Interaction of external H+ with the Na+-H+ exchanger in renal microvillus membrane vesicles

We examined the effects of external H+ on the kinetics of Na+-H+ exchange in microvillus membrane vesicles isolated from the rabbit renal cortex. The initial rate of Na+ influx into vesicles with internal pH 6.0 was optimal at external pH 8.5 and was progressively inhibited as external pH was reduced to 6.0. A plot of 1/V versus [H+]o was linear and yielded apparent KH = 35 nM (apparent pK 7.5). In vesicles with internal pH 6.0 studied at external pH 7.5 or 6.6, apparent KNa was 13 or 54 mM, Ki for inhibition of Na+ influx by external Li+ was 1.2 or 5.2 mM, Ki for inhibition by external NH4+ was 11 or 50 mM, and Ki for inhibition by external amiloride was 7 or 25 microM, respectively. These findings were consistent with competition between each cation and H+ at a site with apparent pK 7.3-7.5. Lastly, stimulation of 22Na efflux by external Na+ (i.e. Na+-Na+ exchange) was inhibited as external pH was reduced from 7.5 to 6.0, also consistent with competition between external H+ and external Na+. Thus, in contrast with internal H+, which interacts at both transport and activator sites, external H+ interacts with the renal microvillus membrane Na+-H+ exchanger at a single site, namely the external transport site, where H+, Na+, Li+, NH4+, and amiloride all compete for binding.     

Mixed type inhibition of the renal Na+/H+ antiporter by Li+ and amiloride. Evidence for a modifier site

We studied the interactions of Na+, Li+, and amiloride on the Na+/H+ antiporter in brush-border membrane vesicles from rabbit renal cortex. Cation-mediated collapse of an outwardly directed proton gradient (pHin = 6.0; pHout = 7.5) was monitored with the fluorescent amine, acridine orange. Proton efflux resulting from external addition of Na+ or Li+ exhibited simple saturation kinetics with Hill coefficients of 1.0. However, kinetic parameters for Na+ and Li+ differed (Km for Li+ = 1.2 +/- 0.1 mM; Km for Na+ = 14.3 +/- 0.8 mM; Vmax for Li+ = 2.40 +/- 0.07 fluorescence units/s/mg of protein; Vmax for Na+ = 7.10 +/- 0.24 fluorescence units/s/mg of protein). Inhibition of Na+/H+ exchange by Li+ and amiloride was also studied. Li+ inhibited the Na+/H+ antiporter by two mechanisms. Na+ and Li+ competed with each other at the cation transport site. However, when [Na+] was markedly higher than [Li+], [( Na+] = 90 mM; [Li+] less than 1 mM), we observed noncompetitive inhibition (Vmax for Na+/H+ exchange reduced by 25%). The apparent Ki for this noncompetitive inhibition was congruent to 50 microM. In addition, 2-30 mM intravesicular Li+, but not Na+, resulted in trans inhibition of Na+/H+ exchange. Amiloride was a mixed inhibitor of Na+/H+ exchange (Ki = 30 microM, Ki' = 90 microM) but was only a simple competitive inhibitor of Li+/H+ exchange (Ki = 10 microM). At [Li] = 1 mM and [amiloride] less than 100 microM, inhibition of Na+/H+ exchange by a combination of the two inhibitors was always less than additive. These results suggest the presence of a cation-binding site (separate from the cation-transport site) which could be a modifier site of the Na+/H+ antiporter.     

High affinity binding of amiloride analogs at an internal site in renal microvillus membrane vesicles.

