Epidermal growth factor up-regulates intestinal Na+/H+ exchange activity.

The present studies were designed to examine the regulation of Na+/H+ exchange activity by epidermal growth factor (EGF) in an in vitro system. Na+/H+ exchange activity was determined in brush-border membranes isolated from rat jejunal enterocytes incubated with epidermal growth factor and a number of second messengers. EGF at physiological concentrations stimulated Na+/H+ exchange activity without affecting vesicle size. The stimulation of Na+/H+ activity was the result of increasing Vmax of Na+/H+ (6.0 +/- 0.4 compared with 3.3 +/- 0.27 nmol/mg protein/5 sec, P < 0.01). Km values of the Na+/H+ exchanger in brush-border membrane from cells stimulated with EGF and controls were similar (16.0 +/- 3.0 vs 13.0 +/- 3.0, respectively). Na+/H+ activity was inhibited by phorbol esters, calmodulin, and cyclic AMP. The effects of EGF, calmodulin, cyclic AMP, and phorbol esters were dependent on ATP, because depleting the cells from ATP masked the effects on Na+/H+ exchange activity. The results suggest that EGF stimulates Na+/H+ exchange activity in the enterocytes. This stimulation is most likely not via activation of the phosphatidylinositol pathway.     

Inhibition of Na+/H+ exchange reduces Ca2+ mobilization without affecting the initial cleavage of phosphatidylinositol 4,5-bisphosphate in thrombin-stimulated platelets

Stimulation of human platelets increases cytoplasmic pH (pHi) via activation of Na+/H+ exchange. We have determined the effect of inhibiting Na+/H+ exchange on (i) thrombin-induced Ca2+ mobilization and (ii) turnover of 32P-labelled phospholipids. Blocking Na+/H+ exchange by removal of extracellular Na+ or by ethylisopropylamiloride (EIPA) inhibited Ca2+ mobilization induced by 0.2 U/ml thrombin, whereas increasing pHi by NH4Cl enhanced the thrombin-induced increase in cytosolic free Ca2+. The effect of EIPA was bypassed after increasing pHi by moneasin. The thrombin-induced cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) was unaffected by treatments that blocked Na+/H+ exchange or increased pHi. It is concluded that activation of Na+/H+ exchange is a prerequisite for Ca2+ mobilization in human platelets but not for the stimulus-induced hydrolysis of PIP2.     

Tumor promoter phorbol 12-myristate 13-acetate inhibits mitogen- stimulated Na+/H+ exchange in human epidermoid carcinoma A431 cells

Addition of polypeptide growth factors to cultured cells results in a rapid stimulation of Na+/H+ exchange, which leads to cytoplasmic alkalinization. We studied the effects of the potent tumor promoter phorbol 12-myristate 13-acetate (PMA) on the Na+/H+ exchange system of A431 cells. Stimulation of Na+/H+ exchange by epidermal growth factor (EGF) and serum as well as by vanadate ions is strongly inhibited after treatment of cells with nanomolar concentrations of PMA. Phorbol esters that have no activity as tumor promoters also do not modulate the activation of Na+/H+ exchange. By contrast, the stimulation of Na+/H+ exchange that is produced upon exposure of cells to hypertonic solution is only slightly inhibited by PMA treatment, indicating that PMA treatment does not directly block the activity of the Na+/H+ antiporter. Furthermore, incubation of cells with PMA causes a weak stimulation of Na+/H+ exchange, although this effect is mostly observed at relatively high PMA concentrations and appears to require external Ca2+. The inhibition BY PMA of EGF-promoted Na+/H+ exchange is not due to inhibition of EGF-binding to the EGF receptor. Since PMA activates protein kinase C, our observations are consistent with the hypothesis that protein kinase C functions to attenuate the stimulation of Na+/H+ exchange by polypeptide growth factors.     

