Na+/H+ antiporter activity in hamster embryos is activated during fertilization

This study characterized the activation of the regulatory activity of the Na+/H+ antiporter during fertilization of hamster embryos. Hamster oocytes appeared to lack any mechanism for the regulation of intracellular pH in the acid range. Similarly, no Na+/H+ antiporter activity could be detected in embryos that were collected from the reproductive tract between 1 and 5 h post-egg activation (PEA). Activity of the Na+/H+ antiporter was first detected in embryos collected at 5.5 h PEA and gradually increased to reach maximal activity in embryos collected at 7 h PEA. Parthenogenetically activated one-cell and two-cell embryos demonstrate Na+/H+ antiporter activity, indicating that antiporter activity is maternally derived and initiated by activation of the egg. The inability of cycloheximide, colchicine, or cytochalasin D to affect initiation of antiporter activity indicates that antiporter appearance is not dependent on the synthesis of new protein or recruitment of existing protein to the cell membrane. In contrast, incubation of one-cell embryos with sphingosine did inhibit the appearance of Na+/H+ antiporter activity, showing that inhibition of normal protein kinase C activity is detrimental to antiporter function. Furthermore, incubation of oocytes with a phorbol ester which stimulates protein kinase C activity induced Na+/H+ antiporter activity in oocytes in which the activity was previously absent. Incubation with an intracellular calcium chelator also reduced the appearance of antiporter activity. Taken together, these data indicate that the appearance of Na+/H+ antiporter activity following egg activation may be due, at least in part, to regulation by protein kinase C and intracellular calcium levels.     

Isolation and properties of a mutant of Escherichia coli possessing defective Na+/H+ antiporter

A mutant of Escherichia coli with defective Na+/H+ antiporter was isolated. The rationale for its isolation was that cells possessing defective Na+/H+ antiporter, which is essential for establishment of a Na+ gradient, could not grow with a carbon source that was taken up with Na+. The mutant had no appreciable Na+/H+ antiporter activity, but its K+/H+ antiporter and Ca2+/H+ antiporter activities were normal. Judging from the reversion frequency, the defect seems to be due to a single mutation. The mutant could not grow at alkaline pH. Therefore, the Na+/H+ antiporter, but not the K+/H+ antiporter or the Ca2+/H+ antiporter, seems to be responsible for pH regulation in alkaline medium. This mutant will be useful for cloning the Na+/H+ antiporter gene and for detection of Na+-substrate cotransport systems.     

Control of the sodium-proton antiporter in human placental microvillous membranes by transport substrates

The microvillous membrane of the human placental syncytiotrophoblast contains an amiloride-inhibitable, electroneutral, Na+/H+ antiporter. The kinetic characteristics of this antiporter have been investigated to determine its response to alterations in intracellular and extracellular H+ and Na+ concentrations. Antiporter activity was measured using a pH-sensitive fluorescent probe entrapped in placental microvillous vesicles. We report here on the kinetic characterization of the antiporter, a transporter which displays simple, saturable kinetics for the external site but complex kinetics at the internal site. Measurement of the external Na+ and H+ dependences demonstrated that Na+ and H+ compete for binding to a single external binding site which displays saturation kinetics. The external Km determined for Na+ was 8.2 +/- 4.0 mM, while the external pK was 7.29 +/- 0.02. The Vmax calculated from these experiments was 0.57 +/- 0.10 nequiv./s per mg membrane protein. By contrast, the internal dependences for both Na+ and H+ showed significant deviations from simple linear kinetics. Decreasing internal pH to 6.0 stimulated Na+/H+ exchange to a greater degree than predicted for a single-site saturable binding model, in a manner which suggested allosteric activation. At the other extreme, Na+/H+ exchange ceased above an internal pH of 7.1, despite the existence of an inwardly-directed Na+ gradient. Increasing intracellular Na+ caused inhibition of Na+/H+ exchange but the intracellular Na+ dependence showed that the effect is due to a mechanism more complex than simple, competitive inhibition between Na+ and H+. These results show that the microvillous Na+/H+ antiporter is insensitive to changes in extracellular Na+ and H+ concentrations in the physiological range. Changes in intracellular Na+ and H+ however are likely to cause marked changes in antiporter activity. These characteristics suggest that cellular Na+ and H+ concentrations are tightly controlled in the placental syncytiotrophoblast and that the Na+/H+ antiporter may play a significant role in their regulation.     