Amiloride analogs with hydrophobic substitutions on the 5-amino nitrogen atom are relatively high affinity inhibitors of the plasma membrane Na(+)-H+ exchanger. We demonstrated that a high affinity-binding site for [3H]5-(N-methyl-N-isobutyl)amiloride ([3H]MIA) (Kd = 6.3 nM, Bmax = 1.2 pmol/mg of protein) is present in microvillus membrane vesicles but not in basolateral membrane vesicles isolated from rabbit renal cortex, in accord with the known membrane localization of the Na(+)-H+ exchanger in this tissue. The rank order potency for inhibition of microvillus membrane [3H]MIA binding by amiloride analogs was: MIA (I50 approximately 10 nM) greater than amiloride (I50 approximately 200 nM) greater than benzamil (I50 approximately 1200 nM). This correlated with a qualitatively similar rank order potency for inhibition of Na(+)-H+ exchange: MIA (I50 approximately 4 microM) greater than amiloride (I50 approximately 15 microM) greater than benzamil (I50 approximately 100 microM), but did not correlate with the rank order potency for inhibition of the organic cation-H+ exchanger in microvillus membrane vesicles: MIA approximately benzamil (I50 approximately 0.5 microM) greater than amiloride (I50 approximately 10 microM). However, tetraphenylammonium, an inhibitor of organic cation-H+ exchange, inhibited the rate of [3H]MIA binding without an effect on equilibrium [3H]MIA binding; the dissociation of bound [3H]MIA was inhibited by preloading the membrane vesicles with tetraphenylammonium. These findings indicated that high affinity [3H]MIA binding to renal microvillus membrane vesicles takes place at an internal site to which access is rate-limited by the tetraphenylammonium-sensitive organic cation transporter. Equilibrium [3H]MIA binding was inhibited by H+ but was unaffected by concentrations of Na+ or Li+ that saturate the external transport site of the Na(+)-H+ exchanger. Binding of MIA to its high affinity binding site had no effect on the rate of Na(+)-H+ exchange. This study suggests that the renal Na(+)-H+ exchanger has a high affinity internal binding site for amiloride analogs that is distinct from the external amiloride inhibitory site.     

Inactivation of the renal microvillus membrane Na+-H+ exchanger by histidine-specific reagents

We examined the effect of histidine-specific reagents on the transport activity of the Na+-H+ exchanger in microvillus (brush-border) membrane vesicles isolated from the rabbit renal cortex. Rose bengal-catalyzed photo-oxidation caused irreversible inhibition of the rate of Na+-H+ exchange but also caused significant loss of vesicle integrity. Treatment of the membrane vesicles with diethylpyrocarbonate caused inactivation of Na+-H+ exchange that could not be attributed to vesicle disruption or collapse of transmembrane H+ gradients. Inactivation of Na+-H+ exchange by diethylpyrocarbonate followed pseudo-first order kinetics to below 10% residual activity, could be reversed by hydroxylamine, was reflected by a decreased Vmax with no change in the Km for Na+, was dependent on external pH but not internal pH, was blocked by amiloride, and was enhanced by Na+. These data are consistent with the hypothesis that a diethylpyrocarbonate-sensitive imidazolium residue is the titratable group found in kinetic studies to bind H+ at the external transport site of the Na+-H+ exchanger.     

3H]Ethylpropylamiloride, a ligand to analyze the properties of the Na+/H+ exchange system in the membranes of normal and hypertrophied kidneys

[3H]Ethylpropylamiloride is a useful radioactive label to identify the Na+/H+ exchange system (Vigne, P., Frelin, C., Audinot, M., Borsotto, M., Cragoe, E. J., and Lazdunski, M. (1984) EMBO J. 3, 2647-2651). This paper extends the analysis of the properties of interaction of [3H]ethylpropylamiloride with the exchanger and describes its use with hypertrophied kidneys. [3H]Ethylpropylamiloride-binding sites copurify with the luminal membrane marker alkaline phosphatase but not with the basolateral membrane marker (Na+,K+)ATPase, thus indicating an asymmetric distribution of the Na+/H+ exchanger. Specific [3H]ethylpropylamiloride binding is dependent on pH. The pH dependency indicates that an ionizable function with a pKapp of 7.0 is essential in the association of the amiloride derivative. H+ acts competitively on [3H]ethylpropylamiloride binding; Na+, Li+, or cholinium ions have no effect on the association. Compensatory adaptation of the kidney to chronic reduction of renal mass is accompanied by a 1.7-fold increase in the activity of the Na+/H+ exchange system. Properties of interaction of internal and external pH with the Na+/H+ exchanger of normal and hypertrophied kidneys are identical. Titration of [3H]ethylpropylamiloride-binding sites in normal and hypertrophied kidneys suggests that the increased activity of the Na+/H+ exchange system is not accompanied by an increased concentration of exchangers.     

