Selective inhibition of sodium-linked and sodium-independent bicarbonate/chloride antiport in Vero cells

The effects of compounds previously described to inhibit anion transport were tested for their ability to inhibit anion antiport in Vero cells as measured by uptake of 36Cl- by chloride self-exchange and as bicarbonate-linked uptake of 22Na+. While 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibited both processes, ethacrynic acid and probenecid selectively inhibited the uptake of 36Cl-. Low concentrations of pyridoxal phosphate and picrylsulfonic acid selectively inhibited the bicarbonate linked uptake of 22Na+, while higher concentrations of these compounds also inhibited the uptake of 36Cl-. Measurements of the internal pH indicated that ethacrynic acid inhibits Na+-independent HCO-3/Cl- exchange, while it has no measurable effect on Na+-linked bicarbonate-dependent regulation of the internal pH. Conversely, picrylsulfonic acid selectively inhibits the latter process. The results indicate that anion antiport in Vero cells occurs by two independent processes.     

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.     

Bicarbonate/chloride antiport in Vero cells: II. Mechanisms for bicarbonate-dependent regulation of intracellular pH

The rates of bicarbonate-dependent uptake and efflux of 22Na+ in Vero cells were studied and compared with the uptake and efflux of 36Cl-. Both processes were strongly inhibited by DIDS. Whereas the transport of chloride increased approximately ten-fold when the internal pH was increased over a narrow range around neutrality, the uptake of Na+ was much less affected by changes in pH. The bicarbonate-linked uptake of 22Na+ was dependent on internal Cl- but not on internal Na+. At a constant external concentration of HCO3-, the amount of 22Na+ associated with the cells increased when the internal concentration of HCO3- decreased and vice versa, which is compatible with the possibility that the ion pair NaCO3- is the transported species and that the transport is symmetric across the membrane. Bicarbonate inhibited the uptake of 36Cl- both in the absence and presence of Na+. At alkaline internal pH, HCO3- stimulated the efflux of 36Cl- from preloaded cells, while at acidic internal pH both Na+ and HCO3- were required to induce 36Cl- efflux. We propose a model for how bicarbonate-dependent regulation of the internal pH may occur. This model implies the existence of two bicarbonate transport mechanisms that, under physiological conditions, transport OH(-)-equivalents in opposite directions across the plasma membrane.     

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.     

Relief from alkaline load in two-cell stage mouse embryos by bicarbonate/chloride exchange

Mouse embryos at the two-cell stage are able to recover from an alkaline load. We found that this recovery is mediated by sodium-independent bicarbonate/chloride exchange: intracellular pH (pHi) recovery from alkaline load is inhibited by the anion exchange inhibitor 4,4'-diisothiocyanostilbene disulfonic acid, lack of bicarbonate, or lack of chloride. The dependence of the pHi recovery on extracellular chloride concentration exhibits Michaelis-Menten kinetics. Furthermore, uptake of chloride is inhibited in a dose-dependent manner by extracellular bicarbonate. The Km for external chloride was found to be about 3 mM, with a Ki for external bicarbonate of about 2 mM. The exchanger is active above approximately pH 7.15. These results demonstrate that mouse embryos at the two-cell stage possess a sodium-independent bicarbonate/chloride exchange mechanism that is similar to that found in other mammalian cells. This bicarbonate/chloride exchanger appears to be the sole pHi-regulatory mechanism in the two-cell stage mouse embryo, since our previous results have shown that there are apparently no specific mechanisms active in these cells for relieving acid loads.     

Properties of the Na+-dependent Cl-/HCO3- exchange system in U937 human leukemic cells