Na+/H+ antiporters

Na+/H+ antiports or exchange reactions have been found widely, if not ubiquitously, in prokaryotic and eukaryotic membranes. In any given experimental system, the multiplicity of ion conductance pathways and the absence of specific inhibitors complicate efforts to establish that the antiport observed actually results from the activity of a specific secondary porter which catalyzes coupled exchanged of the two ions. Nevertheless, a large body of evidence suggests that at least some prokaryotes possess a delta psi-dependent, mutable Na+/H+ antiporter which catalyzes Na+ extrusion in exchange for H+; in other bacterial species, the antiporter my function electroneutrally, at least at some external pH values. The bacterial Na+/H+ antiporter constitutes a critical limb of Na+ circulation, functioning to maintain a delta mu Na+ for use by Na+-coupled bioenergetic processes. The prokaryotic antiporter is also involved in pH homeostasis in the alkaline pH range. Studies of mutant strains that are deficient in Na+/H+ antiporter activity also indicate the existence of a relationship, e.g., a common subunit or regulatory factor, between the Na+/H+ antiporter and Na+/solute symporters in several bacterial species. In eukaryotes, an electroneutral, amiloride-sensitive Na+/H+ antiport has been found in a wide variety of cell and tissue types. Generally, the normal direction of the antiport appears to be that of Na+ uptake and H+ extrusion. The activity is thus implicated as part of a complex system for Na+ circulation, e.g., in transepithelial transport, and might have some role in acidification in the renal proximal tubule. In many experimental systems, the Na+/H+ antiport appears to influence intracellular pH. In addition to a role in general pH homeostasis, such Na+-dependent changes in intracellular pH could be part of the early events in a variety of differentiating and proliferative systems. Reconstitution and structural studies, as well as detailed analysis of gene loci and products which affect the antiport activity, are in their very early stages. These studies will be important in further clarification of the precise structural nature and role(s) of the Na+/H+ antiporters. In neither prokaryotes nor eukaryotes systems is there yet incontrovertible evidence that a specific protein carrier, that catalyzes Na+/H+ antiport, is actually responsible for any of the multitude of effects attributed to such antiporters. The Na+-H+ exchange might turn out to be side reactions of other porters or the additive effects of several conductance pathways; or, as appears most likely in at least some bacteria and in renal tissue, the antiporter may be a discrete, complex carr     

Inhibitory effect of local anesthetics on Na+/H+ antiporter in brush border membrane-reconstituted vesicles

The effects of three local anesthetics, lidocaine, dibucaine, and tetracaine, on Na+/H+ antiporter activity were examined in brush border membrane-reconstituted vesicles. Lidocaine at 10 microM inhibited H+ efflux in the presence of an inward Na+ gradient, suggesting that this anesthetic specifically inhibits the Na+/H+ antiporter. On the other hand, dibucaine and tetracaine decreased H+ efflux even in the absence of a Na+ gradient.     

Inhibition of cardiac sarcolemmal Na+/H+ antiporter by opioids.

In our routine screening of chemicals that would inhibit cardiac sarcolemmal Na+/H+ antiporter, we discovered that some of the opioids produced inhibition of cardiac sarcolemmal Na+/H+ antiporter in micromolar concentrations. Using U-50,488H, a selective kappa-opioid agonist, we characterized the nature of interaction between opioids and the Na+/H+ antiporter. The inhibitory effect of U-50,488H on Na+/H+ antiporter was immediate and reversible, and was not mediated through the interaction with the opioid receptors but due to the direct interaction of U-50,488H with the Na+/H+ antiporter. The kinetic data show that in the presence of U-50,488H the Km for Na+ was increased from 2.5 +/- 0.2 to 5.0 +/- 0.3 mM, while the Vmax (52.0 +/- 5.0 nmol.mg-1.min-1) remained the same. These results suggest that U-50,488H and Na+ compete for the same site on the antiporter. When testing the effect of U-50,488H on other transport systems of cardiac sarcolemma, we found that U-50,488H also inhibited Na+/Ca2+ antiporter and Na+/K+ pump but at much higher concentrations suggesting that U-50,488H shows some degree of selectivity for cardiac sarcolemmal Na+/H+ antiporter. When we compared the inhibitory potency of U-50,488H with amiloride and its analog, namely 5-(N,N-hexamethylene)amiloride, we found that U-50,488H (IC50 = 100 +/- 15 microM) was threefold more potent than amiloride (IC50 = 300 +/- 20 microM) but it was three-fold less potent than the amiloride analog (IC50 = 30 +/- 10 microM) in inhibiting cardiac sarcolemmal Na+/H+ antiporter. These results show that although U-50,488H is more potent than amiloride, the inhibitory characteristics of U-50,488H on cardiac sarcolemmal Na+/H+ antiporter are similar to amiloride.     