Two uncompetitive, activated, and transport sites of the Na+/H+ exchanger for pH regulation in perfused rat kidney

The purpose of this study is to assess the effect of an apparent alteration in intracellular pH and the effect of amiloride on the activity of the Na+/H+ antiporter in perfused rat kidney. Rat kidney-Na+ retention was determined using tracer 22Na in perfusate composed of HCl-glycine buffer (pH 3.80 to pH 5.92) or NH4OH-glycine buffer (pH 6.22-7.95) containing Na+ to match physiologic concentrations. Plotting renal Na+ retention for 10 min versus pH in absence of amiloride showed two classical uncompetitive activator curves for H+, one curve from pH 4.19 to 5.10 and another from pH 6.22 to 7.95. H+ acts as an uncompetitive reversible binding substrate with the receptor triggering activation of the exchanger already sequestered with Na+, thus yielding two Ka values for the exchanger suggesting non-first order kinetics. Using an equation derived for uncompetitive-activation binding of Nao+ and Hi+, plotting [mM Na+ mg protein-1 10 min-1]-1 versus [H+], two linear plots are observed on Cartesian coordinates with abscissa intersecting at 47 +/- 1 microM, pKa = 4.32 +/- 0.02 (pH 4.19-5.10) and 4.21 +/- 0.02 microM, pKa = 5.38 +/- 0.01 (pH 6.22-7.95), respectively. Perfusing buffer containing 2 mM amiloride, completely inactivated the antiporter showing stronger inhibition between pH 3.80 and 5.92. Results suggest the presence of two uncompetitive binding sites for H+ with the Na+/H+ exchanger. One is a high affinity binding site at physiological intracellular apparent pH, and another is a low affinity binding site at ischaemic apparent pH, implying the existence of two titration sites for intracellular pH regulation.     

Estimation of the number and turnover rate of Na+/H+ exchangers in lymphocytes. Effect of phorbol ester and osmotic shrinking

Rat thymic lymphocytes possess an amiloride-sensitive Na+/H+ exchanger in their plasma membrane. Kinetic studies revealed that 5-(N-methyl-N-isobutyl)amiloride (MIA) was a more potent inhibitor of the antiport than amiloride (cf. apparent Ki of 174 nM and 6 microM, respectively). Inhibition by MIA was rapid (less than 5 s) and readily reversible. [3H]MIA binding to whole cells was assayed by rapid centrifugation following short (5 s) incubations to minimize nonspecific binding. A saturable binding component (Kd approximately equal to 170 nM) which was displaced by amiloride was detected. In contrast, there was no significant amiloride-displaceable binding to human erythrocytes, which have comparatively little Na+/H+ exchange activity. In lymphocytes, the ability of amiloride and several of its analogs to displace [3H]MIA correlated with their potency as inhibitors of the antiport. Both kinetic and binding studies revealed that extracellular H+, but not Na+, inhibited the interaction of MIA with its receptor(s). Taken together, these data suggest that [3H]MIA binds to the Na+/H+ exchanger. Scatchard analysis revealed that [3H]MIA bound to a maximum of 8000 high affinity sites/cell. Activation of Na+/H+ exchange by osmotic shrinking or by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate was not accompanied by a significant change in [3H]MIA binding. Given an upper limit of 8000 functional sites/thymocyte, we estimate that the turnover number of each maximally activated exchanger is at least 2000 cycles/s.     