U937 cell possess two mechanisms that allow them to recover from an intracellular acidification. The first mechanism is the amiloride-sensitive Na+/H+ exchange system. The second system involves bicarbonate ions. Its properties have been defined from intracellular pH (pHi) recovery experiments, 22Na+ uptake experiments, 36Cl- influx and efflux experiments. Bicarbonate induced pHi recovery of the cells after a cellular acidification to pHi = 6.3 provided that Na+ ions were present in the assay medium. Li+ or K+ could not substitute for Na+. The system seemed to be electroneutral. 22Na+ uptake experiments showed the presence of a bicarbonate-stimulated uptake pathway for Na+ which was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate. The bicarbonate-dependent 22Na+ uptake component was reduced by depleting cells of their internal Cl- and increased by removal of external Cl-. 36Cl- efflux experiments showed that the presence of both external Na+ and bicarbonate stimulated the efflux of 36Cl- at a cell pHi of 6.3. Finally a 36Cl- uptake pathway was documented. It was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonate (K0.5 = 10 microM) and bicarbonate (K0.5 = 2 mM). These results are consistent with the presence in U937 cells of a coupled exchange of Na+ and bicarbonate against chloride. It operates to raise the intracellular pH. Its pHi and external Na+ dependences were defined. No evidence for a Na+-independent Cl-/HCO3- exchange system could be found. The Na+-dependent Cl-/HCO3- exchange system was relatively insensitive to (aryloxy)alkanoic acids which are potent inhibitors of bicarbonate-induced swelling of astroglia and of the Li(Na)CO3-/Cl- exchange system of human erythrocytes. It is concluded that different anionic exchangers exist in different cell types that can be distinguished both by their biochemical properties and by their pharmacological properties.     

Proton transport catalyzed by the sodium pump. Ouabain-sensitive ATPase activity and the phosphorylation of Na,K-ATPase in the absence of sodium ions

The possibility that H+ might substitute for Na+ at Na+ sites of Na+,K+-ATPase was studied. Na+,K+-ATPase purified from pig kidney showed ouabain-sensitive K+-dependent ATPase activity in the absence of Na+ at acid pH (H+,K+-ATPase). The specific activity was 1.1 mumol Pi/mg/min at pH 5.7, whereas the specific activity of Na+,K+-ATPase was 14 mumol Pi/mg/min at pH 7.5. The enzyme was phosphorylated from ATP in the absence of Na+ at the acid pH. The initial rate of the phosphorylation was also accelerated at the acid pH in the absence of Na+, and the maximal rate obtained at pH 5.5 without Na+ was 9% of the rate at pH 7.0 with Na+. The phosphoenzyme was sensitive to K+ but almost insensitive to ADP. The phosphoenzyme was sensitive to hydroxylamine treatment and the alpha-subunit of the enzyme was found to be phosphorylated. H+,K+-ATPase was inhibited as effectively as Na+,K+-ATPase by N-ethylmaleimide but was less inhibited by oligomycin or dimethyl sulfoxide. These results indicate that protons have an Na+-like effect on the Na+ sites of Na+,K+-ATPase and suggest that protons can be transported by the sodium pump in place of Na+.     

The sodium/proton antiport system in a newly isolated alkalophilic Bacillus sp.

The pH homeostasis and the sodium/proton antiport system have been studied in the newly isolated alkalophilic Bacillus sp. strain N-6, which could grow on media in a pH range from 7 to 10, and in its nonalkalophilic mutant. After a quick shift in external pH from 8 to 10 by the addition of Na2CO3, the delta pH (inside acid) in the cells of strain N-6 was immediately established, and the pH homeostatic state was maintained for more than 20 min in an alkaline environment. However, under the same conditions, the pH homeostasis was not observed in the cells of nonalkalophilic mutant, and the cytoplasmic pH immediately rose to pH 10. On the other hand, the results of the rapid acidification from pH 9 to 7 showed that the internal pH was maintained as more basic than the external pH in a neutral medium in both strains. The Na+/H+ antiport system has been characterized by either the effect of Na+ on delta pH formation or 22Na+ efflux in Na+-loaded right-side-out membrane vesicles of strain N-6. Na+- or Li+-loaded vesicles exhibited a reversed delta pH (inside acid) after the addition of electron donors (ascorbate plus tetramethyl-p-phenylenediamine) at both pH 7 and 9, whereas choline-loaded vesicles generated delta pHs of the conventional orientation (inside alkaline). 22Na+ was actively extruded from 22Na+-loaded vesicles whose potential was negative at pH 7 and 9. The inclusion of carbonyl cyanide m-chlorophenylhydrazone inhibited 22Na+ efflux in the presence of electron donors. These results indicate that the Na+/H+ antiport system in this strain operates electrogenically over a range of external pHs from 7 to 10 and plays a role in pH homeostasis at the alkaline pH range. The pH homeostasis at neutral ph was studied in more detail. K+ -depleted cells showed no delta pH (acid out) in the neutral conditions in the absence of K+, whereas these cells generated a delta pH if K+ was present in the medium. This increase of internal pH was accompanied by K+ uptake from the medium. These results suggest that electrogenic K+ entry allows extrusion of H+ from cells by the primary proton pump at neutral pH.     

