Biochemical properties of the Na+/H+ exchange system in rat brain synaptosomes. Interdependence of internal and external pH control of the exchange activity

Properties of the Na+/H+ exchange system in synaptosomes have been studied primarily by using acridine orange fluorescence to follow H+ efflux. Results obtained from 22Na+ uptake experiments and [3H]ethylpropylamiloride binding experiments are also presented for comparison. The basal properties of the Na+/H+ antiport in synaptosomes are similar to those found in other systems; (i) the stoichiometry of Na+/H+ exchange is 1:1; (ii) Li+ can be successfully substituted for Na+; its affinity for the exchanger (KLi+ = 3 mM) is higher than that of Na+ (KNa+ = 12 mM), but the maximal rate of H+ efflux in the presence of Li+ is about 3 times lower than the maximal rate of H+ efflux in the presence of Na+; and (iii) the Na+/H+ antiport is inhibited by amiloride derivatives with the rank order:ethylisopropylamiloride greater than ethylpropylamiloride greater than amiloride greater than benzamil. The most important finding of this paper is that the external pH dependence of the synaptosomal Na+/H+ antiport is controlled by the value of internal pH and vice versa. For example apparent pHo values for half-maximum activation of the Na+/H+ exchanger are pHo = 7.12 when pHi = 6.4 and pHo = 7.95 when pHi = 7.3. Therefore, a 0.9 pH unit increase in internal pH produces a shift of at least a 0.83 pH unit in the external pH dependence. In addition, changing pHo from 7.75 to 8.50 also shifts the half-maximum pHi value for activation of the Na+/H+ antiport from 6.67 to 7.54.     

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.     

[3H]ethylpropylamiloride, a radio-labelled diuretic for the analysis of the Na+/H+ exchange system. Its use with kidney cell membranes.

The interaction of amiloride and several amiloride derivatives with the Na+/H+ exchange system in Madin-Darby canine kidney cells and in rabbit renal microvillus membrane vesicles was studied from 22Na+ uptake experiments. On both types of preparation, the order of potency of the different molecules tested is: ethylisopropylamiloride greater than ethylpropylamiloride (EPA) greater than amiloride greater than benzamil. 3H-labelled EPA was prepared and used to titrate amiloride binding sites in solubilized microvillus membranes. Kinetics experiments, equilibrium binding studies and competition experiments between [3H]EPA and unlabelled EPA indicate that EPA recognizes a single family of binding sites with a Kd value of 45 nM and a maximum binding capacity of 2 pmol/mg of protein. The order of potency of different amiloride analogs tested in [3H]EPA competition experiments is identical to that found for the inhibition of 22Na+ uptake by the Na+/H+ exchange system, suggesting that [3H]EPA binding sites are associated with the Na+/H+ exchange system. [3H]EPA binding sites are pharmacologically distinct from those of [3H]benzamil and [3H]bumetanide in kidney membranes.     

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.     

The role of the Na+/H+ exchange system in the regulation of the internal pH in cultured cardiac cells

The Na+/H+ exchange system is not the major mechanism that regulates the internal pH value (pHi) of chick cardiac cells in culture under normal physiological conditions in the absence of carbonate. In cardiac cells in which the internal pH has been lowered to 6.6-6.7, the Na+/H+ exchanger becomes the major mechanism to bring back pHi to normal values (pHi = 7.3). The blockade of the Na+/H+ exchange activity with an active amiloride derivative, ethylisopropylamiloride, prevents internal pH recovery. The internal pH dependence of the Na+/H+ exchanger activity has been carefully studied. The [H+]i-dependence is very cooperative. For an external pH of 7.4, the system is nearly completely inactive at pHi 7.8 and nearly completely active at pHi 6.9-7.0 with half-maximum activation at pHi = 7.35. The increased activity of the Na+/H+ exchange system which follows the acidification of the internal medium produces an activation of the (Na+,K+)-ATPase.     

