Roos, 1983). There is also evidence in microvillus
membrane vesicles (Kinsella and Aronson, 1981a;
Aronson et al., 1983; Gunther and Wright, 1983)
that ammonium ion (NH^+) can share the Na + -H +
exchanger. Taken together, these observations suggest that the Na + -H + exchanger has appreciable
affinity for Ii + and NH4"1" in addition to Na+ and
H+, and can function in multiple exchange modes
involving these four cations, as illustrated schematically in Figure 2. In several preparations, the Ky, for
interaction of external H+ with the Na + -H + exchanger is in the range, 10~8 to 10~7 M, far lower than
the Kv, values (10~3 to 10"2 M) for U + , NHL,+, and
Na+ (Aronson, 1985). The apparent selectivity sequence for binding of external cations is H+ » Li+
> NH«+ > Na+; there is no appreciable affinity for
K+, Rb+, Cs+, or organic cations such as choline or
tetramethylammonium (Aronson, 1985).
Given the model in Figure 2, one would predict
that the plasma membrane Na + -H + exchanger could
mediate Na+-Li+ exchange. However, this has yet to
be conclusively demonstrated. In erythrocytes, a
one-for-one exchange of Na+ for Li+ is easily demonstrated, and this system has been well characterized as having many features in common with the
Na + -H + exchange system (Aronson, 1982; Funder et
al., 1984). Specifically, both systems mediate electroneutral monovalent cation exchange, both systems are ATP-independent and ouabain-insensitive
and are energized by the downhill cation gradients,
both systems can transport Ii + and actually have a
higher affinity for Li+ than for Na+, and, both systems can operate in a Na+-Na+ exchange mode.
Furthermore, external H+ interacts competitively
with the erythrocyte Na + -Ii + exchanger, just as Ii +
interacts competitively with the Na + -H + exchanger
OUTSIDE
Na, NH4 , Li
INSIDE
or H
Ami I
FIGURE 2. Schematic model summarizing properties of the plasma
membrane Na+-H* exchanger.
775
+
+
(Funder et al., 1984). Coupled Na -H exchange has
been demonstrated in mammalian erythrocytes
(Dissing and Hoffman, 1982; Parker, 1983; Parker
and Glosson, 1984), but whether the same antiport
system that mediates Na+-Li+ exchange also mediates the Na + -H + exchange in these erythrocytes is
not yet clear.
Inhibitors
Sensitivity of the plasma membrane Na + -H + exchanger to a variety of transport inhibitors has been
evaluated. Ouabain, furosemide, disulfonic acid stilbene derivatives, and acetazolamide have no direct
effects on this pathway (Kinsella and Aronson,
1980). The best inhibitors identified to date are the
diuretic drug amiloride and its analogues. With the
exception of one study (Ives et al., 1983a), amiloride
has generally been found to be a purely competitive
inhibitor with respect to external Na+(K| ~ 10~6 to
10~5 M) (Kinsella and Aronson, 1981b; Vigne et al.,
1982; Paris and Pouyssegur, 1983), implying that
amiloride interacts at or near the Na+ transport site.
Inhibition by this agent is immediate in onset and is
rapidly reversible. Note that, compared to its effect
on Na + -H + exchangers, amiloride is 10-100 times
more potent as an inhibitor of the Na+ channels
found in the apical membranes of such epithelia as
the colon and the cortical collecting tubule of the
kidney (Benos, 1982). The effects of amiloride as a
diuretic employed clinically probably arise solely
from inhibition of apical Na+ channels in the distal
nephron, rather than from inhibition of Na + -H +
exchange.
Nevertheless, amiloride has been employed extensively as an experimental probe of Na + -H + exchange activity in various tissues. Inhibition of H+
efflux or Na+ influx along with inhibition of a particular cell function is often taken as .evidence for a
specific role of Na + -H + exchange in that cell function. Although amiloride is useful and informative
in this regard, it is not an ideal agent when employed
in this manner, and such experiments need to be
interpreted carefully. Because amiloride and Na+
compete for access to the external transport site of
the Na + -H + exchanger, and because the Km for Na+
is quite low (Km ~ 3-50 HIM), amiloride is not an
effective inhibitor of Na + -H + exchange in the presence of physiological Na+ concentrations (140-150
HIM) unless relatively high concentrations of the
drug (>10~4 to 10~3 M) are used. At these concentrations, amiloride may have additional effects, such
as inhibiting paracellular cation permeability in epithelia (Balaban et al., 1979), and inhibiting Na+,K+ATPase (Soltoff and Mandel, 1983). In addition, at
high concentrations, amiloride can act as a weak
base and nonspecifically alter transmembrane pH
gradients (Dubinsky and Frizzell, 1983). Finally,
high concentrations of amiloride can inhibit protein
synthesis in cells and in cell-free systems, implying
that, under certain circumstances, this drug has a
direct toxic action (Leffert et al., 1982). In spite of
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these constraints, amiloride has been useful as an
experimental probe of Na + -H + exchange activity,
particularly when used in the presence of lower than
physiological Na+ concentrations. Recently, amiloride analogues with substitution of the 5-amino
group by alkyl or alkenyl groups have been found
to be up to 100 times more potent than amiloride in
inhibiting Na + -H + exchangers (Vigne et al., 1984).
These analogues should be more useful than amiloride for studying the functions of Na + -H + exchangers under physiological conditions.
Other inhibitors of the Na + -H + exchanger include
the alkaloids, harmaline and quinidine, which are
less specific than amiloride (Kinsella and Aronson,
1980; Parker, 1983), the carboxyl-activating reagent
N - ethoxycarbony 1 - 2 - ethoxy -1,2- dihy droquinoline
(EEDQ) (Burnham et al., 1982), and the histidinespecific reagent diethylpyrocarbonate, an irreversible inhibitor (Grillo and Aronson, 1983). Whether
any of these agents are useful for probing the function and roles of the Na + -H + exchanger under physiological conditions is yet to be determined.
Regulation by Intracellular H+
A general observation in most studies reviewed is
that raising the internal H+ concentration dramatically stimulates the rate of Na + -H + exchange. Since
this system is thermodynamically energized by the
Nao+/Nai+ and Hi+/Ho+ gradients, it is predicted that
as the outward H+ gradient steepens in the face of
an unchanging inward Na+ gradient, the rate of
antiport should increase proportionally. However,
in renal microvillus membrane vesicles, the rate of
Na+ influx by Na + -H + exchange has a greater than
linear dependence on internal H+ concentration
(Aronson et al., 1982), and, in whole renal cells and
in human lymphocytes, efflux of H+ via Na + -H +
exchange shows a steeper dependence on internal
pH than expected for a simple Michaelis-Menten
process (Boron and Boulpaep, 1983; Grinstein et al.,
1984). In view of the considerable data indicating
that the stoichiometry of Na + -H + exchange is onefor-one, these observations have led to the proposal
that internal H+ can allosterically activate the exchanger through an interaction that is independent
of its role as a transportable ion (Aronson et al.,
1982). The evidence in support of this concept derives from two observations: first, that raising the
internal H+ concentration actually stimulates Na+Na+ exchange in vesicles, an effect opposite to that
expected if internal H+ were to interact (and compete) only at the internal transport site, and second,
that raising internal H+ stimulates net Na+ efflux in
exchange for external H+, also an effect opposite to
that expected if internal H+ were interacting only at
the transport site (Aronson et al., 1982). Conceptually, one explanation for the striking stimulatory
effect of internal H+ is that the transport system
possesses one or more functional groups on its inner,
Circulation Research/Vo/. 56, No. 6, June 1985
cytoplasmic surface, the protonation of which causes
activation of the exchanger.