Differential regulation of Na+/H+ antiporter gene expression in vascular smooth muscle cells by hypertrophic and hyperplastic stimuli

The Na+/H+ antiporter is a ubiquitous transmembrane protein that plays a vital role in cell growth via regulation of intracellular Na+ and H+. In vascular smooth muscle cells (VSMC), vasoconstrictors and mitogens rapidly activate the antiporter, suggesting that both should have growth promoting effects. Indeed, angiotensin II increases VSMC protein and volume (hypertrophy), but does not increase cell number (hyperplasia). In the present work we investigated whether alterations in the steady state levels of Na+/H+ antiporter mRNA might differentiate these VSMC growth responses. Differences in function of the Na+/H+ antiporter appeared likely because exposure of growth-arrested VSMC for 24 h to 100 nM angiotensin II decreased intracellular pH from 7.08 to 6.99, while exposure to 10% calf serum caused an increase to 7.18. Simultaneous measurement of Na+/H+ antiporter mRNA levels, using the human c28 cDNA, revealed a 25-fold increase in response to serum (as well as to platelet-derived and fibroblast growth factors), but no change in response to angiotensin II. All agonists increased mRNA levels of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase approximately 3-fold. The increase in Na+/H+ antiporter mRNA induced by serum was first apparent within 2 h and peaked 24 h after treatment. These results demonstrate that expression of Na+/H+ antiporter mRNA in VSMC is dependent on growth state: hyperplastic agonists (serum, platelet-derived and fibroblast growth factor) increase the steady state levels of Na+/H+ antiporter mRNA while a hypertrophic agonist (angiotensin II) does not.     

Development and polarization of the Na+/H+ antiport system during reorganization of LLC-PK1A cells into an epithelial membrane

Changes in Na+/H+ antiport activity and transepithelial electrical resistance were analyzed in a clone of LLC-PK1 cells as the dispersed cells became organized into an epithelial membrane. The clone designated LLC-PK1A showed a 250% increase in Na+/H+ exchange activity as compared with the parent cell line. Na+ influx induced by an outwardly oriented H+ gradient is almost completely abolished during active cell proliferation or after cell dispersion. The activity of the Na+/H+ antiport system increases after plating the cells at high density. This increase precedes the increase in the transepithelial electrical resistance. The increase in the Na+/H+ antiport activity was not observed when the cells were plated at low density in the presence of an antimitotic agent indicating that close cell contact is an absolute requirement for the development of the system. The increase in Na+ influx correlated with an increase in Vmax, while the Km for Na+ remained essentially unchanged. Unidirectional Na+ influx measured from the apical or basolateral side as the dispersed cells became reorganized into an epithelial membrane indicated that the insertion of the Na+/H+ antiporter proteins occurred directly in the apical membrane of the epithelial cells. This finding is consistent with the hypothesis that the sorting of native proteins occurs intracellularly prior to their insertion in the apical membrane of the epithelial cells. The delay in the increase of transepithelial electrical resistance as compared with the increase in Na+ influx indicates that the settlement of the limits between the apical and basolateral membrane (fence function) precedes the closing of the intercellular space (barrier function) during the development of the occluding junctions. Further, the development of the Na+/H+ antiporter was inhibited by cycloheximide but not by actinomycin D, suggesting that the expression of epithelial cell polarization is a translational or posttranslational event.     

Interactions of chloride and amiloride with the renal Na+/H/ antiporter

Amiloride is a reversible inhibitor of the Na+/H+ antiporter which acts at the external aspect of the transport system. The kinetics of inhibition of the Na+/H+ antiporter with amiloride have been controversial, with the usual finding of simple competitive inhibition, but with other reports of mixed and noncompetitive inhibition of the transporter by amiloride. The present experiments demonstrate that the chloride content of the external transport buffer affects the kinetics of amiloride inhibition. Either simple competitive or mixed inhibition by amiloride was observed in the same vesicle preparations depending on the presence of chloride or gluconate in the buffer. The effect of chloride on the inhibitory effect of amiloride was dependent on the concentration of chloride and amiloride. Similar effects were observed with more potent analogues of amiloride. These findings suggest that the external aspect of the antiporter has a site or sites at which the inhibitory effects of amiloride on the Na+/H+ antiporter can be modified by chloride, even though chloride has only slight effects on the kinetics of the Na+/H+ antiporter in the absence of amiloride.     

Properties of two different Na+/H+ antiport systems in alkaliphilic Bacillus sp. strain C-125.