Nerve growth factor inhibits HCO3- absorption in renal thick ascending limb through inhibition of basolateral membrane Na+/H+ exchange

Nerve growth factor (NGF) inhibits transepithelial HCO3- absorption in the rat medullary thick ascending limb (MTAL). To investigate the mechanism of this inhibition, MTALs were perfused in vitro in Na+-free solutions, and apical and basolateral membrane Na+/H+ exchange activities were determined from rates of pHi recovery after lumen or bath Na+ addition. NGF (0.7 nM in the bath) had no effect on apical Na+/H+ exchange activity, but inhibited basolateral Na+/H+ exchange activity by 50%. Inhibition of basolateral Na+/H+ exchange activity with ethylisopropyl amiloride (EIPA) secondarily reduces apical Na+/H+ exchange activity and HCO3- absorption in the MTAL (Good, D. W., George, T., and Watts, B. A., III (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 12525-12529). To determine whether a similar mechanism could explain inhibition of HCO3- absorption by NGF, apical Na+/H+ exchange activity was assessed in physiological solutions (146 mM Na+) by measurement of the initial rate of cell acidification after lumen EIPA addition. Under these conditions, in which basolateral Na+/H+ exchange activity is present, NGF inhibited apical Na+/H+ exchange activity. Inhibition of HCO3- absorption by NGF was eliminated in the presence of bath EIPA or in the absence of bath Na+. Also, NGF blocked inhibition of HCO3- absorption by bath EIPA. We conclude that NGF inhibits basolateral Na+/H+ exchange activity in the MTAL, an effect opposite from the stimulation of Na+/H+ exchange by growth factors in other systems. NGF inhibits transepithelial HCO3- absorption through inhibition of basolateral Na+/H+ exchange, most likely as the result of functional coupling in which primary inhibition of basolateral Na+/H+ exchange activity results secondarily in inhibition of apical Na+/H+ exchange activity. These findings establish a role for basolateral Na+/H+ exchange in the regulation of renal tubule HCO3- absorption.     

Early effect of growth factors (EGF+insulin) upon ATP turnover in 3T3 cells

Various early biochemical events have been observed after the addition of growth factors to quiescent cultures of 3T3 cells; however, the cascade of events which take place in the cells after growth-factor addition is not yet entirely known. Our results show that the addition of a mixture of two growth factors, i.e., Epidermal Growth Factor (EGF) and insulin, to quiescent cultures of 3T3 cells rapidly stimulated phosphate uptake and ATP turnover. Our present and previous results suggest that the increase in phosphate uptake is the consequence of the stimulation of ATP synthesis. This stimulation was not simply a consequence of an increase in oxidative phosphorylation or in glucose transport and metabolism. The change in ATP turnover was an early event observed as soon as 5 min after growth-factor addition; furthermore, it was not dependent on protein synthesis. This change may therefore be the result of post-synthetic modification of enzymes by phosphorylation. We do not know what cellular process is responsible for the increase in ATP turnover. Since growth-factor addition rapidly enhanced ATP degradation in quiescent 3T3 cell cultures, we assumed that this increase is the result of an increase in ATP degradation. We know that it was not due to a stimulation of an oligomycin-sensitive ATPase. We verified that it was not the consequence of early biochemical events like an increase in Na+/K+ ATPase or a stimulation of RNA or protein synthesis. However, it is of interest to note that the stimulation of ATP turnover due to the growth-factor addition was inhibited by quercetin.     

Activation of 45Ca2+ influx and 22Na+/H+ exchange by epidermal growth factor and vanadate in A431 cells is independent of phosphatidylinositol turnover and is inhibited by phorbol ester and diacylglycerol

Both epidermal growth factor (EGF) and vanadate can activate 45Ca2+ influx into A431 epidermal carcinoma cells, without a detectable lag period possibly via a voltage-independent calcium channel. 22Na+/H+ exchange and 45Ca2+ uptake are mutually independent. Neither EGF nor vanadate induce any significant change in the steady-state levels of [1,3-3H]glycerol-labeled diacylglycerol, myo-[2-3H]inositol-labeled inositol trisphosphate or in 32P-labeled polyphosphoinositides or phosphatidic acid over the first 10 min of treatment, suggesting that the EGF receptor is not directly coupled to phosphatidylinositol turnover and that the two ion fluxes are not induced via a kinase C-dependent pathway. An increase in turnover of polyphosphoinositides can be detected in EGF-stimulated cells by nonequilibrium labeling with [32P]phosphate, but the increase shows a lag of about 1 min under the conditions used to detect 45Ca2+ influx. Chelation of free Ca2+ decreases but does not abolish the EGF-stimulated turnover. Preincubation with tetradecanoylphorbol acetate or 1-oleoyl-2-acetylglycerol inhibits the increase in 45Ca2+ uptake by both EGF and vanadate. Tetradecanoylphorbol acetate alone does not alter the basal rate of influx when added together with 45Ca2+. Surprisingly, the activation by vanadate and its inhibition by phorbol 12-myristate 13-acetate are unaffected by down-regulation of the EGF receptors through prior incubation with growth factor. Therefore, in A431 cells the activation of Na+/H+ exchange and Ca2+ influx appear to be independent of phosphatidylinositol turnover, and the EGF receptor does not itself function as a Ca2+ channel. Vanadate apparently activates influx through a mechanism distinct from or distal to the EGF receptor.     