This kinetic regulation of Na + -H + exchange activity by internal protons has important consequences
for the cell. Activation of Na + -H + exchange by a
falling cytoplasmic pH would greatly enhance the
ability of plasma membrane Na+-H+ exchangers to
extrude protons promptly, thereby protecting
against intracellular acid loads. Conversely, this
property is reflected by the existence of an apparent
'activity threshold,' because, as pH| rises to or above
a certain value, the rate of H+ extrusion is sharply
curtailed in most tissues. This kinetically protects
against the generation of an intracellular alkalosis
inasmuch as the average Na<,+/Nai+ gradient of approximately 10 could theoretically drive pH 1.0 unit
higher than extracellular pH if there were not some
mechanism to prevent the system from reaching
thermodynamic equilibrium. It appears that the kinetic dependence on internal H + activity is just such
a 'brake' for this system. Of interest in this regard
is recent evidence that suggests that stimulation of
the plasma membrane Na + -H + exchanger by purified growth factors results from a shift in its sensitivity to intracellular pH (Moolenaar et al., 1983).
Hence, altering the apparent affinity of the transporter for internal H+ ions may be an important
mechanism for physiological regulation of this transport system.
Possible Regulation by Intracellular Ca**
In addition to the regulation of Na + -H + exchange
by internal pH, it has been proposed that antiport
activity might also be importantly influenced by
intracellular calcium (Ca++) activity, perhaps
through the mediation of calmodulin (Benos, 1982;
Owen and Villereal, 1982a, 1982b). This hypothesis
is founded on the observation in fibroblasts that
elevations in free intracellular Ca++ induced by the
calcium ionophore A23187 markedly stimulate amiloride-sensitive Na+ influx, and that treatment of
these cells with intracellular Ca++ antagonists or
with calmodulin antagonists blocks activation of
Na + -H + exchange in a dose-dependent manner (Villereal, 1981; Owen and Villereal, 1982a; 1982b,
1983; Mix et al., 1984). Furthermore, in sea urchin
eggs, fertilization is associated with an immediate
rise in free intracellular Ca++ and a subsequent
activation of Na + -H + exchange (see next section).
Since treatment of the unfertilized egg with A23187
also induces egg activation and Na"^-dependent H+
extrusion, it has been proposed that intracellular
Ca++ might be a modulator of Na + -H + exchange in
these cells (Whitaker and Steinhardt, 1982; Busa and
Nuccitelli, 1984). However, some doubt has been
cast upon the hypothesis that Ca++ is a regulator of
Na + -H + exchange activity. Treatment of either
mouse neuroblastoma cells (Moolenaar et al., 1981a)
or mouse fibroblast cells (Frelin et al., 1983) with a
range of A23187 concentrations in the presence of
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external Ca++ does not modify the rate of amiloridesensitive Na+ uptake, nor does it trigger mitogenesis.
Also, Grinstein et al. (1984) have reported that the
Ca++ ionophore ionomycin does not activate Na+H+ exchange in human lymphocytes, and preloading renal microvillus membrane vesicles with 10~7
to 10~3 M calcium has no significant effect on renal
Na + -H + exchange activity (Aronson et al., 1982).
Thus, the exact interplay between intracellular Ca++
activity, calmodulin, and the Na + -H + exchanger is
uncertain.
An alternative mechanism for Ca++-dependent
regulation of the plasma membrane Na + -H + exchanger is via the action of the calcium and phospholipiddependent protein kinase (i.e., protein kinase C). In
several different cell types, phorbol esters, which
are relatively specific activators of protein kinase C,
stimulate the rate of plasma membrane Na + -H +
exchange (Burns and Rozengurt, 1983; Moolenaar
et al., 1984). If Na+-H+ exchange activity is regulated
by protein kinase C -induced phosphorylation under
physiological conditions, then it may be sensitive
not only to changes in intracellular Ca++ activity,
but also to changes in availability of diacylglycerol,
a potent endogenous activator of protein kinase C
(Berridge, 1984). Diacylglycerol is a product of phosphoinositide hydrolysis. Another such product is
inositol rriphosphate, which is known to stimulate
the release of Ca++ from intracellular storage pools.
Thus, receptor-mediated hydrolysis of phosphoinositides to diacylglycerol and inositol rriphosphate
may be an important mechanism for regulating activity of the Na+-H+ exchanger through the action
of protein kinase C (Berridge, 1984).
Physiological Roles
In almost all animals cells, a steep inwardly directed Na+ gradient is maintained across the plasma
membrane due to the primary active extrusion of
Na+ from the cell via the Na ,K+-ATPase (sodium
pump). Because Na^/Na^ exceeds Ho+/Hi+ under
most physiological conditions, the net thermodynamic driving force acting on plasma membrane
Na + -H + exchangers generally favors the net exchange of external Na+ for internal H+. Thus, it is
easy to conceptualize how this transport system
could function in the protection of intracellular pH
against intracellular acid loads, especially in light of
the demonstrated ability of internal protons to activate proton extrusion. Of great interest are the recent
observations that the plasma membrane Na+-H+
exchange system serves a variety of functions in
addition to merely defending pHi against acid loads.
These roles can be broadly distinguished into three
categories: (1) intracellular pH change is caused by
activation of Na + -H + exchange, and the ensuing pHi
rise is permissive or serves as a signal for generating
a biological response; (2) changes in activity of
plasma membrane Na + -H + exchange alter the intra-
777
cellular content of Na+, as in cell volume regulation;
and (3) plasma membrane Na + -H + exchange participates in net transepithelial movement of Na and
H+, as in the processes of acid secretion and Na+
reabsorption by gallbladder, intestine, and kidney.
Regulation of Intracellular pH
Since the development of accurate means for intracellular pH and voltage measurements, there has
been general agreement that H+ activity is lower
inside cells than would be predicted if H + were
simply passively distributed in equilibrium with the
membrane potential. In most cell systems studied to
date, active H+ extrusion is accomplished by a Na+coupled ion exchange mechanism, whereby downhill Na+ influx is coupled either singly to H+ efflux
or Na+ influx is coupled to Cl~ efflux and HCO3"
influx (Roos and Boron, 1981). Which system is
present or predominates in any given tissue appears
to depend at first approximation on animal class and
perhaps tissue type: with one exception, invertebrate
cells seem to rely on the Na+-dependent Cr-HCO 3 ~
exchange system, whereas vertebrate cells appear to
rely more on Na + -H + exchange with or without an
independently operating Cr-HCO 3 ~ exchange. The
systems are distinguished by external and internal
ionic requirements and by their sensitivity to inhibitors: Na+-H+ exchange is inhibited by amiloride,
and Cr-HCO 3 ~ exchange but not Na+-H+ exchange
is inhibited by disulfonic stilbene derivatives (DIDS,
SITS).