Na+/H+ antiport was studied in alkaliphilic Bacillus sp. strain C-125, its alkali-sensitive mutant 38154, and a transformant (pALK2) with recovered alkaliphily. The transformed was able to maintain an intracellular pH (pHin) that was lower than that of external milieu and contained an electrogenic Na+/H+ antiporter driven only by delta psi (membrane potential, interior negative). The activity of this delta psi-dependent Na+/H+ antiporter was highly dependent on pHin, increasing with increasing pHin, and was found only in cells grown at alkaline pH. On the other hand, the alkali-sensitive mutant, which had lost the ability to grow above pH 9.5, lacked the delta psi-dependent Na+/H+ antiporter and showed defective regulation of pHin at the alkaline pH range. However, this mutant, like the parent strain, still required sodium ions for growth and for an amino acid transport system. Moreover, another Na+/H+ antiporter, driven by the imposed delta pH (pHin > extracellular pHout), was active in this mutant strain, showing that the previously reported delta pH-dependent antiport activity is probably separate from delta psi-dependent antiporter activity. The delta pH-dependent Na+/H+ antiporter was found in cells grown at either pH 7 or pH 9. This latter antiporter was reconstituted into liposomes by using a dilution method. When a transmembrane pH gradient was applied, downhill sodium efflux was accelerated, showing that the antiporter can be reconstituted into liposomes and still retain its activity.     

Introduction of a Na+/H+ antiporter gene from Atriplex gmelini confers salt tolerance to rice

We engineered a salt-sensitive rice cultivar (Oryza sativa cv. Kinuhikari) to express a vacuolar-type Na+/H+ antiporter gene from a halophytic plant, Atriplex gmelini (AgNHX1). The activity of the vacuolar-type Na+/H+ antiporter in the transgenic rice plants was eight-fold higher than that in wild-type rice plants. Salt tolerance assays followed by non-stress treatments showed that the transgenic plants overexpressing AgNHX1 could survive under conditions of 300 mM NaCl for 3 days while the wild-type rice plants could not. These results indicate that overexpression of the Na+/H+ antiporter gene in rice plants significantly improves their salt tolerance.     

The effect of beta 2-adrenoceptor stimulation and blockade of L-type calcium channels on in vivo Na+/H+ antiporter activity in rat skeletal muscle.

We have studied the in vivo response of the Na+/H+ antiporter in skeletal muscle to beta 2-adrenoceptor stimulation with isoprenaline and the effect of blocking L-type calcium channels with nifedipine. Na+/H+ antiporter activity in skeletal muscle in vivo increased after beta 2-adrenoceptor stimulation with isoprenaline; nifedipine attenuated that effect. This suggests that opening of L-type calcium channels is necessary for full activation of the Na+/H+ antiporter in skeletal muscle. Bleeding also increased Na/H+ antiporter activity, which we believe could be explained by an increase in sympathetic nervous system activity as a result of hypotension. This may be one of the mechanisms by which animals under stress prepare their skeletal muscle for exercise as part of the 'fright and flight' reaction.     

Transport by the (Na+,K+) ATPase: modulation by differentiation inducers and inhibition of protein synthesis in the MDCK kidney epithelial cell line

MDCK kidney epithelial cell cultures exposed to the differentiation inducer hexamethylene bisacetamide (HMBA) for 24 hours exhibited a 50% decrease in transport activity per (Na+,K+)-ATPase molecule (turnover number) but an unchanged number of pump sites (Kennedy and Lever, 1984). Inhibition of protein synthesis by either 10 microM cycloheximide or 2 microM emetine blocked the inhibitory effects of HMBA on Na+/K+ pump efficiency assessed by measurements of [3H]-ouabain binding to intact cells, (Na+,K+) ATPase activity of detergent-activated cell extracts, and ouabain-sensitive Rb+ uptake. In the absence of inducer treatment, inhibition of protein synthesis increased Na+/K+ pump turnover number by twofold while maintaining Na+/K+ pump activity per cell at a constant level. Intracellular Na+ levels were decreased after cycloheximide treatment; therefore, pump stimulation was not due to substrate effects. Furthermore, cycloheximide effects of Rb+ uptake could be dissociated from effects on tight junctions. These observations suggest that the transport activity of the (Na+,K+) ATPase is tightly regulated by factors dependent on protein synthesis.     

Amiloride-sensitive Na+-H+ antiporter in Escherichia coli.

In everted vesicles of Escherichia coli, delta pH caused by H+ efflux through the Na+/H+ antiporter was measured by using a fluorescent dye. Amiloride inhibited the activity of the Na+/H+ antiporter. Kinetic studies showed that amiloride competed with Na+. The inhibition constant of 40 microM was obtained.     