Kidney epithelial cells of monkey origin (BSC-1) express a sodium bicarbonate cotransport. Characterization by 22Na+ flux measurements

Na movement across the plasma membranes of confluent monolayers of monkey kidney epithelial cells (BSC-1) was studied using 22Na+ uptake and efflux techniques in the presence of 10(-4) M ouabain. In the presence of 28 mM bicarbonate, uptake was inhibited by both 10(-3) M amiloride and 10(-3) M 4,4'diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). In DIDS-pretreated cells, 10(-3) M amiloride led to a further reduction of 22Na+ uptake, while 10(-5) furosemide was ineffective. DIDS also inhibited sodium efflux, indicating that the DIDS-sensitive pathway mediates both influx and efflux of 22Na+. DIDS-sensitive 22Na+ uptake, as studied in the presence of both 10(-4) M ouabain and 10(-3) M amiloride, was abolished by the absence of bicarbonate, which could not be substituted by other plasma membrane-permeable buffers. In 28 mM HCO3-, DIDS-sensitive uptake of 28 mM Na+ was cis-inhibited by 124 mM Na+, but no significant inhibition by K+ or Li+ was found. DIDS-sensitive 22Na+ uptake was a saturable function of both Na+ concentration (apparent Km between 20 and 40 mM at 28 mM HCO3-) and HCO3- concentration (apparent Km between 7 and 14 mM at 151 mM Na+). Intracellular microelectrode measurements showed that net Na+ transport in the presence of HCO3- is electrogenic, i.e. that there is anion cotransport with Na+. This effect is abolished by 1 mM DIDS. It is concluded that monkey kidney epithelial cells possess a stilbene-sensitive, electrogenic sodium bicarbonate symport, which may play an important role in bicarbonate reabsorption in the mammalian kidney.     

Regulation of internal pH of sea urchin sperm. A role for the Na/K pump

In the absence of sodium, sea urchin sperm have an acidic internal pH. The addition of sodium, lithium, or ammonium, but not of potassium ions, induces an internal alkalization. If potassium is added in the presence of sodium, a further alkalization is obtained; in contrast, potassium addition in presence of Li+ or NH+4 does not change the internal pH. The K+-induced pHi change is inhibited by ouabain and when sperm are depleted of their ATP. A large part of the potassium influx is stimulated by Na+, but not Li+, and inhibited by ouabain and cellular ATP depletion. We conclude that activity of Na/K-ATPase pumps located in the plasma membrane of sea urchin sperm could play a role in regulating the internal pH of sea urchin sperm by recycling sodium ions that enter the cell through Na/H countermovements.     

Na+/H+ exchange regulates intracellular pH in a cell clone derived from bovine pigmented ciliary epithelium

The regulation of intracellular pH (pHi) was monitored in a virus-transformed cell clone derived from bovine ciliary body exhibiting characteristics of pigmented ciliary epithelium. Data were obtained from confluent monolayers grown on plastic coverslips in nominally bicarbonate-free media using the pH-sensitive absorbance of 5- (and 6-) carboxy-4',5'-dimethylfluorescein. Under resting conditions, pHi averaged 6.98 +/- 0.01 (SEM; n = 57). When cells were acid loaded by briefly exposing them to Ringer containing NH4+ and then withdrawing the NH4+, pHi spontaneously regained its initial value. In the presence of 1 mM amiloride or in the absence of Na+, this process was blocked, indicating the involvement of an Na+/H+ exchanger in the regulation of pHi after an acid load. Removing Na+ during resting conditions decreased cytoplasmatic pH. This acidification could be slowed by amiloride, which is evidence for reversal of the Na+/H+ countertransport exchanging intracellular Na+ for extracellular protons. Application of 1 mM amiloride during steady state led to a slow acidification. Thus the Na+/H+ exchanger is operative during resting conditions extruding protons, derived from cellular metabolism, or from downhill leakage into the cell. Addition of Na+ to Na+ -depleted cells led to an alkalinization, which was sensitive to amiloride, with an IC50 of about 20 microM. This alkalinization was attributed to the Na+/H+ exchanger and exhibited saturation kinetics with increasing Na+ concentrations, with an apparent KM of 29.6 mM Na+. It is concluded that Na+/H+ exchange regulates pHi during steady state and after an acid load.     