For example, in the snail neuron (Thomas, 1977),
the squid giant axon (Boron and Russell, 1983), and
the giant barnacle muscle fiber (Boron et al., 1981),
effective recovery of pHi after the cell interior is
acidified depends on the presence of external Na+
and HCO3~ and internal Cl~. Based on experiments
with either ion-sensitive electrodes or radioisotope
tracers, the actual stoichiometry of ion fluxes appears to be 1 Na+ in, 1 Cl~ out, and 2 equivalents
of HCO3" in (Boron and Russell, 1983). The entire
process is electroneutral and can be inhibited by
SITS, a selective anion inhibitor, but not by amiloride. Although a Na+-H+ exchange may occur along
with the C1~-HCO3~ exchange in these tissues, an
independent Na + -H + exchange has not been discerned. Unlike other tissues to be discussed, there is
an absolute dependence on Cl~ and HCO3~, and
deletion of any one ion prevents pHi recovery. In
another invertebrate preparation, the crayfish neuron, Moody (1981) found that regulation of pHi
occurs somewhat differently. In this case, two operations were needed to effect pHi recovery: Na+H+ exchange, and a Na+-dependent Cr-HCO 3 ~ exchange.
A more prominent role for Na + -H + exchange in
pHi regulation has been documented in a variety of
vertebrate cells. Aickin and Thomas (1977) recorded
an intracellular pH of 7.07 in mouse skeletal muscle
situated in buffered Ringers at pHo 7.40. After intra-
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cellular acid loading, pHi recovered spontaneously
over about 30 minutes, and was associated with an
influx of Na+. Recovery of pH could be blocked
more than 90% by removal of outside Na+ or application of 10~4 M amiloride. A less active Cr-HCO 3 ~
exchange process was also noted in this tissue, and
it made a minor contribution to pHi recovery. Other
similar types of investigations in vertebrate skeletal
muscle have documented that Na + -H + exchange
and Cr-HCO 3 ~ exchange co-exist independently in
the plasma membranes of this tissue (Roos and
Boron, 1978; Abercrombie et al., 1983). Although
both systems appear to function importantly toward
pHj recovery when the cell is acid loaded, activity
of the amiloride-sensitive Na + -H + exchanger appears to be more significant, particularly when the
muscle cell is depolarized (Roos and Boron, 1978;
Abercrombie et al., 1983).
Regulation of internal pH in mammalian cardiac
tissue also depends importantly on Na + -H + exchange. Ellis and Thomas (1976) were the first to
document that both mammalian Purkinje tissue and
ventricular muscle cells possess active mechanisms
for pHj maintenance. Resting intracellular pH was
7.27, and after cytoplasmic acidification, pHi recovery was prompt. This recovery process was dependent upon external Na+, was accompanied by Na+
influx, was amiloride-sensitive, and was unaffected
by Cl~ or HCO3~ deletion or SITS addition (Deitmer
and Ellis, 1980). This strongly suggests that Na + -H +
exchange is the chief if not sole pHi regulator in this
tissue. A similar conclusion was reached by PiwnicaWorms and lieberman (1983), who investigated
regulation of intracellular pH in spontaneously beating cardiac muscle cells in culture by using the pHsensitive chromophore, 6-carboxyfluorescein. Significantly, either lowering external Na+ from 144
DIM to 0.8 mM or application of 1 DIM amiloride
caused a rapid cytosolic acidification. These data
strongly imply that the Na + -H + exchanger is continuously active in the contracting cardiac cell, where
it compensates for the generation of metabolic acid
or for the passive entry of H+ or exit of HCO3~
across the cell membrane. A C1~-HCO3~ exchange
system that is independent of Na + -H + antiport also
exists in the Purkinje fiber (Vaughan-Jones, 1979).
However, this anion transport system does not appear to play a major part in acid extrusion in the
heart, but, rather, it may serve to facilitate Cl~ entry
into the cell and thereby regulate intracellular Cl~
activity (Vaughan-Jones, 1979).
The Na + -H + exchange system has been identified
as the principal mechanism for pHi regulation in a
number of other tissues and cells. In such epithelia
as the gallbladder (Weinman and Reuss, 1982) and
the renal proximal tubule (Boron and Boulpaep,
1983), Na + -H + exchange is the principal mechanism
for controlling intracellular pH. Grinstein et al.
(1984) have found that lymphocytes activate an
amiloride-sensitive electroneutral Na + -H + exchange
Circulation Research/Vol. 56, No. 6, June 1985
following cytoplasmic acidification, and Betz (1983)
has identified Na + -H + exchange in isolated brain
capillary endothelium from the rat. Finally, a variety
of cultured animal cells possess a relatively quiescent
Na + -H + antiporter that is dramatically stimulated
when the cell interior is acidified slightly, indicating
a direct link between activity of the Na + -H + exchanger and intracellular pH (Moolenaar et al., 1981b;
1983; Rindler and Saier, 1981; Vigne et al., 1982;
Schuldiner and Rozengurt, 1982; Rothenberg et al.,
1983; Paris and Pouyssegur, 1983; Frelin et al.,
1983).
hi general, acid-loaded cells recover only to the
baseline pHi and then seem to curtail H+ transport
sharply, even though the driving force for Na + -H +
exchange, the inwardly directed Na+ gradient, still
remains favorable for additional H + extrusion. This
phenomenon probably reflects the kinetic sensitivity
of the transport system to internal H + as discussed
earlier (Aronson et al., 1982; Boron and Boulpaep,
1983). Thus, H+ extrusion rates are dramatically
increased as internal H + rises, and are dramatically
slowed as internal H+ falls in spite of a persistently
steep thermodynamic driving force for continued
H + extrusion. The physiological importance of this
phenomenon resides in the fact that the cell must
maintain its pHi within narrowly defined limits for
optimal enzyme and organelle functioning. If there
were not a kinetic modulator for this system, and if
activity depended solely on thermodynamic forces,
the mechanism would be sluggish in defense of acid
loads, and would tend to drive steady state pHi to
excessively alkaline values.
Intracellular pH as Mediator of Cell Response to
Activating Stimuli
Cell Proliferation
Within the first few minutes after fertilization of
an oocyte, there is a dramatic increase in DNA and
protein synthesis, an increase in metabolic rate, and
initiation of cell division (Whitaker and Steinhardt,
1982; Busa and Nuccitelli, 1984). One of the earliest
events triggered by sperm-egg fusion is an increase
in intracellular pH. In studies with sea urchin eggs,
this cytoplasmic alkalinization appears to be due to
an active proton extrusion process that is quiescent
in the unfertilized egg but is activated 5-10 minutes
after the fertilization event. Both the proton efflux
and the increase in pHj depend upon the presence
of external Na+ and have been linked to Na+ influx;
as well, these Na + and H + fluxes are amiloridesensitive. Thus, it is postulated that activation of sea
urchin eggs by sperm-egg fusion involves activation
of Na + -H + exchange, which is critical to further
development. The subsequent alkalinization of the
cytoplasm may act as a regulator of the biosynthetic
events (Whitaker and Steinhardt, 1982; Busa and
Nuccitelli, 1984). Of additional interest in this regard
is that initiation of motility in sea urchin and rat
Mahnensmith and Aronson /Plasma Membrane Na+-H+ Exchanger
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sperm also appears to rely upon a Na+-dependent
H+-efflux system and an associated rise in pHj
(Wong et al., 1981; Lee et al., 1982).