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.     

The Na+/H+ antiport is activated by serum and phorbol esters in proliferating myoblasts but not in differentiated myotubes. Properties of the activation process

The properties of the Na+/H+ exchange system have been studied with 22Na+ uptake techniques at two stages of muscle development: proliferating myoblasts and differentiated myotubes. The characteristics of the interactions of the exchanger with external H+, with external Na+, and with amiloride or its more potent analogs are the same at both stages of development. Differences between the two stages of development concern: (i) the internal pH (pHi) dependence of the Na+/H+ exchanger, and (ii) the activation of the Na+/H+ exchanger by serum and phorbol ester which is observed in myoblasts but not in myotubes. Properties of the Na+/H+ exchanger in myoblasts after serum activation seem to be identical to those observed in myotubes with or without serum as if myotube formation stabilized a fully activated state of the exchanger. The activation of the myoblast Na+/H+ exchange system by serum is due to a shift of the pHi dependence towards alkaline pHi values and to an increase in the maximal activity of the Na+/H+ exchange system at acidic pH. Phorbol esters which are well-known activators of protein kinase C can only partially mimic the effects of serum on the Na+/H+ exchanger: they produce a shift of the pH dependence, but they do not increase the maximal activity at acidic pH.     

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.     

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.     

Stimulation of the Na+/H+ exchanger in human endothelial cells activated by granulocyte- and granulocyte-macrophage-colony-stimulating factor. Evidence for a role in proliferation and migration

It has been shown that human endothelial cells (HEC) are stimulated to migrate and proliferate by granulocyte (G)- and granulocyte-macrophage (GM)-colony-stimulating factor (CSF) (Bussolino, F., Wang, J. M., Defilipii, P. Turrini, F., Sanavio, F., Edgell, C.-J. S., Aglietta, M., Arese, P., and Mantovani, A. (1989) Nature 337, 471-473). The rapid intracellular events initiated by these cytokines on binding to their receptors on HEC are not defined. Addition of G- or GM-CSF to HEC produced a rapid activation of Na+/H+ exchanger resulting in an increase in intracellular pH (pHi). Both cytokines induced an alkaline displacement in the pHi dependence of the exchanger without affecting the affinity for external Na+ (Nao) and the rate of exchanger. Ethylisopropylamiloride, a selective inhibitor of the Na+/H+ exchanger, inhibited the intracellular alkalinization, the migration, and proliferation induced by G- and GM-CSF. The data indicate that G- and GM-CSF initiate a rapid exchange of Na+ and H+ by means of the Na+/H+ exchanger and that this ethylisopropylamiloride-sensitive ions flux is important to the biological effects of these cytokines on HEC.     

Cytoplasmic pH regulation in thymic lymphocytes by an amiloride- sensitive Na+/H+ antiport

The mechanisms underlying cytoplasmic pH (pHi) regulation in rat thymic lymphocytes were studied using trapped fluorescein derivatives as pHi indicators. Cells that were acid-loaded with nigericin in choline+ media recovered normal pHi upon addition of extracellular Na+ (Nao+). The cytoplasmic alkalinization was accompanied by medium acidification and an increase in cellular Na+ content and was probably mediated by a Nao+/Hi+ antiport. At normal [Na+]i, Nao+/Hi+ exchange was undetectable at pHi greater than or equal to 6.9 but was markedly stimulated by internal acidification. Absolute rates of H+ efflux could be calculated from the Nao+-induced delta pHi using a buffering capacity of 25 mmol X liter-1 X pH-1, measured by titration of intact cells with NH4+. At pHi = 6.3, pHo = 7.2, and [Na+]o = 140 mM, H+ extrusion reached 10 mmol X liter-1 X min-1. Nao+/Hi+ exchange was stimulated by internal Na+ depletion and inhibited by lowering pHo and by addition of amiloride (apparent Ki = 2.5 microM). Inhibition by amiloride was competitive with respect to Nao+. Hi+ could also exchange for Lio+, but not for K+, Rb+, Cs+, or choline+. Nao+/Hi+ countertransport has an apparent 1:1 stoichiometry and is electrically silent. However, a small secondary hyperpolarization follows recovery from acid-loading in Na+ media. This hyperpolarization is amiloride- and ouabain-sensitive and probably reflects activation of the electrogenic Na+-K+ pump. At normal Nai+ values, the Nao+/Hi+ antiport of thymocytes is ideally suited for the regulation of pHi. The system can also restore [Na+]i in Na+-depleted cells. In this instance the exchanger, in combination with the considerable cytoplasmic buffering power, will operate as a [Na+]i- regulatory mechanism.     