Mitogen-independent activation of Na+/H+ exchange in human epidermoid carcinoma A431 cells: regulation by medium osmolarity

Previous studies have documented the activation of Na+/H+ exchange in A431 cells by the addition of epidermal growth factor or serum (Rothenberg et al., 1983b). Here we show that exposure of A4 31 cells to medium of increased osmolarity also leads to activation of Na+/H+ exchange and to an increase in intracellular pH (pHi), which under a variety of conditions displays similar kinetics to that observed upon addition of mitogens to the cells. Measurements of cell volume using the 3-0-methylglucose equilibration technique clearly show that mitogens do not activate Na+/H+ exchange by an osmotic mechanism (i.e., a decrease in cell volume). In fact, mitogens can induce further intracellular alkalinization if added to cells which have been shrunken in hypertonic medium. Activation of the Na+/H+ antiport does not lead to an obligatory change in pHi. Addition of epidermal growth factor of hypertonic solution to A431 cells in bicarbonate buffer activates Na+/H+ exchange without a concomitant increase in pHi. Under these conditions the increased proton efflux via Na+/H+ exchange must therefore be compensated by other mechanisms that control cytoplasmic pH.     

Effects of glucocorticoids on Na+/H+ exchange and growth in cultured vascular smooth muscle cells

We have examined the effects of hydrocortisone on growth and Na+/H+ exchange in cultured rat aortic vascular smooth muscle cells (VSMC). Hydrocortisone (2 microM) treatment of growth-arrested VSMC significantly decreased VSMC growth in response to 10% calf serum assayed by 3H-thymidine incorporation and cell number at confluence. This effect was associated with the appearance of an altered cell phenotype characterized by large, flat VSMC that did not form typical "hillocks." Na+/H+ exchange was also altered in hydrocortisone-treated cells assayed by dimethylamiloride-sensitive 22Na+ influx into acid-loaded cells or by intracellular pH (pHi) change using the fluorescent dye BCECF. Resting pHi was 7.25 +/- 0.04 and 7.15 +/- 0.05 in control and hydrocortisone-treated cells, respectively (0.1 less than P less than 0.05). Following intracellular acidification in the absence of external Na+, pHi recovery upon addition of Na+ was increased 89% in hydrocortisone-treated cells relative to control. This was due to an increase in the Vmax for the Na+/H+ exchanger from 17.5 +/- 2.4 to 25.9 +/- 2.0 nmol Na+/mg protein x min (P less than 0.01) without a significant change in Km. Treatment of VSMC with actinomycin D (1 microgram/ml) or cycloheximide (10 microM) completely inhibited the hydrocortisone-mediated increase in Na+/H+ exchange, indicating a requirement for both RNA and protein synthesis. Because hydrocortisone altered the Vmax for Na+/H+ exchange, in contrast to agonists such as serum or angiotensin II which alter the Km for intracellular H+ or extracellular Na+, respectively, we studied the effect of hydrocortisone on activation of Na+/H+ exchange by these agonists. In cells maintained at physiological pHi (7.2), the initial rate (2 min) of angiotensin II-stimulated alkalinization was increased 66 +/- 39% in hydrocortisone-treated compared with control cells. Hydrocortisone caused no change in angiotensin II-stimulated phospholipase C activity assayed by measurement of changes in intracellular Ca2+ or diacylglycerol formation. However, angiotensin II and serum stimulated only small increases in Na+/H+ exchange in acid-loaded (pHi = 6.8) hydrocortisone-treated cells. These findings suggest that hydrocortisone-mediated increases in VSMC Na+/H+ exchange occur in association with a nonproliferating phenotype that has altered regulation of Na+/H+ exchange activation. We propose that hydrocortisone-mediated growth inhibition may be a useful model for studying the role of Na+/H+ exchange in cell growth responsiveness.     