Inhibition and labeling of the rat renal Na+/H+-exchanger by an antagonist of muscarinic acetylcholine receptors

A covalently binding label for muscarinic acetylcholine receptors, propylbenzilylcholine mustard (PrBCM), irreversibly inhibits the Na+/H+ exchanger in rat renal brush-border membrane vesicles. Substrates of the antiporter, Na+ and Li+, as well as inhibitors, amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and propranolol, protect the antiporter from inactivation by PrBCM. With [3H]PrBCM a band with an app. Mr of 65 kDa is predominantly labeled. Amiloride protects this band from labeling with [3H]PrBCM and [14C]-N,N'-dicyclohexylcarbodiimide (DCCD) proving its identity with the renal Na+/H+ exchanger. Our data reveal a specific interaction of PrBCM with the Na+/H+ exchanger and suggest structural relations between antiporter and receptors.     

Ontogeny of the Na(+)-H+ exchanger in rat ileal basolateral membrane vesicles.

This study was designed to examine the activity of the Na(+)-H+ exchanger across the basolateral membranes of the ileal enterocyte and its developmental pattern. The function of the Na(+)-H+ exchanger was studied using a well validated basolateral membrane vesicle technique. Na+ uptake represented transport into the vesicle rather than binding as validated by initial rate studies. Na+ uptake represented an electroneutral process as shown by studies in which negative membrane potential was induced by the ionophore valinomycin. Various outwardly directed pH gradients significantly stimulated Na+ uptake compared with no pH gradient conditions at all age groups. However, the magnitude of stimulation was significantly different between the age groups with more marked stimulation of amiloride-sensitive Na+ uptake occurring in adolescent rats as compared to weanling or suckling rats. The amiloride sensitivity of the pH stimulated Na+ uptake was investigated using [Amiloride] = 10(-2)-10(-5) M at pHi/pHo = 5.2/7.5. At 10(-2) M amiloride concentration, Na+ uptake was inhibited by 80%, 70%, 77%, in the basolateral membranes of adolescent, weanling and suckling rats, respectively. Dixon plot analysis in both adolescent and weanling rats was consistent with two amiloride binding sites, a low affinity system and a high affinity system. In the suckling rat, on the other hand, the data supported a single high affinity binding site. Kinetic studies revealed a Km for amiloride-sensitive Na+ uptake of 12.6 +/- 6.6, 10.2 +/- 1.77, 9.46 and Vmax of 4.83 +/- 1.22, 4.47 +/- 0.36 and 8.08 +/- 1.92 n.mol.mg.protein-1.7 s-1 in suckling, weanling and adolescent rats, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of Na+-H+ exchange by N,N'-dicyclohexylcarbodiimide in isolated rat renal brush border membrane vesicles

The inactivation of rat renal brush border membrane Na+-H+ exchange by the covalent carboxylate reagent N,N'-dicyclohexylcarbodiimide (DCCD) was studied by measuring 1 mM Na+ influx in the presence of a pH gradient (pHi = 5.5; pHo = 7.5) and H+ influx in the presence of a Na+ or Li+ gradient ([Na+]i = 150 mM; [Na+]o = 1.5 mM). In the presence of DCCD, the rate of Na+ uptake decreased exponentially with time and transport inhibition was irreversible. At all DCCD concentrations the loss of activity was described by a single exponential, consistent with one critical DCCD-reactive residue within the Na+-H+ exchanger. Among several carbodiimides the most hydrophobic carbodiimide, DCCD, was also the most effective inhibitor of Na+-H+ exchange. With 40 nmol of DCCD/mg of protein, at 20 degrees C for 30 min, 75% of the amiloride-sensitive 1 mM Na+ uptake was inhibited. Neither the equilibrium Na+ content nor the amiloride-insensitive Na+ uptake was significantly altered by the treatment. The Na+-dependent H+ flux, measured by the change in acridine orange absorbance, was also decreased 80% by the same DCCD treatment. If 150 mM NaCl, 150 mM LiCl, or 1 mM amiloride was present during incubation of the brush border membranes with 40 nmol of DCCD/mg of protein, then Li+-dependent H+ flux was protected 50, 100, or 100%, respectively, compared to membranes treated with DCCD in the absence of Na+-H+ exchanger substrates. The combination of DCCD and an exogenous nucleophile, e.g. ethylenediamine and glycine methyl ester, increased Na+-dependent H+ flux in the presence of 80 nmol of DCCD/mg of protein, compared to the transport after DCCD treatment alone. These findings suggest that the Na+-H+ exchanger contains a single carboxylate residue in a hydrophobic region of the protein, and the carboxylate and/or a nearby endogenous nucleophilic group is critical for exchange activity.     