An even more convincing role for Na + -H + antiport
and alterations of cytoplasmic pH in the regulation
of cell proliferation and growth has been documented with a variety of mammalian cells in culture.
When quiescent cells in culture are stimulated to
grow and divide by the application of serum, a
dramatic and persistent increase in Na+ influx occurs
(Smith and Rozengurt, 1978). The cell growth response can be suppressed by either a reduction in
external Na+ (Smith and Rozengurt, 1978) or by the
presence of amiloride (Koch and Leffert, 1979; Rozengurt and Mendoza, 1980; Moolenaar et al., 1982).
Similarly, in a number of cultured cell preparations
it has been observed that (1) serum-activated Na+
influx is simultaneous with a rise in pH| due to H+
extrusion, (2) serum and purified growth factors
stimulate a rise in pl-^, and this is strictly dependent
on external Na+; (3) the pHi increase, like Na+ influx,
is inhibited by amiloride, and (4) the fluxes of Na+
and H+ are electroneutral (Moolenaar et al., 1981a,
1981b, 1982, 1983; Pouyssegur et al., 1982; Schuldiner and Rozengurt, 1982; Cassel et al., 1983; Paris
and Pouyssegur, 1983; Frelin et al., 1983). Moreover,
when mutant fibroblasts which specifically lack
Na + -H + exchange activity are exposed to mitogens,
cytoplasmic pH does not rise and DNA synthesis is
not activated to the same magnitude as in the wild
type fibroblast (L'Allemain et al., 1984).
The biochemical steps by which the binding of
growth factors to their membrane receptors is linked
to activation of Na + -H + exchange are unknown at
present. In this regard, Villereal (1981) has suggested
that the serum activation of Na + -H + exchange seen
in human fibroblasts might be secondary to an elevation in intracellular free Ca++ activity. When
quiescent fibroblasts are activated by growth factors,
there is a prompt mobilization of Ca++ from intracellular Ca++ storage pools, causing a rise in free
intracellular Ca++ activity, as measured by quin-2
fluorescence (Mix et al., 1984); this occurs at the
same time that Na + -H + exchange activity is stimulated by serum. In the absence of serum growth
factors, the calcium ionophore A23187 induces a
dose-dependent increase in amiloride-sensitive Na+
influx and cell proliferation (Villereal, 1981). Amiloride blocks serum-stimulated Na+ influx but does
not block Ca++ mobilization, suggesting that the
serum-induced rise in Cai++ is an event that is independent of Na+ influx; treatment with agents that
block intracellular Ca++ mobilization or with agents
that inactivate Ca++-calmodulin complexes inhibits
serum-stimulated Na+ influx and blocks cell activation (Owen and Villereal, 1982a, 1982b). Therefore,
it has been proposed that serum growth factors act
primarily by mobilizing Ca++ from intracellular
pools, and the consequent formation of Ca++-calmodulin complexes then activates Na + -H + ex-
779
change, causing a rise in pHi. However, as discussed
earlier, activation of Na -H+ exchange by calcium
ionophores is an inconsistent finding. Also as discussed earlier, the critical event may instead be a
receptor-mediated hydrolysis of phosphophinositides to yield diacylglycerol, a potent stimulator of
protein kinase C. A rise in Cai+ may not be necessary or sufficient for stimulation of Na + -H + exchange via the mediation of protein kinase C (Moolenaar et al., 1984). It appears that, perhaps, as the
result of protein kinase C-mediated phosphorylation, growth factors alter the proton modifier site of
the Na + -H + exchanger, resulting in an increased
affinity for cytoplasmic H+, and, thus, a shift in the
curve for activation by internal H+ (Moolenaar et
al., 1983).
Whatever the precise biochemical steps that result
in the activation of Na + -H + exchange by serum
growth factors, it is the resulting cytoplasmic alkalinization that is responsible, at least in part, for the
subsequent biosynthetic and mitotic events. For example, the early phosphorylation of certain proteins
that occurs with mitogen activation of quiescent
fibroblasts is strongly influenced by pH| and seems
to be a limiting prerequisite for further activation of
biosynthetic events (Pouyssegur et al., 1982). Likewise, a small rise in pHt accelerates other critical
operations within the cell, such as glycolysis (Fidelman et al., 1982; Moolenaar et al., 1983), DNA
synthesis (Zetterberg and Engstrom, 1981; Gerson
et al., 1982), and protein synthesis (Winkler et al.,
1980). Mitosis can be induced in serum-starved fibroblasts simply by alkalinizing the cells, while
serum-induced mitosis can be halted by a small
acidification (Busa and Nuccitelli, 1984). Furthermore, mutant fibroblasts that lack Na + -H + exchangers do not alkalinize when exposed to serum mitogens, and their rate of DNA synthesis and cell
growth rises very slowly compared with wild type
cells that possess Na + -H + exchangers (L'Allemain et
al., 1984). Thus, it is dear that an internal pH shift
brought about by activation of Na + -H + exchange is
probably a critical feature of cell activation, but other
regulatory signals are probably equally important
and may not be related to pHi shifts at all. This point
has been emphasized by those workers who have
demonstrated the parallel importance of internal
Ca++ activity (Winkler et al., 1980; Mix et al., 1984)
and by those investigators who have noted that the
protein phosphorylation events induced by mitogens were only partially suppressible by inhibiting
Na+-H+ exchange activity (Pouyssegur et al., 1982).
Moreover, new DNA synthetic activity can occur
upon cell exposure to growth factors in the absence
of a pHi rise, albeit at a much slower rate (L'Allemain
et al., 1984). An interesting observation in this context is that the calcium-calmodulin interaction appears to be pH sensitive, such that a rise in pH from
7.0 to 7.5 might increase calcium binding to calmodulin by more than 60% (Busa and Nuccitelli, 1984).
780
This implies that calmodulin-mediated reactions
might be pHi sensitive and that activity of the Na+H + exchanger might have a modulating function for
calmodulin-mediated responses. An alkaline pHj
shift might serve to amplify Ca++-calmodulin signals
involved in cell activation, and some calmodulin
responses may depend upon this alkaline amplification. Furthermore, if Na + -H + exchange itself is
indeed modulated by a calmodulin-dependent
mechanism, then an enhanced Ca++-calmodulin interaction might serve to sustain Na + -H + exchange
activity in the face of a higher than normal pHt.