A membrane potential-sensitive Na+-H+ exchange system in flagella isolated from sea urchin spermatozoa

Sea urchin sperm motility is activated by a Na+-dependent increase of internal pH. A flagellar preparation was used in the present study to investigate this ionic mechanism. Using 22Na and a pH electrode, the stoichiometry of Na+ uptake to H+ release in the isolated flagella was found to be 1.09 +/- 0.11. Reversing the Na+ gradient induced reacidification of the intraflagellar pH as measured by [14C]methylamine, while reversal of the H+ gradient resulted in a Na+ efflux. Furthermore, a parallel inhibition of both ionic movements was observed with increasing external [K+]. These results indicate that Na+ and H+ are coupled through an exchanger. Measurements of the membrane potential (psi) with [3H]tetraphenylphosphonium showed depolarization by K+, suggesting its inhibitory effect on the exchanger is through changes in psi. This is further supported by the following experiments. (a) Cs+ by itself had little effect on either psi or the Na+/H+ exchange, but in the presence of the ionophore valinomycin it depolarized psi and inhibited the exchange. (b) Tetraphenylphosphonium a highly permeant cation, at 2.5 mM caused depolarization and inhibition of the exchange, and these effects were reversible by repolarization of psi with valinomycin. The inhibitory effect of depolarization was not due to the electrogenicity of the exchange since both directions of the exchange were inhibited. It is proposed that the flagellar exchange is basically a electroneutral process but has a charged regulatory component (a gate or a conformational change) which confers the observed potential sensitivity.     

Ion specificity of cardiac sarcolemmal Na+/H+ antiporter

In bovine cardiac sarcolemmal vesicles, an outward H+ gradient stimulated the initial rate of amiloride-sensitive uptake of 22Na+, 42K+, or 86Rb+. Release of H+ from the vesicles was stimulated by extravesicular Na+, K+, Rb+, or Li+ but not by choline or N-methylglucamine. Uptakes of Na+ and Rb+ were half-saturated at 3 mM Na+ and 3 mM Rb+, but the maximal velocity of Na+ uptake was 1.5 times that of Rb+ uptake. Na+ uptake was inhibited by extravesicular K+, Rb+, or Li+, and Rb+ uptake was inhibited by extravesicular Na+ or Li+. Amiloride-sensitive uptake of Na+ or Rb+ increased with increase in extravesicular pH and decrease in intravesicular pH. In the absence of pH gradient, there were stimulations of Na+ uptake by intravesicular Na+ and K+ and of Rb+ uptake by intravesicular Rb+ and Na+. Similarly, there were trans stimulations of Na+ and Rb+ efflux by extravesicular alkali cations. The data suggest the existence of a nonselective antiporter catalyzing either alkali cation/H+ exchange or alkali cation/alkali cation exchange. Since increasing Na+ caused complete inhibition of Rb+/H+ exchange, but saturating K+ caused partial inhibitions of Na+/H+ exchange and Na+/Na+ exchange, the presence of a Na(+)-selective antiporter is also indicated. Although both antiporters may be involved in pH homeostasis, a role of the nonselective antiporter may be in the control of Na+/K+ exchange across the cardiac sarcolemma.     