Evidence for histidyl and carboxy groups at the active site of the human placental Na+-H+ exchanger.

The Na+-H+ exchanger of the human placental brush-border membrane was inhibited by pretreatment of the membrane vesicles with a histidyl-group-specific reagent, diethyl pyrocarbonate and with a carboxy-group-specific reagent, N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline. In both cases the inhibition was irreversible and non-competitive in nature. But, if the membrane vesicles were treated with these reagents in the presence of amiloride, cimetidine or clonidine, there was no inhibition. Since amiloride, cimetidine and clonidine all interact with the active site of the exchanger in a mutually exclusive manner, the findings provide evidence for the presence of essential histidyl and carboxy groups at or near the active site of the human placental Na+-H+ exchanger. This conclusion was further substantiated by the findings that Rose Bengal-catalysed photo-oxidation of histidine residues as well as covalent modification of carboxy residues with NN'-dicyclohexylcarbodi-imide irreversibly inhibited the Na+-H+ exchanger and that amiloride protected the exchanger from inhibition caused by NN'-dicyclohexylcarbodi-imide.     

Kinetic properties of the Na+/H+ antiport of heart mitochondria

The fluorescence of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) has been used to follow the Na+/H+ antiport activity of isolated heart mitochondria as a Na+-dependent extrusion of matrix H+. The antiport activity measured in this way shows a hyperbolic dependence on external Na+ or Li+ concentration when the external pH (pHo) is 7.2 or higher. The apparent Km for Na+ decreases with increasing pHo to a limit of 4.6 mM. The Ki for external H+ as a competitive inhibitor of Na+/H+ antiport averages 3.0 nM (pHo 8.6). The Vmax at 24 degrees C is 160 ng ion of H+ min-1 (mg of protein)-1 and does not vary with pHo. Li+ reacts with the antiporter with higher affinity, but much lower Vmax, and is a competitive inhibitor of Na+/H+ antiport. The rate of Na+/H+ antiport is optimal when the pHi is near 7.2. When pHo is maintained constant, Na+-dependent extrusion of matrix H+ shows a hyperbolic dependence on [H+]i with an apparent Km corresponding to a pHi of 6.8. The Na+/H+ antiport is inhibited by benzamil and by 5-N-substituted amiloride analogues with I50 values in the range from 50 to 100 microM. The pH profile for this inhibition seems consistent with the availability of a matrix binding site for the amiloride analogues. The mitochondrial Na+/H+ antiport resembles the antiport found in the plasma membrane of mammalian cells in that Na+, Li+, and external H+ appear to compete for a common external binding site and both exchanges are inhibited by amiloride analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Na+-dependent regulation of the internal pH in chick skeletal muscle cells. The role of the Na+/H+ exchange system and its dependence on internal pH.