Circulation Research/Vo/. 56, No. 6, June 1985
dose-response curve for thrombin-induced serotonin secretion is nearly identical to that of the
thrombin-induced pHi rise. Taken together, these
data imply a primary role for Na + -H + exchange in
platelet activation by thrombin but do not exclude
other parallel events. For example, activation of
platelets and neutrophils also involves a rise in
intracellular free Ca , and protein phosphorylations are prerequisite steps for the full physiological
cellular response (Kaibuchi et al., 1983; Korchak et
al., 1984). The relationships among Na+ influx, elevation of pHi, and these events are presently unclear.
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Stimulus-Response Coupling in Neutrophils,
Lymphoq/tes, and Platelets
Effects of Hormones
Stimulus-response coupling in certain differentiated cells also appears to bear a relationship to
activation of Na -H+ exchange and subsequent
cytoplasmic alkalinization. When mature lymphocytes are activated by exposure to bacterial lipopolysaccharide or concanavalin A, there is an abrupt rise
in intracellular pH that is associated with a burst of
RNA and protein synthesis, followed by a second
higher and more protracted rise in pHi that strongly
correlates with the rate of RNA synthesis (Gerson et
al., 1982, 1983). An early surge of amiloride-inhibitable Na+ influx occurs during lymphocyte activation (Rosoff and Cantley, 1983), and lymphocyte
activation and subsequent DNA synthesis can be
inhibited by micromolar doses of amiloride, an effect
due to inhibition of cation flux and not nonspecific
toxiciry (Heikkila et al., 1983; Rosoff and Cantley,
1983).
Similarly, activation of mammalian neutrophils
by complement component C5a and by the synthetic
chemotactic agent formyl-methionyl-leucyl-phenylalanine causes a significant increase in Na+ influx
and cytoplasmic pH (Naccache et al., 1977a; Molski
et al., 1981). These events have been correlated with
the functional ability of the neutrophil to migrate,
degranulate, and generate antibacterial granular superoxides (Naccache et al., 1977b). Both the Na+
influx and the rise in pH( induced by f-met-leu-phe
are amiloride-sensitive but DIDS-insensitive, suggesting that this response is mediated by Na + -H +
exchange (Molski et al., 1981).
Platelet activation by thrombin also appears to
involve activation of plasma membrane Na -H+ antiport (Home et al., 1981; Greenberg-Sepersky and
Simons, 1984). With the use of internalized pHsensitive fluorescent indicators, it has been observed
that thrombin stimulation of washed human platelets leads to a dose-dependent increase in 22Na influx
and a concomitant rise in cytoplasmic pH. The rise
in pHi produced by a saturating dose of thrombin is
reduced 50% by 10"4 M amiloride and is prevented
completely by 10~3 M amiloride. The secretion of
serotonin by the platelet, which serves to attract and
aggregate other platelets, is also thrombin-dependent and is partially inhibited by amiloride. The
Rather convincing evidence has been presented
which suggests that cytoplasmic alkalinization secondary to activation of Na + -H + exchange is one of
the early events mediating the action of insulin to
stimulate glycolysis in frog skeletal muscle. The
addition of insulin to muscle strips increases pHi by
0.1 to 0.15 pH unit (Moore, 1981; Fidelman et al.,
1982). The possibility that activation of plasma
membrane Na + -H + exchange is responsible for this
rise in pHj is suggested by the observations that the
elevation in pHi is associated with an increased
influx of Na+, that lowering extracellular Na+ prevents the insulin-induced pHi rise, and that both the
insulin-induced Na+ influx and the pHi rise can be
inhibited by 0.5 mM amiloride (Moore, 1981). That
insulin stimulation of glycolysis results from the rise
in pHi per se is indicated by the findings that
changes in glycolytic activity closely parallel the
changes in pHi produced by insulin, and that glycolysis is significantly reduced when the insulininduced rise in pHi is inhibited by amiloride or by
lowering extracellular Na+ (Fidelman et al., 1982). It
was known earlier that glycolysis in cell-free extracts
from rat diaphragm and in guinea pig leukocytes is
increased by small increases in medium pH (Ui,
1966; Halperin et al., 1969). The likely explanation
for this effect is that the activity of the rate-limiting
enzyme of glycolysis, phosphofructokinase, is very
sensitive to small changes in ambient pH and can
be maximally stimulated by a pH elevation as small
as 0.1-0.2 pH unit (Trivedi and Danforth, 1966).
Other hormones also appear to influence Na + -H +
exchange activity. For example, when rats are adrenalectomized, Na + -H + exchange activity as measured in isolated renal microvillus membrane vesicles
is diminished compared to control rats (Freiberg et
al., 1982). In these adrenalectomized rats, Na + -H +
exchange activity can be restored by the administration of dexamethasone but not aldosterone. Hence,
it appears that glucocorticoids but not mineralocortoicoids stimulate renal Na + -H + exchange activity.
That this effect can be demonstrated in isolated
membrane vesicles studied in vitro indicates that
there has been an intrinsic change in the number or
activity of the Na + -H + exchangers in the membrane
Mahnensmith and Aronson /Plasma Membrane Na+-H+ Exchanger
+
+
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(Freiberg et al., 1982). Stimulation of renal Na -H
exchange by glucocorticoids may explain the effect
of these hormones to augment renal acid excretion
(Wilcox et al., 1982). Whether glucocorticoids stimulate Na + -H + exchange in other target tissues or
whether changes in pHi are important for mediating
the metabolic effects of glucocorticoids is not
known.
Preliminary investigations also indicate an interaction between thyroid hormone and Na + -H + exchange activity. Induction of a hypothyroid state
with thiouracil in rats causes a decrease in renal
brush border Na + -H + exchange activity (Kinsella
and Sacktor, 1984). As thyroid extract is given back
to hypothyroid animals, a dose-related increase in
renal Na + -H + exchange is observed. A physiological
correlation is suggested by the fact that hypothyroidism in the intact rat is associated with urinary
sodium and bicarbonate wasting (Michael et al.,
1979). Again, it is not known to what extent changes
in Na + -H + exchange and pHi may be important for
mediating the effects of thyroid hormone on other
tissues.
It is interesting to note that the three hormones
just discussed—insulin, glucocorticoids and thyroid
hormone—all are known to enhance the specific
activity of Na+,K+-ATPase in a variety of target
tissues (Edelman, 1974; Asano et al., 1976; Resh et
al., 1980; Kaji et al., 1981). The increased specific
activity of Na+,K+-ATPase induced by mineralocorticoids in certain target tissues appears to be secondary to stimulation of Na+ entry into the cell via an
increased number of Na+ channels (Marver, 1984).
Analogously, it is possible that insulin, glucocorticoids, and thyroid hormone stimulate Na+,K+ATPase activity secondary to increased Na+ entry
via Na + -H + exchange.
In addition, at least one hormone has been described that inhibits Na + -H + exchange activity.
Renal membrane vesicles isolated from parathyroidectomized dogs have enhanced Na + -H + exchange (Cohn et al., 1983), and parathyroid hormone inhibits Na + -H + exchange and reduces pHi in
cultured renal cells (Pollock et al., 1984). Inhibition
of Na + -H + exchange may explain the ability of
parathyroid hormone to reduce the rate of proximal
tubular acidification (lino and Burg, 1979). It is not
known whether alterations in Na + -H + exchange and
pHi result from the action of parathyroid hormone
in other target tissues such as bone.