Epidermal growth factor and cyclic AMP stimulate Na+/H+ exchange in isolated rat hepatocytes

Na+/H+ exchange in acid-loaded isolated hepatocytes was measured using the intracellular pH indicator biscarboxyethyl-carboxyfluorescein (BCECF) to follow intracellular pH (pHi). The rate of amiloride-sensitive Na(+)-dependent recovery from cytoplasmic-acid-loading was found to be increased in cells treated with epidermal growth factor (EGF), 8-(4-chlorophenylthio)adenosine 3',5'-monophosphate (ClPhScAMP) or phorbol 12-myristate 13-acetate (PMA). These three agents increased the rate of Na+/H+ exchange to similar extents and their effects were not additive. The stimulation was shown in all three cases to be due an alkaline shift of 0.1 in the set point pH of the Na+/H+ exchanger. Experiments measuring the uptake of 22Na+ into acid-loaded primary hepatocyte monolayer cultures confirmed these results. EGF, ClPhScAMP and PMA significantly increased the amiloride-inhibitable accumulation of 22Na+, thus providing further evidence that Na+/H+ exchange is stimulated by these effectors.     

The Na+/H+ exchanger in eukaryotic cells: biochemical and pharmacological properties and physiological role

A membrane mechanism that catalyses the electroneutral exchange of Na+ for H+ has recently been characterized in a variety of eukaryotic cells. This exchanger is inhibited by amiloride, a potent diuretic drug. It has been implicated in a number of important physiological processes such as the regulation of the intracellular pH, the reabsorption of Na+ by the renal proximal tubule, the regulation of the cell volume and the fertilization of the sea urchin egg. The Na+/H+ exchanger seems able to mediate the action of growth factors. The biochemical and pharmacological properties of the Na+/H+ exchange system are reviewed. They are very similar in the different cell types that have been studied. Yet the Na+/H+ exchange system can fulfil different functions in different cell types depending i) on its properties of interaction with intracellular H+, ii) on the presence of other membrane structures that are involved in the maintenance of transmembrane Na+ and H+ gradients and iii) on the presence of extracellular messages that modify its catalytic properties and, among them, its interaction with internal H+.     

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+, K+, and Cl- transport in resting pancreatic acinar cells

To understand the role of Na+, K+, and Cl- transporters in fluid and electrolyte secretion by pancreatic acinar cells, we studied the relationship between them in resting and stimulated cells. Measurements of [Cl-]i in resting cells showed that in HCO3(-)-buffered medium [Cl- ]i and Cl- fluxes are dominated by the Cl-/HCO3- exchanger. In the absence of HCO3-, [Cl-]i is regulated by NaCl and NaK2Cl cotransport systems. Measurements of [Na+]i showed that the Na(+)-coupled Cl- transporters contributed to the regulation of [Na+]i, but the major Na+ influx pathway in resting pancreatic acinar cells is the Na+/H+ exchanger. 86Rb influx measurements revealed that > 95% of K+ influx is mediated by the Na+ pump and the NaK2Cl cotransporter. In resting cells, the two transporters appear to be coupled through [K+]i in that inhibition of either transporter had small effect on 86Rb uptake, but inhibition of both transporters largely prevented 86Rb uptake. Another form of coupling occurs between the Na+ influx transporters and the Na+ pump. Thus, inhibition of NaK2Cl cotransport increased Na+ influx by the Na+/H+ exchanger to fuel the Na+ pump. Similarly, inhibition of Na+/H+ exchange increased the activity of the NaK2Cl cotransporter. The combined measurements of [Na+]i and 86Rb influx indicate that the Na+/H+ exchanger contributes twice more than the NaK2Cl cotransporter and three times more than the NaCl cotransporter and a tetraethylammonium-sensitive channel to Na+ influx in resting cells. These findings were used to develop a model for the relationship between the transporters in resting pancreatic acinar cells.     