Na+/H+ exchange involvement in colony-stimulating factor-1-stimulated macrophage proliferation. Evidence for a requirement during late G1 of the cell cycle but not for early growth factor responses

Na+/H+ exchange activation by growth factors is proposed to be an important early signal for mitogenesis; however, little is known of its duration and requirement during later stages of the cell cycle. Macrophage-specific colony factor (CSF-1) rapidly activates murine bone marrow-derived macrophage Na+/H+ exchange, resulting in stimulation of Na+,K(+)-ATPase activity. The response to CSF-1 is maintained for at least 24 h. Inhibition of Na+/H+ exchange with 5-N,N-dimethylamiloride prevents CSF-1-stimulated DNA synthesis and cell growth. This is unlikely to be due to cytoplasmic acidosis, but more likely reflects a requirement for Na+/H+ exchange-mediated Na+ influx. DMA addition even up to 8 h after the growth factors suppresses S-phase progression. Na+/H+ exchange appears not to be involved in the induction of other early growth factor responses (c-fos and c-myc mRNA induction and general RNA and protein synthesis). We propose that growth factor-stimulated Na+/H+ exchange late in G1 of the cell cycle is required for S-phase progression but not for certain early growth factor responses.     

Growth factors activate the Na+/H+ antiporter in quiescent fibroblasts by increasing its affinity for intracellular H+

Growth factors (alpha-thrombin and insulin) activate a Na+/H+ antiport in G0/G1-arrested Chinese hamster lung fibroblasts (CCL39). In this report, we have examined the influence of intracellular pH on this exchange activity, measured by initial rates of amiloride-sensitive 22Na+ uptake, in the absence and presence of growth factors. Our results indicate the following. 1) In quiescent as in mitogen-stimulated cells, Na+/H+ antiport is regulated by internal H+ in an allosteric way, whereas, in contrast, interactions with external H+ and Na+ obey simple saturation kinetics. 2) The growth factor-induced activation of Na+/H+ exchange, which, under physiological conditions, is responsible for a sustained cytoplasmic alkalinization, is due to an increased affinity for internal H+ (the apparent pK is shifted by approximately 0.3 pH unit towards alkaline pH values). Therefore, we propose that growth factors promote a conformational change of the Na+/H+ antiporter, possibly at the level of an internal modifier site(s).     

Photosensitized irreversible binding of estrone to protein via a hydroperoxide intermediate: an explanation of (photo-) allergic side-effects of estrogens

Recent studies have established that polypeptide growth factors cause an elevation of the cytoplasmic pH (pHi) in cultured mammalian cells by stimulating Na+/H+ exchange. We show that vanadate, previously found to act as a mitogen for a number of cells, reversibly activates Na+/H+ exchange at micromolar concentrations in A431 cells, leading to a large increase of pHi. The stimulation of Na+/H+ exchange by vanadate is not due to inhibition of the Na+/K+ ATPase and is unrelated to possible effects of vanadate on cAMP levels. Elevation of pHi by vanadate and by epidermal growth factor (EGF) both display similar kinetics, and both EGF and vanadate stimulate the rate of pHi recovery following an acute acid load, suggesting that vanadate stimulates Na+/H+ exchange by a mechanism similar to that of polypeptide growth factor stimulation. Thus, stimulation of Na+/H+ exchange may be a common property not only of polypeptide growth factors but also of other, chemically unrelated mitogens.     