The Na+/H+ antiporter is not involved in potentiation of thrombin-induced responses by epinephrine

Stimulation of platelets with thrombin, ADP and epinephrine has recently been shown to activate a Na+/H+ antiporter, with a resulting alkalinization of the cytoplasm. Unlike thrombin, however, epinephrine is incapable of directly activating phospholipase C, but is well known to potentiate the effects of thrombin on this enzyme and other subsequent steps of platelet activation. Therefore, we have studied the involvement of the Na+/H+ antiporter in this aspect of epinephrine action to see whether alkalinization of platelet cytosol could be a requirement for agonists to stimulate inositol phospholipid hydrolysis and mobilize cellular Ca2+ stores. Alpha-thrombin induced the rapid formation of inositol trisphosphate with a parallel mobilization of intracellular Ca2+ stores. Epinephrine alone had no effect on either of these parameters. The response to thrombin desensitized over a period of minutes, and after this had occurred, epinephrine was able to activate phospholipase C and induce the release of intracellular Ca2+. This showed that epinephrine was able to recouple thrombin receptors to phospholipase C, and this appeared to be mediated by the same mechanism which is involved in potentiation by epinephrine of thrombin-stimulation of phospholipase C. These effects of epinephrine were not altered by inhibition of the Na+/H+ antiporter with ethylisopropylamiloride or by use of the Na+/H+ ionophore, monensin. We conclude that epinephrine potentiates thrombin-induced responses by a mechanism which is unrelated to its effects on the Na+/H+ antiporter, and this is not a requirement for thrombin-induced phospholipase C activation.     

Na+/H+ 逆向转运蛋白与植物耐盐性关系

Na+/H+ 逆向转运蛋白与植物的耐盐性有密切的关系。在高等植物体内,主要存在两种Na+/H+ 逆向转运蛋白,分别为位于细胞质膜上的逆向转运蛋白SOS1,以及存在于液泡膜上的AtNHX1。质膜Na+/H+ 逆向转运蛋白主要负责Na+ 的外排,液泡膜Na+/H+ 逆向转运蛋白主要负责把Na+ 区隔化入液泡。过量表达质膜Na+/H+ 逆向转运蛋白SOS1或液泡膜Na+/H+ 逆向转运蛋白AtNHX1能够明显提高植物的耐盐性。本文对植物中Na+/H+ 逆向转运蛋白及其与植物耐盐性之间的关系研究最新进展作一概述。     

Relationship among activation of the Na+/H+ antiporter, ornithine decarboxylase induction, and DNA synthesis

The relationship among activation of the Na+/H+ antiporter, ornithine decarboxylase, and DNA synthesis was examined with bovine small lymphocytes stimulated by concanavalin A (Con A). The Na+/H+ antiport activity was activated immediately after addition of concanavalin A; the maximum was reached 1 h after Con A addition and the activation continued at least 6 h. With increasing concanavalin A concentrations, the activities of the Na+/H+ antiporter, ornithine decarboxylase, and DNA synthesis increased in a parallel manner. In the presence of HCO3- in the medium, the internal alkalinization of lymphocytes was not induced by Con A. Ornithine decarboxylase and DNA synthetic activities were not inhibited by 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a specific inhibitor of the Na+/H+ antiporter. In contrast, in the absence of HCO3- in the medium, the internal pH was alkalinized approximately 0.06 pH units by Con A. EIPA did inhibit the alkalinization of the internal pH or DNA synthesis significantly. Ornithine decarboxylase activity was not inhibited by EIPA. These results indicate that the activation of a Na+/H+ antiporter is not a trigger for cell proliferation, but its activation is important probably through the maintenance of the internal pH optimum, especially in HCO3(-)-free medium.     

Characterization and mapping of a major Na+/H+ antiporter gene of Escherichia coli.

Using in vivo assays, we show that the Na+/H+ antiporter activity of the Escherichia coli mutant HIT-1 is reduced dramatically compared with activity in wild-type cells. An isogenic nhaA (formerly antA) deletion strain, however, is not significantly different from wild type in this respect. We call the locus affecting Na+/H+ antiporter activity of the HIT-1 mutant nhaB. The nhaB activity exhibits no pH dependence in the range between 7.0 and 8.5, whereas that of the nhaA gene increases considerably at pH levels above 8.0. Mutants with defects in nhaB grow normally on agar media containing 0.5 M NaCl, but nhaA mutants are sensitive to 0.5 M NaCl. We have mapped the nhaB mutation of HIT-1 to 25.6 min on the E. coli map. It is unlinked to the nhaA region, which is located at about 0.5 min. Since a cell with a mutation in nhaB alone is essentially Na+/H+ antiporter negative up to pH 8.0, we conclude that nhaB is required for the major Na+/H+ antiporter activity in the usual physiological pH range.