Finally, it has been reported that angjotensin peptides stimulate amiloride-sensitive Na+ influx in vascular smooth muscle cells (Smith and Brock, 1983).
The possibility that this may represent an activation
of plasma membrane Na + -H + exchange has not been
examined.
Cell Volume Regulation
In the steady state, most cells appear to maintain
their cell volume through primary active extrusion
+
781
+
+
of Na via the Na ,K -ATPase system (MacKnight
and Leaf, 1977). However, when cells are exposed
to anisotonic media, ouabain- insensitive ion flux
pathways are activated, which restore cell volume
to original size within minutes (Kregenow, 1981;
Spring and Ericson, 1982). Based on specific ionic
requirements and inhibitor sensitivity, two types of
electroneutral transport mechanisms have been proposed to mediate the regulatory volume increase
that occurs after cell shrinkage in hyperosmotic media: (1) a Na+-K+-2C1~ cotransport system that is
inhibited by loop diuretics such as furosemide (Kregenow, 1981); (2) an amiloride-sensitive Na+-H+
antiport system, which when activated in parallel
with C1~-HCO3~ antiport, results in net cellular gain
of Na+, Cl~, and H2O. Activation of Na + -H + exchange by cell shrinkage has been described in
salamander and dog erythrocytes, and Necturus gallbladder (Cala, 1980; Ericson and Spring, 1982; Parker, 1983). For example, after Amphiuma erythrocytes shrink in hypertonic media, cell re-expansion
occurs within 3 hours with a dramatic increase in
Na+ influx that is amiloride-sensitive, and accompanied by an increase in intracellular pH and a
decrease in medium pH (Cala, 1980). Similar volume-responsive sodium and proton movements are
observed in dog erythrocytes (Parker, 1983) and in
human lymphocytes (Grinstein et al., 1983). The
process of cell volume regulation by gallbladder
epithelial cells in hypertonic media appears to be
much the same, although actual H+ fluxes have not
been measured (Ericson and Spring, 1982).
What actually activates Na -H+ exchange at the
initiation of regulatory volume increase is an open
question. Evidence has been presented that cell
shrinkage per se is not the trigger (Cala, 1980; Spring
and Ericson, 1982). Alternative proposals have included a shrinkage-associated fall in intracellular pH
(Cala, 1980) and/or a change in intracellular Ca++
(Cala, 1983). However, affirmative data for either
one of these mechanisms are lacking, and measurements in hypertonically shrunken lymphocytes have
failed to show any significant shift in pH| or in free
cytoplasmic Ca++ (Grinstein et al., 1983).
Transepithelial Ion Transport
Epithelial cells mediate the net transport of solutes
and water from one extracellular compartment to
another, and thereby play a determining role in the
ionic composition and volume of extracellular fluids.
Although the involvement of Na + -H + exchange in
transepithelial fluxes of Na+ and H+ would seem
most obvious, this transport system also indirectly
but importantly contributes to the transport of Cl~
and organic anions in several epithelia.
Studies both in isolated membranes vesicles
(Murer et al., 1976; Kinsella and Aronson, 1980) and
in isolated perfused tubules (Boron and Boulpaep,
1983) have clearly demonstrated the presence of
Na + -H + exchange across the luminal membrane of
Circulation Research/Vol. 56, No. 6, June 1985
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the renal proximal tubular cell. In the amphibian
(Boron and Boulpaep, 1983), but not the mammalian
proximal tubule (Ives et al., 1983b; Sabolic and
Burckhardt, 1983), Na + -H + exchange also occurs in
the basolateral membrane. Along the mammalian
proximal tubule, over 90% of the filtered bicarbonate load is reabsorbed and a lumen-acid transrubular
pH gradient is generated. The role of luminal membrane Na + -H + exchange in facilitating the process
of H+ secretion and HCC>3~ absorption along the
mammalian proximal tubule is illustrated in Figure
3A. The 'primary active* transport of Na+ across the
basolateral membrane via the Na,K-ATPase generates an inward electrochemical gradient for Na+
across the luminal membrane. This Na+ gradient
then serves as the driving force for secondary active
H + secretion via the Na -H+ exchanger. Normally,
luminal HCO3~ acts as the principal acceptor for
secreted H+, with its eventual disappearance after
conversion of H2CO3 to CO2 and H2O, a reaction
catalyzed by carbonic anhydrase. The intracellular
OH~ generated by H+ secretion combines with CO2
to form intracellular HCO3~, another reaction catalyzed by carbonic anhydrase. Thus, the net effect of
active H+ secretion by the Na + -H + exchanger is to
cause the intracellular accumulation of HCO3~
against its electrochemical gradient. Hence, there is
a large passive driving force for the exit of HCO3~
across the basolateral membrane of the cell. As
recently reviewed elsewhere, current evidence suggests that at least 80% of active H+ secretion and
HCO3~ reabsorption in the proximal tubule takes
place via the luminal membrane Na + -H + exchanger
(Aronson, 1983).
Lumen
Ctfl
HjO
FIGURE 3. Role of the Na+-H+ exchanger in facilitating active
transport of HCO3" (part A), Cl~ (part B), and organic anions (parts
C and D). See text for details. Abbreviations: c.a., carbonic anhydrase.
The presence of luminal membrane Na + -H + exchange has also been clearly demonstrated in the
small intestine (Murer et al., 1976; Gunther and
Wright, 1983; Knichelbein et al., 1983) and gall
bladder (Weinman and Reuss, 1982). There is acidification of the luminal fluid in both the jejunum
(Turnberg et al., 1970) and gall bladder (Whitlock
and Wheeler, 1969). Thus, the model shown in
Figure 3A is probably applicable to the process of
H secretion in these epithelia as well.
In other epithelia, such as pancreas (Swanson and
Solomon, 1975) and choroid plexus (Wright, 1977),
there is net secretion of HCO3~. In these cases, there
is probably Na + -H + exchange across the basolateral
membrane of the cell and downhill exit of HCO3~
across the luminal membrane.
When a C1~-OH~ (or HCO3") exchange system is
present, Na + -H + exchange can promote active
transepithelial transport of Cl~. For example, Figure
3B illustrates the case in which both Na + -H + and
C1~-HCO3~ exchangers are present on the luminal
membrane of an epithelial cell. In this scheme, secondary active H + secretion via Na + -H + exchange is
driven by the luminal membrane Na+ gradient and
serves to maintain intracellular OH~ above the concentration at which it would be in electrochemical
equilibrium across the luminal membrane. In the
presence of CO2, intracellular HCO3~ is generated
from OH~, a reaction accelerated by carbonic anhydrase. The resulting electrochemical HCO3~ gradient from cell to lumen then drives Cl~ uptake
across the luminal membrane by Cr-HCO 3 ~ exchange. Such tertiary active Cl~ accumulation elevates the intracellular Cl~ concentration above equilibrium so that there is a favorable driving force for
passive Cl~ exit across the basolateral membrane.