Na+-H+ exchange in isolated renal brush-border membrane vesicles in response to metabolic acidosis. Kinetic effects

Chronic metabolic acidosis increased the Na+-H+ exchange activity in isolated renal brush-border membrane vesicles. Treatment altered the initial rate of Na+ uptake by increasing Vm (acidotic, 15.3 +/- 0.7 nmol of Na+ X mg-1 X 2 s-1; normal, 11.3 +/- 0.9 nmol of Na+ X mg-1 X 2 s-1), and not the apparent affinity KNa+ (acidotic, 10.2 +/- 0.5 mM; normal 10.2 +/- 0.6 mM). Metabolic acidosis resulted in the proportional increase in 1 mM Na+ uptake at every intravesicular pH measured. A positive cooperative effect on Na+ uptake was found with increased intravesicular acidity in vesicles from both normal and acidotic rats. When the data were analyzed by the Hill equation, it was found that metabolic acidosis did not change the n (acidotic, 1.33 +/- 0.13; normal, 1.43 +/- 0.07) or the K'H+ (acidotic, 0.27 +/- 0.05 microM; normal, 0.28 +/- 0.06 microM), but increased the apparent Vm (acidotic, 1.10 +/- 0.08 nmol of Na+ X mg-1 X 2 s-1; normal, 0.81 +/- 0.07 nmol of Na+ X mg-1 X 2 s-1). The uptake of Na+ in exchange for H+ in membrane vesicles from normal and acidotic animals was not influenced by membrane potential. We conclude that metabolic acidosis leads to either an increase in the number of functioning exchangers or an increase in the turnover rate of the limiting step in the exchange.     

Vasopressin, insulin and peroxide(s) of vanadate (pervanadate) influence Na+ transport mediated by (Na+, K+)ATPase or Na+/H+ exchanger of rat liver plasma membrane vesicles

Uptake of 22Na+ by liver plasma membrane vesicles, reflecting Na+ transport by (Na+, K+)ATPase or Na+/H+ exchange was studied. Membrane vesicles were isolated from rat liver homogenates or from freshly prepared rat hepatocytes incubated in the presence of [Arg8]vasopressin or pervanadate and insulin. The ATP dependence of (Na+, K+)ATPase-mediated transport was determined from initial velocities of vanadate-sensitive uptake of 22Na+, the Na(+)-dependence of Na+/H+ exchange from initial velocities of amiloride-sensitive uptake. By studying vanadate-sensitive Na+ transport, high-affinity binding sites for ATP with an apparent Km(ATP) of 15 +/- 1 microM were observed at low concentrations of Na+ (1 mM) and K+ (1mM). At 90 mM Na+ and 60 mM K+ the apparent Km(ATP) was 103 +/- 25 microM. Vesiculation of membranes and loading of the vesicles prepared from liver homogenates in the presence of vasopressin increased the maximal velocities of vanadate-sensitive transport by 3.8-fold and 1.9-fold in the presence of low and high concentrations of Na+ and K+, respectively. The apparent Km(ATP) was shifted to 62 +/- 7 microM and 76 +/- 10 microM by vasopressin at low and high ion concentrations, respectively, indicating that the hormone reduced the influence of Na+ and K+ on ATP binding. In vesicles isolated from hepatocytes preincubated with 10 nM vasopression the hormone effect was conserved. Initial velocities of Na+ uptake (at high ion concentrations and 1 mM ATP) were increased 1.6-1.7-fold above control, after incubation of the cells with vasopressin or by affinity labelling of the cells with a photoreactive analogue of the hormone. The velocity of amiloride-sensitive Na+ transport was enhanced by incubating hepatocytes in the presence of 10 nM insulin (1.6-fold) or 0.3 mM pervanadate generated by mixing vanadate plus H2O2 (13-fold). The apparent Km(Na+) of Na+/H+ exchange was increased by pervanadate from 5.9 mM to 17.2 mM. Vesiculation and incubation of isolated membranes in the presence of pervanadate had no effect on the velocity of amiloride-sensitive Na+ transport. The results show that hormone receptor-mediated effects on (Na+, K+)ATPase and Na+/H+ exchange are conserved during the isolation of liver plasma membrane vesicles. Stable modifications of the transport systems or their membrane environment rather than ionic or metabolic responses requiring cell integrity appear to be involved in this regulation.     