The internal pH (pHi) of chick muscle cells is determined by the transmembrane Na+ gradient. Li+, but not K+, Rb+ or Cs+, can substitute for Na+ for regulating the internal pH of chick muscle cells. Pharmacological evidence using amiloride and amiloride analogs has shown that the Na+/H+ exchange system is the membrane mechanism that couples the pHi to the transmembrane Na+ gradient. The pHi dependence of the amiloride-sensitive Na+/H+ exchange mechanism was defined. Internal H+ interacts cooperatively with the Na+/H+ exchange system, in contrast with external H+, thus indicating an asymmetrical behaviour of this exchanger. The half-maximum effect for the activation by the internal H+ of the Na+ transporting activity of the amiloride-sensitive Na+/H+ exchange was observed at pH 7.4. The Hill coefficient of the H+ concentration dependence is higher than 3. Insulin was shown to have no effect on the pHi of chick muscle cells.     

Identification and purification of a renal amiloride-binding protein with properties of the Na+-H+ exchanger

The aim of this study was to identify and purify the Na+-H+ exchanger from rabbit renal brush border membranes by use of affinity chromatography. Triton-solubilized membranes were equilibrated with an affinity matrix consisting of the amiloride analogue A35 (5-N-(3-aminophenyl)amiloride) covalently coupled to Sepharose CL-4B beads through a triglycine spacer arm. The matrix was then washed extensively with buffer and sequentially eluted with buffer, buffer containing 5 mM amiloride, and 1% sodium dodecyl sulfate (SDS). Eluates were concentrated and subjected to SDS-polyacrylamide gel electrophoresis. The silver-stained gel revealed a 25-kDa protein that was not visible in the initial solubilized brush border membrane extract, was not eluted from the affinity matrix by buffer alone, but was eluted with 5 mM amiloride. A subsequent elution with 1% SDS did not release any more of the 25-kDa protein, indicating that it had been completely eluted from the affinity matrix by amiloride. The presence of 5 mM amiloride during equilibration of the solubilized brush border extract with the affinity matrix completely blocked adsorption of the 25-kDa protein. The relative abundance of this protein correlated closely with Na+-H+ exchange activity when preparations of cortical brush border membrane vesicles, outer medullary brush border membrane vesicles, and cortical basolateral membrane vesicles were compared. Moreover, binding of the protein to the affinity matrix was inhibited by amiloride and amiloride analogues with a rank order identical to that for inhibition of Na+-H+ exchange activity. These findings strongly suggest that the 25-kDa protein is a structural component of the Na+-H+ exchanger.     

Characterization of the mitochondrial Na+-H+ exchange. The effect of amiloride analogues

The kinetic properties and inhibitor sensitivity of the Na+-H+ exchange activity present in the inner membrane of rat heart and liver mitochondria were studied. (1) Na+-induced H+ efflux from mitochondria followed Michaelis-Menten kinetics. In heart mitochondria, the Km for Na+ was 24 +/- 4 mM and the Vmax was 4.5 +/- 1.4 nmol H+/mg protein per s (n = 6). Basically similar values were obtained in liver mitochondria (Km = 31 +/- 2 mM, Vmax = 5.3 +/- 0.2 nmol H+/mg protein per s, n = 4). (2) Li+ proved to be a substrate (Km = 5.9 mM, Vmax = 2.3 nmol H+/mg protein per s) and a potent competitive inhibitor with respect to Na+ (Ki approximately 0.7 mM). (3) External H+ inhibited the mitochondrial Na+-H+ exchange competitively. (4) Two benzamil derivatives of amiloride, 5-(N-4-chlorobenzyl)-N-(2',4'-dimethyl)benzamil and 3',5'-bis(trifluoromethyl)benzamil were effective inhibitors of the mitochondrial Na+-H+ exchange (50% inhibition was attained by approx. 60 microM in the presence of 15 mM Na+). (5) Three 5-amino analogues of amiloride, which are very strong Na+-H+ exchange blockers on the plasma membrane, exerted only weak inhibitory activity on the mitochondrial Na+-H+ exchange. (6) The results indicate that the mitochondrial and the plasma membrane antiporters represent distinct molecular entities.     