The simultaneous presence of Na + -H + and C1~-OH~
exchangers on the same membrane was first shown
by liedtke and Hopfer (1982) using luminal membrane vesicles from rat small intestine. Na + -H + and
C1~-OH~ (or HCO3~) exchangers have subsequently
been demonstrated in luminal membrane vesicles
isolated from Necturus proximal tubule (Seifter and
Aronson, 1984) and from rabbit ileum (Knickelbein
et al., 1983), but not from rabbit or rat jejunum
(Gunther and Wright, 1983; Cassano et al., 1984).
Stimulation of Cl~ uptake by H + gradients has been
observed in luminal membrane vesicles isolated
from the rabbit proximal tubule, a finding interpreted to indicate the presence of CP-OH" exchange
in this preparation (Warnock and Yee, 1981). However, when adequate precautions are taken to avoid
electrical coupling of Cl~ and H+ fluxes through
independent conductive pathways, actual C1~-OH~
exchange cannot be documented in rabbit or rat
renal microvillus membranes (Seifter et al., 1984;
Sabolic and Burckhardt, 1983). Similarly, studies on
the intact proximal tubule of the rabbit have failed
to demonstrate measurable C1~-OH~ exchange
across the luminal membrane (Schwartz, 1983; Sasaki et al., 1984). Experiments employing ion-spe-
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cific microelectrodes have documented the simultaneous presence of Na + -H + and CP-OH" (or HCO3~)
exchangers in the apical membrane of the Necturus
gall bladder (Reuss 1984), but there is dispute as to
whether these exchangers contribute the major fraction of NaCl entry under basal conditions (Reuss
1984) or only during cell volume regulation (Ericson
and Spring, 1982). Simultaneous Na + -H + and Cl~HCO3~ exchange probably also occurs across the
apical membrane of the mouse renal cortical thick
ascending limb of Henle, although this has not yet
been directly demonstrated (Friedman and Andreoli,
1982).
Clearly, double ion exchange is a commonly
found but not ubiquitous mechanism for mediating
Na+-coupled Cl~ transport in diverse epithelia. It is
interesting to note that in many of the epithelia in
which this mechanism is found, such as the gall
bladder, ileum, and renal cortical thick ascending
limb, Na+-coupled Cl~ transport is stimulated by
CO2 and is sensitive to inhibitors of carbonic anhydrase (Fisher et al., 1981; Heintze et al., 1981; Nellans et al., 1975; Friedman and Andreoli, 1982).
These characteristics probably arise from the fact
that at physiological pH values, Cl~ exchanges with
HCO3~ much more rapidly than with OH~ (Seifter
and Aronson, 1984; Knicklebein et al., 1984). Thus,
the generation of HCOs" from intracellular OH~
(Fig. 3B) may, under certain conditions, be ratelimiting for Na+-coupled Cl~ transport occurring by
the parallel operation of Na + -H + and CP-HCOr
exchangers.
There are at least two mechanisms whereby Na++
H exchange can promote the transepithelial transport of organic anions. First, the processes of active
H+ secretion and HCO3~ absorption resulting from
luminal membrane Na + -H + exchange can drive the
net absorption of lipid-soluble weak acids that cross
cell membranes predominantly by nonionic diffusion (Fig. 3C). This mechanism probably accounts
for the absorption of organic anions such as glycodiazine, butyrate, propionate, and acetate in the
mammalian proximal tubule (Ullrich et al., 1971).
Similar mechanisms probably account for absorption of lipid-soluble weak acids in the jejunum (Jackson and Morgan, 1975) and for their secretion in the
choroid plexus (Wright, 1977). A second mechanism
for organic anion transport indirectly coupled to
Na + -H + exchange is shown in Figure 3D. In this
case, the transport of organic anion across the luminal membrane occurs by a carrier-mediated process of OH~-anion exchange or H+-anion cotransport.
Such a mechanism probably contributes at least in
part to urate reabsorpu'on in the mammalian proximal tubule (Blomstedt and Aronson, 1980).
Pathophysiological Roles
To be sure, a clear-cut role for Na + -H + exchange
in any specific pathophysiological state is yet to be
proved with certainty. Nevertheless, in view of the
783
several important physiological roles of the plasma
membrane Na+-H exchanger just discussed, it requires only a small leap of imagination to invoke a
role for this transport system in various pathological
states. A few examples will be described briefly.
Renal Acid-Base Disorders
As mentioned earlier, Na + -H + exchange appears
to be the principal mechanism for mediating H+
secretion and HCO3~ reabsorption in the proximal
tubule. Factors affecting the number or activity of
Na+-H+ exchangers in the proximal tubule may
therefore influence the efficiency of reclamation of
filtered bicarbonate, and may thereby contribute to
determining the steady state concentration of
plasma bicarbonate. For example, the elevated renal
threshold for bicarbonate excretion during hypercapnia may result from the fact that elevating Pcc>2
lowers intracellular pH (Struyvenberg et al., 1968),
an effect leading to allosteric stimulation of the
luminal membrane Na + -H + exchanger by intracellular H + (Aronson et al., 1982). Another example is
the increased number or activity of Na + -H + exchangers in the luminal membranes of proximal tubular
cells during potassium depletion (Seifter and Harris,
1984), an effect that may contribute to the maintenance of metabolic alkalosis in this disorder. Addi
tional examples include the mild metabolic acidosis
of hyperparathyroidism and the metabolic alkalosis
of hyperglucocorticoid states. These two disorders
may at least in part reflect the effects of parathyroid
hormone and glucocorticoids to inhibit and stimulate, respectively, the number or activity of Na + -H +
exchangers in the luminal membrane of proximal
tubular cells (Freiberg et al., 1982; Cohn et al., 1983).
Essential Hypertension
As recently reviewed elsewhere (Haddy, 1983;
Parker and Berkowitz, 1983; Blaustein, 1984), several different alterations of transmembrane Na+
transport in patients with hypertension have been
described. One of the more consistent and reproducible findings has been an increased activity of
the red cell transport system mediating Na + -Ii +
exchange in certain subgroups of patients with essential hypertension and their first degree relatives
(see Smith et al., 1984, for a comprehensive compilation of these data). Under physiological conditions
in the absence of Li+, the red cell Na -Ii + exchanger
mediates 1:1 Na+-Na+ exchange and, thus, has no
impact on intracellular electrolyte content (Duhm
and Becker, 1979). The role of abnormal Na + -Ii +
countertransport in the pathogenesis of hypertension has therefore been obscure.
One possibility is that the transport system assayed for its Na + -Ii + exchange activity in red cells
is really a Na + -H + exchanger. As discussed above,
the plasma membrane Na -H+ exchanger has several features in common with the pathway mediating Na+-Li+ exchange in red cells, and Na + -H + exchange has been demonstrated in human red cells
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(Dissing and Hoffman, 1982).
Several mechanisms have been suggested by
which increased activity of the plasma membrane
Na + -H + exchanger in cells other than erythrocytes
could play a pathophysiological role in essential
hypertension (Aronson, 1982; Funder et al., 1984).