Na+/H+ exchange activity in the plasma membrane of Arabidopsis

In plants, Na+/H+ exchangers in the plasma membrane are critical for growth in high levels of salt, removing toxic Na+ from the cytoplasm by transport out of the cell. The molecular identity of a plasma membrane Na+/H+ exchanger in Arabidopsis (SOS1) has recently been determined. In this study, immunological analysis provided evidence that SOS1 localizes to the plasma membrane of leaves and roots. To characterize the transport activity of this protein, purified plasma membrane vesicles were isolated from leaves of Arabidopsis. Na+/H+ exchange activity, monitored as the ability of Na to dissipate an established pH gradient, was absent in plants grown without salt. However, exchange activity was induced when plants were grown in 250 mm NaCl and increased with prolonged salt exposure up to 8 d. H+-coupled exchange was specific for Na, because chloride salts of other monovalent cations did not dissipate the pH gradient. Na+/H+ exchange activity was dependent on Na (substrate) concentration, and kinetic analysis indicated that the affinity (apparent Km) of the transporter for Na+ is 22.8 mm. Data from two experimental approaches supports electroneutral exchange (one Na+ exchanged for one proton): (a) no change in membrane potential was measured during the exchange reaction, and (b) Na+/H+ exchange was unaffected by the presence or absence of a membrane potential. Results from this research provide a framework for future studies into the regulation of the plant plasma membrane Na+/H+ exchanger and its relative contribution to the maintenance of cellular Na+ homeostasis during plant growth in salt.     

Protons as substitutes for sodium and potassium in the sodium pump reaction

The role of protons as substitutes for Na+ and/or K+ in the sodium pump reaction was examined using inside-out membrane vesicles derived from human red cells. Na+-like effects of protons suggested previously (Blostein, R. (1985) J. Biol. Chem. 260, 829-833) were substantiated by the following observations: (i) in the absence of extravesicular (cytoplasmic) Na+, an increase in cytoplasmic [H+] increased both strophanthidin-sensitive ATP hydrolysis (nu) and the steady-state level of phosphoenzyme, EP, and (ii) as [H+] is increased, the Na+/ATP coupling ratio is decreased. K+-like effects of protons were evidenced in the following results: (i) an increase in nu, decrease in EP, and hence increase in EP turnover (nu/EP) occur when intravesicular (extracellular) [H+] is increased; (ii) an increase in the rate of Na+ influx into K+(Rb+)-free inside-out vesicles and (iii) a decrease in Rb+/ATP coupling occur when [H+] is increased. Direct evidence for H+ being translocated in place of cytoplasmic Na+ and extracellular K+ was obtained by monitoring pH changes using fluorescein isothiocyanate-dextran-filled vesicles derived from 4',4-diisothiocyano-2',2-stilbene disulfonate-treated cells. With the initial pHi = pHo = pH 6.2, a strophanthidin-sensitive decrease in pHi was observed following addition of ATP provided the vesicles contained K+. This pH gradient was abolished following addition of Na+. With alkali cation-free inside-out vesicles, a strophanthidin-sensitive increase in pH was observed upon addition of both ATP and Na+. The foregoing changes in pHi were not affected by the addition of tetrabutylammonium to dissipate any membrane potential and were not observed at pH 6.8. These ATP-dependent cardiac glycoside-sensitive proton movements indicate Na,K-ATPase mediated Na+/H+ exchange in the absence of extracellular K+ as well as H+/K+ exchange in the absence of cytoplasmic Na+.     

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.     

Mechanism of activation of lymphocyte Na+/H+ exchange by concanavalin A. A calcium- and protein kinase C-independent pathway