Cytosolic pH regulation in osteoblasts. Interaction of Na+ and H+ with the extracellular and intracellular faces of the Na+/H+ exchanger

The interaction of Na and H ions with the extracellular and intracellular sites of the Na+/H+ exchanger of the osteosarcoma cell line UMR-106 was investigated. Na ions interact with a single, saturable extracellular transport site. H+ and amiloride appear to compete with Na+ for binding to this site. The apparent affinity for extracellular Na+ (Nao+) and amiloride was independent of intracellular H+ (Hi+), Nai+, or an outwardly directed H+ gradient. The interaction of H+ with the intracellular face of the exchanger had a sigmoidal characteristic with a Hill coefficient of approximately 2. The apparent affinity for Hi+ was independent of Nao+ between 25 and 140 mM. The apparent affinity for Hi+, but not the number of intracellular sites, increased with the increase in the outwardly directed H+ gradient across the membrane. Nai+/Ho+ exchange (reverse mode) is an electroneutral process with a Na+/H+ stoichiometry of 1. The dependence of Nai+/Ho+ exchange on Nai+ was sigmoidal, with a Hill coefficient of 2.16. Nai+ competes with Hi+ for binding to at least the transport site. The apparent affinity for Nai+ decreased with the increase in the outwardly directed H+ gradient. High Ho+ inhibited exchange activity in the reverse mode. We conclude that intracellular Na+ and H+ can activate the exchanger. The exchanger has two separate and asymmetric extracellular and intracellular transport sites. The relative apparent affinities of the internal transport site for Na+ and H+ are determined by the direction and magnitude of the H+ gradient across the membrane. Kinetic characterization of the exchanger suggests that Na+/H+ exchange is compatible with a simultaneous transport model, although a ping-pong transport model could not be excluded.     

Na+-H+ exchange at the apical membrane of Necturus gallbladder. Extracellular and intracellular pH studies

The mechanism of luminal solution acidification was studied in Necturus gallbladder by measurement of mucosal solution and intracellular pH with glass electrodes. When the gallbladder was bathed by a Na-Ringer's solution it acidified the luminal side by a Na+-dependent, amiloride- inhibitable process. In the presence of ouabain, acidification was reduced but could be stimulated to a rate greater than that under control conditions by the imposition of an inwardly directed Na+ gradient. These results suggest that luminal acidification results from Na+-H+ exchange at the apical membrane and not by diffusion of metabolic CO2. Li+ can substitute for Na+ but K+, Rb+, Cs+, and tetramethylammonium (TMA+) cannot. The maximal rate of exchange was about five times greater for Na+ than for Li+. Intracellular pH (pHi) was measured with recessed-tip glass microelectrodes; with the tissue bathed in Na-Ringer's solution (pH 7.75), pHi was 7.51 +/- 0.04. After inhibition of Na+-H+ exchange by mucosal perfusion with amiloride (1 mM) or by complete Na+ replacement with TMA+, phi fell reversibly by 0.15 and 0.22 pH units, respectively. These results support the conclusion that Na+-H+ exchange at the apical membrane is the mechanism of luminal acidification and is involved in the maintenance of steady state pHi.     

Intracellular acidification-induced alkali metal cation/H+ exchange in human neutrophils