First, increased Na + -H + exchange across the luminal
membrane of cells in the proximal tubule or cortical
thick ascending limb of Henle would tend to enhance Na+ reabsorption and thereby cause a defect
in renal salt excretion. By analogy with other abnormalities that tend to impair renal Na+ excretion,
such as chronic renal insufficiency or hyperaldosteronism, a defect of this type would require that the
kidney be perfused at greater than normal arterial
pressure in order to maintain salt balance (Guyton
et al., 1974). In addition, the secondary release of
natriuretic, digitalis-like, humoral factors could lead
to an increase in peripheral vascular resistance
(Hamlyn et al., 1982; Poston et al., 1982). In the
steady state condition with sustained hypertension,
'escape' from the primary defect in salt excretion
would prevent a detectable increase in extracellular
fluid volume. In addition, the expected tendency of
augmented Na + -H + exchange to generate metabolic
alkalosis would be offset by inhibition of distal
nephron H + secretion, which occurs by an ATPdriven H+ pump, rather than by Na + -H + exchange
(Koeppen and Steinmetz, 1983). There is considerable evidence that a defect in renal salt excretion is
a cause of essential hypertension in at least some
patients (Haddy, 1980; de Wardener and MacGregor, 1982).
Increased activity of the plasma membrane Na++
H exchanger in vascular smooth muscle cells could
alternatively play a pathophysiological role in essential hypertension. Blaustein (1977, 1984) has suggested that Na+-Ca++ exchange across the plasma
membrane is an important determinant of the steady
state cytosolic concentration of Ca++ in vascular
smooth muscle cells, and thus of steady state vessel
wall tension. According to this view, any factor that
elevates intracellular Na+ will tend to elevate intracellular Ca++ and increase vascular tone. Thus, increased activity of the Na + -H + exchanger in vascular
smooth muscle could cause an elevated peripheral
resistance by virtue of a rise in intracellular Na+. In
addition, it is likely that vascular tone is sensitive to
changes in intracellular pH. For example, in a variety
of vascular beds, hypercapnia induces vasodilation,
probably secondary to a fall in intracellular pH
(Shepherd, 1983). Thus, increased activity of the
Na + -H + exchanger in vascular smooth muscle might
also lead to an elevated peripheral resistance by
virtue of a rise in intracellular pH.
Cancer
As outlined previously, activation of plasma membrane Na+-H exchange is an early event in the
response of cells to mitogenic growth factors. One
Circulation Research/VoZ. 56, No. 6, June 1985
such mitogenic substance known to activate Na+H+ exchange is platelet-derived growth factor
(PDGF) (Burns and Rozengurt, 198,3; Cassel et al.,
1983). PDGF-like molecules are produced by a number of transformed cell lines, including naturally
occurring human tumors (Bowen-Pope et al., 1984).
Moreover, the oncogene product of the simian sarcoma virus is extensively homologous with PDGF
(Waterfield et al., 1983). Tumor promoters such as
phorbol esters have been recently found to stimulate
plasma membrane Na + -H + exchange (Burns and
Rozengurt, 1983). Thus, it appears likely that activation of plasma membrane Na + -H + exchange plays
an important part in oncogenic transformation.
Activation of plasma membrane Na + -H + exchange could explain the high rates of lactic acid
production by malignant tumors, as first described
by Warburg 60 years ago. Glycolysis is limited by
the availability of inorganic phosphate and adenosine diphosphate (ADP), and thus, by the rate of
breakdown of adenosine triphosphate (ATP) to ADP
and P, by cellular ATPases (Racker, 1983). The increased Na+ influx associated with enhanced Na+H + exchange in transformed cells could therefore
promote glycolysis by stimulating the Na+,K+ATPase, which is highly sensitive to changes in
intracellular Na + concentration (Smith and Rozengurt, 1978). Furthermore, the elevation of intracellular pH resulting from activation of Na + -H + exchange would also stimulate lactic acid production,
inasmuch as the rate-limiting enzyme in glycolysis,
phosphofructokinase, is extremely pH sensitive (Trivedi and Danforth, 1966).
Organ Growth and Hypertrophy
Another implication of the important role of Na+H+ exchange in the response of cells to mitogenic
stimuli is the possible involvement of this transport
system in nonmalignant forms of cell growth and
proliferation. For example, after loss of renal mass,
the remaining renal tissue undergoes compensatory
growth (Hayslett, 1979). Recent studies indicate that
Na + -H + exchange activity is enhanced in plasma
membrane vesicles prepared from remnant renal
tissue after renal ablation in the dog or rat (Cohn et
al., 1982; Harris et al., 1984). Whether stimulation
of Na + -H + exchange is a general feature of such
other examples of tissue growth and proliferation as
cardiac or vascular smooth muscle hypertrophy remains to be examined. The possible effect of angiotensins to stimulate Na + -H + exchange as noted
earlier is of interest in this regard.
Conclusions
The plasma membrane Na + -H + exchanger is a
ubiquitous transport system that participates in diverse cell functions involving extrusion of H+ from
the cell or uptake of Na+. Intracellular pH is probably the most important physiological factor regulating the Na + -H + exchanger, a property that en-
Mahnensmith and Aronson/Plasma Membrane Na+-H+ Exchanger
Downloaded from http://circres.ahajournals.org/ by guest on July 31, 2017
ables the antiporter to play a critical role in the
regulation of intracellular pH. As a consequence of
this role, the Na + -H + exchanger functions as an
important signal transducer for a broad spectrum of
stimuli that alter cell function through changes in
intracellular pH. As a transporter of Na+, the antiporter participates in the regulation of cell volume.
The plasma membrane Na + -H + exchanger helps
mediate the transport of not only Na+ and H+, but
also Cl~ and organic anions across epithelia. The
Na + -H + exchanger thus plays a major role in determining the volume and ionic composition of the
extracellular fluid and of various epithelial secretions.
Given the diversity of its physiological functions,
it would not be surprising if the Na + -H + exchanger
participated in pathophysiological processes, as well.
Very preliminary evidence raises the possibility that
this may indeed be the case. Disorders of whole
body acid-base balance have traditionally been considered to fall within the domain of nephrology.
Clearly, the study of acid-base homeostasis at the
cellular level is of great relevance to all disciplines.
It is not altogether unreasonable to suggest that
pharmacological manipulation of plasma membrane
Na + -H + exchange activity may eventually find clinical use in the treatment of a spectrum of diseases
ranging from cancer to hypertension.
The excellent secretarial assistance of Carol Robinson is gratefully
acknowledged. Critical review of the manuscript by F. Gregory Grillo
and Peter Igarashi is also appreciated.
Support was provided by U.S. Public Health Service Grants AM17433 and AM-33793, and an Established Investigatorship from the
American Heart Association (P.S.A.).
Address for reprints: Peter S. Aronson, M.D., Department of Physiology, Yale University School of Medicine, 333 Cedar Street, New
Haven, Connecticut 06510.
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INDEX TERMS: Acid-base transport • Bicarbonate transport •
Cell growth • Cell volume regulation • pH homeostasis • Stimulus-response coupling
The plasma membrane sodium-hydrogen exchanger and its role in physiological and
pathophysiological processes.
R L Mahnensmith and P S Aronson
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Circ Res. 1985;56:773-788
doi: 10.1161/01.RES.56.6.773
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