Treatment of thymic lymphocytes with the mitogenic lectin concanavalin A (ConA) increases the intracellular free Ca2+ concentration and stimulates phosphoinositide turnover. ConA also induced a rapid, amiloride-sensitive, Na+-dependent increase in cytosolic pH of 0.13 +/- 0.01, indicative of stimulation of the Na+/H+ antiport. To investigate the mechanism underlying activation of Na+/H+ exchange by ConA, the intracellular free Ca2+ concentration changes induced by this lectin were precluded by loading the cells with Ca2+-buffering agents and suspension in Ca2+-free media. Under these conditions, the ConA-induced cytoplasmic alkalinization proceeded normally. Two approaches were used to assess the role of protein kinase C. First, this enzyme was inhibited by the addition of 1-(5-isoquinolinysulfonyl)-2-methylpiperazine. In the presence of this potent antagonist, stimulation of the antiport by 12-O-tetradecanoylphorbol-13-acetate was greatly inhibited. In contrast, stimulation by ConA was unaffected. Second, protein kinase C was depleted by overnight incubation with phorbol esters. Following this treatment, Na+/H+ exchange was no longer activated by 12-O-tetradecanoyl-13-acetate, but was still stimulated by ConA. These data suggest that a Ca2+- and protein kinase C-independent mechanisms mediates the activation of Na+/H+ exchange by ConA. The possible role of GTP-binding proteins in the activation was also studied. The antiport was not stimulated by either fluoroaluminate or vanadate. Moreover, pretreatment with pertussis toxin failed to inhibit the ConA-induced cytoplasmic alkalinization. In contrast, preincubation with cholera toxin partially inhibited activation. Under these conditions, cholera toxin significantly elevated intracellular cAMP levels. Inhibition was also observed in cells treated with forskolin at concentrations that increased [cAMP]. The data suggest that a novel cAMP-sensitive signaling mechanism not involving Ca2+ and protein kinase C is involved in the stimulation of Na+/H+ exchange by mitogens in T lymphocytes.     

Reduced contribution from Na+/H+ exchange to acid extrusion during anoxia in adult rat hippocampal CA1 neurons

The effect of anoxia on Na+/H+ exchange activity was examined in acutely isolated adult rat hippocampal CA1 neurons loaded with the H+-sensitive fluorophore, BCECF. Five-minute anoxia imposed under nominally HCO3-/CO2-free conditions induced a fall in pHi, the magnitude of which was smaller following prolonged exposure to medium in which N-methyl-D-glucamine (NMDG+) was employed as an extracellular Na+ (Na(+)(o)) substitute. Also consistent with the possibility that Na+/H+ exchange becomes inhibited soon after the induction of anoxia, rates of Na(+)(o)-dependent pHi recovery from internal acid loads imposed during anoxia were slowed, compared to rates of Na(+)(o)-dependent pHi recovery observed prior to anoxia. At the time at which rates of pHi recovery were reduced during anoxia, cellular adenosine triphosphate (ATP) levels had fallen to 35% of preanoxic levels, suggesting that ATP depletion might contribute to the observed inhibition of Na+/H+ exchange. In support, incubation of neurons with 2-deoxyglucose and antimycin A under normoxic conditions induced a fall in cellular ATP levels that was also associated with reduced Na(+)(o)-dependent rates of pHi recovery from imposed acid loads; conversely, pre-treatment with 10 mm creatine attenuated the effects of anoxia to reduce both ATP levels and Na(+)(o)-dependent rates of pHi recovery from internal acid loads. Taken together, the results are consistent with the possibility that functional Na+/H+ exchange activity in adult rat CA1 neurons declines soon after the onset of anoxia, possibly as a result of anoxia-induced falls in intracellular ATP.     

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.     

Characterization of potent Na+/H+ exchange inhibitors from the amiloride series in A431 cells

Na+/H+ exchange is stimulated in a variety of cell types by addition of mitogenic polypeptides such as epidermal growth factor or platelet-derived growth factor. In order to assess the importance of Na+/H+ exchange in the mitogenic response, it is desirable to have available inhibitors of this process which exhibit high affinity and good specificity. We characterize in this report a number of 5-alkylamino-substituted derivatives of amiloride [3,5-diamino-6-chloro-N-(diaminomethylene)pyrazinecarboxamide++ +] which show much higher affinity than the parent compound for the Na+/H+ antiporter in A431 cells. High affinity is conferred by substitution with two alkyl groups and is increased by introducing a branched alkyl chain. An analogue bearing a 5-anilino group is also very potent. These analogues effectively inhibit the elevation of intracellular pH upon stimulation of Na+/H+ exchange by growth factors. We have assessed other potential inhibitory effects of these compounds on cellular metabolism. In agreement with previous reports, we find that amiloride inhibits protein synthesis both in cells and in cell-free translation systems. While amiloride and its analogues show similar inhibition of protein synthesis in a cell-free system, most analogues inhibit cellular protein synthesis at much lower concentrations than does amiloride. These analogues are also potent inhibitors of purified Na,K-ATPase and cause a profound decrease in intracellular K+ as well as ATP content. These latter effects, however, require analogue concentrations which are 5-7 times higher than those inhibiting cellular protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)