Differentiation of human promyelocytic HL 60 cells by retinoic acid is accompanied by an increase in the intracellular pH. The role of the Na+/H+ exchange system

Retinoic acid, which induces the differentiation of HL 60 cells to granulocytes, produces a cell alkalinization from pHi = 7.03 to pHi = 7.37. The half-maximum effect of retinoic acid is observed at 10 nM. The effect of retinoic acid on the pHi develops slowly, and it precedes the differentiation of the cells. A cell alkalinization is also observed after differentiation of the cells by dimethyl sulfoxide. It is not observed using etretinate, a synthetic retinoid that does not promote the differentiation of HL 60 cells. Two pHi regulating mechanisms coexist in HL 60 cells. The Na+/H+ exchange system is the major mechanism that allows HL 60 cells to recover from an intracellular acidosis. A second mechanism is a Na-HCO3 cotransport system. During differentiation of the cells by retinoic acid, a 2-fold increase in the activity of the Na+/H+ exchange system is observed, while the activity of the NaHCO3 cotransport remains constant. The properties of interaction of the Na+/H+ exchanger with internal H+, external Na+, and Li+ as well as with amiloride and its derivatives are defined. The Na+/H+ exchanger of HL 60 cells is characterized by unusually low affinities for alkali cations and a high affinity for amiloride and its derivatives. The pHi dependence of the exchanger is not modified after differentiation by retinoic acid. It is concluded that the mechanism of activation of the Na+/H+ exchanger by retinoic acid is distinct from the short-term effect produced by mitogens and phorbol esters which change the pHi dependence of the system.     

Inhibition of the rat renal Na+/H+ exchanger by beta-adrenergic antagonists.

The beta-adrenergic antagonists, alprenolol and propranolol, inhibit the Na+/H+ exchanger in rat renal brush-border membrane vesicles. Half-maximal inhibition occurs at 86 microM alprenolol and 36 microM propranolol. Similar to amiloride and Na+, propranolol protects the Na+/H+ exchanger from irreversible inhibition by the carboxyl group reagent, N,N'-dicyclohexyl-carbodiimide (DCCD). Protection is incomplete, depends on propranolol concentration, and reaches a maximum at 0.4 mM propranolol. With a comparable dose dependence, propranolol protects a 65 kDa band from labeling with [14C]DCCD. The data indicate that beta-adrenergic antagonists specifically interact with the proximal tubular Na+/H+ exchanger.     

Characterization of Na+-linked and Na+-independent Cl-/HCO3- exchange systems in Chinese hamster lung fibroblasts

The PS120 variant of Chinese hamster lung fibroblasts which lacks Na+/H+ exchange activity was used to investigate bicarbonate transport systems and their role in intracellular pH (pHi) regulation. When pHi was decreased by acid load, bicarbonate caused pHi increase and stimulated 36Cl- efflux from the cells, both in a Na+-dependent manner. These results together with previous findings that bicarbonate stimulates 22Na+ uptake in PS120 cells (L'Allemain, G., Paris, S., and Pouyssegur, J. (1985) J. Biol. Chem. 260, 4877-4883) demonstrate the presence of a Na+-linked Cl-/HCO3- exchange system. In cells with normal initial pHi, bicarbonate caused Na+-independent pHi increase in Cl(-)-free solutions and stimulated Na+-independent 36Cl- efflux, indicating that a Na+-independent Cl-/HCO3- exchanger is also present in the cell. Na+-linked and Na+-independent Cl-/HCO3- exchange is apparently mediated by two distinct systems, since a [(tetrahydrofluorene-7-yl)oxy]acetic acid derivative selectively inhibits the Na+-independent exchanger. An additional distinctive feature is a 10-fold lower affinity for chloride of the Na+-linked exchanger. The Na+-linked and Na+-independent Cl-/HCO3- exchange systems are likely to protect the cell from acid and alkaline load, respectively.