Pretreatment of isolated human neutrophils (resting pHi congruent to 7.25 at pHo 7.40) with 30 mM NH4Cl for 30 min leads to an intracellular acidification (pHi congruen to 6.60) when the NH4Cl prepulse is removed. Thereafter, in 140 mM Na+ medium, pHi recovers exponentially with time (initial rate, approximately 0.12 pH/min) to reach the normal resting pHi by approximately 20 min, a process that is accomplished mainly, if not exclusively, though an exchange of internal H+ for external Na+. This Na+/H+ countertransport is stimulated by external Na+ (Km congruent to 21 mM) and by external Li+ (Km congruent to 14 mM), though the maximal transport rate for Na+ is about twice that for Li+. Both Na+ and Li+ compete as substrates for the same translocation sites on the exchange carrier. Other alkali metal cations, such as K+, Rb+, or Cs+, do not promote pHi recovery, owing to an apparent lack of affinity for the carrier. The exchange system is unaffected by ouabain or furosemide, but can be competitively inhibited by the diuretic amiloride (Ki congruent to 8 microM). The influx of Na+ or Li+ is accompanied by an equivalent counter-reflux of H+, indicating a 1:1 stoichiometry for the exchange reaction, a finding consistent with the lack of voltage sensitivity (i.e., electroneutrality) of pHi recovery. These studies indicate that the predominant mechanism in human neutrophils for pHi regulation after intracellular acidification is an amiloride-sensitive alkali metal cation/H+ exchange that shares a number of important features with similar recovery processes in a variety of other mammalian cell types.     

Interaction of guanidinium and guanidinium derivatives with the Na+/H+ exchange system

Guanidinium, a small organic monovalent cation that is permeant through voltage-dependent cationic channels cannot be transported by the cardiac Na+/H+ exchange system. Yet it recognizes the exchanger and is able to block its activity (K0.5 = 30 mM). Guanidinium derivatives that do not belong to the amiloride series and which possess potent antihypertensive properties also block the activity of the Na+/H+ exchange system in various cell types with a greater potency than unsubstituted guanidinium. The most potent compound found, guanochlor, has an affinity for the exchanger ranging between 0.5 microM and 6 microM in different systems and is more potent than amiloride in all systems studied. Guanochlor has the same action as amiloride derivatives on the cardiac cells; it prevents intracellular pH recovery in cardiac cells that have been acidified and also antagonizes the effect of ouabain on 45Ca2+ uptake by chick cardiac cells. Guanochlor does not compete with [3H]ethylpropylamiloride for its binding to the Na+/H+ exchange system of rabbit kidney brush border membrane. It is suggested that guanochlor recognizes a binding site on the Na+/H+ exchanger that is distinct from the amiloride binding site.     

Biochemical characterization of the amiloride-sensitive Na+/H+ antiport in Chinese hamster lung fibroblasts

Net H+ fluxes across the plasma membrane of Chinese hamster lung fibroblasts (CC139) were monitored by pH-stat titration. Na+-depleted cells release H+ upon addition of Na+. Conversely Na+- or Li+-loaded cells take up H+ from the medium when shifted to a Na+,Li+-free medium. This reversible Na+ (or Li+)-dependent H+ flux is inhibited by amiloride and does not occur in digitonin-permeabilized cells. A similar Na+/H+ exchanger was identified in vascular smooth muscle cells, corneal and aortic endothelial cells, lens epithelial cells of bovine origin, and human platelets. Kinetic studies carried out with CC139 cells indicate the following properties: 1) half-saturation of the system is observed at pH = 7.8, in the absence of Na+; 2) external Na+ stimulates H+ release and inhibits H+ uptake in a competitive manner (Ki = 2-3 mM); 3) amiloride is a competitive inhibitor for Na+ (Ki congruent to 1 microM) and a noncompetitive inhibitor for H+; 4) a coupling ratio of 1.3 +/- 0.3 for the H+/Li+ exchange suggests a stoichiometry of 1:1. We conclude that CC139 cells possess in their plasma membrane a reversible, electroneutral, and amiloride-sensitive Na+/H+ antiporter, with two distinct and mutually exclusive binding sites for Na+ and H+. The rapid stimulation of the Na+/H+ antiporter in G0/G1-arrested CC139 cells upon addition of growth factors, together with the fact that intracellular H+ concentration is, under physiological conditions, around the apparent K0.5 of the system, strongly suggests a key role of this antiport in pHi regulation and mitogen action.