Inhibition of Na(+)-independent H+ pump by Na(+)-induced changes in cell Ca2+

Apical membrane H+ extrusion in the renal outer medullary collecting duct, inner stripe, is mediated by a Na(+)-independent H+ pump. To examine the regulation of this transporter, cell pH and cell Ca2+ were measured microfluorometrically in in vitro perfused tubules using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein and fura-2, respectively. Apical membrane H+ pump activity, assayed as cell pH recovery from a series of acid loads (NH3/NH+4 prepulse) in the total absence of ambient Na+, initially occurred at a slow rate (0.06 +/- 0.02 pH units/min), which was not sufficient to account for physiologic rates of H+ extrusion. Over 15-20 min after the initial acid load, the rate of Na(+)-independent cell pH recovery increased to 0.63 +/- 0.09 pH units/min, associated with a steady-state cell pH greater than the initial pre-acid load cell pH. This pattern suggested an initial suppression followed by a delayed activation of the apical membrane H+ pump. Replacement of peritubular Na+ with choline or N-methyl-D-glucosamine resulted in an initial spike increase in cell Ca2+ followed by a sustained increase in cell Ca2+. The initial rate of Na(+)-independent cell pH recovery could be increased by elimination of the Na+ removal-induced sustained cell Ca2+ elevation by: (a) performing studies in the presence of 135 mM peritubular Na+ (1 mM peritubular amiloride used to inhibit basolateral membrane Na+/H+ antiport); (b) clamping cell Ca2+ low with dimethyl-BAPTA, an intracellular Ca2+ chelating agent; or (c) removal of extracellular Ca2+. Cell acidification induced a spike increase in cell Ca2+. The late acceleration of Na(+)-independent cell pH recovery was independent of Na+ removal and of the method used to acidify the cell, but was eliminated by prevention of the cell Ca2+ spike and markedly delayed by the microfilament-disrupting agent, cytochalasin B. This study demonstrates that peritubular Na+ removal results in a sustained elevation in cell Ca2+, which inhibits the apical membrane H+ pump. In addition, rapid cell acidification associated with a spike increase in cell Ca2+ leads to a delayed activation of the H+ pump. Thus, cell Ca2+ per se, or a Ca(2+)-activated pathway, can modulate H+ pump activity.     

Na+ requirement for growth, photosynthesis, and pH regulation in the alkalotolerant cyanobacterium Synechococcus leopoliensis

We have found that Na+ is required for the alkalotolerance of the cyanobacterium Synechococcus leopoliensis. Cell division did not occur at any pH in the absence of Na+, but cells inoculated into Na+-free growth medium at pH 6.8 did continue metabolic activity, and over a period of 48 h, the cells became twice their normal size. Many of these cells remained viable for at least 59 h and formed colonies on Na+ -containing medium. Cells grown in the presence of Na+ and inoculated into Na+ -free growth medium at pH 9.6 rapidly lost viability. An Na+ concentration of ca. 0.5 milliequivalents X liter-1 was required for sustained growth above pH 9.0. The Na+ requirement could be only partially met by Li+ and not at all by K+ or Rb+. Cells incubated in darkness in growth medium at pH 6.8 had an intracellular pH near neutrality in the presence or absence of Na+. When the external pH was shifted to 9.6, only cells in the presence of Na+ were able to maintain an intracellular pH near 7.0. The membrane potential, however, remained high (-120 mV) in the absence or presence of Na+ unless collapsed by the addition of gramicidin. Thus, the inability to maintain a neutral intracellular pH at pH 9.6 in the absence of Na+ was not due to a generalized disruption of membrane integrity.Even cells containing Na+ still required added Na+ to restore photosynthetic rates to normal after the cells had been washed in Na+ -free buffer at pH 9.6. This requirement was only partially met by Li+ and was not met at all by K+, Rb+, Cs+ Mg2+, or Ca2+. The restoration of photosynthesis by added Na+ occurred within 30 s and suggests a role for extracellular Na+. Part of our results can be explained in terms of the operation of an Na+/H+ antiporter activity in the plasma membrane, but some results would seem to require other mechanisms for Na+ action.     

Intracellular pH and Catecholamine Secretion from Bovine Adrenal Chromaffin Cells

To study the role of intracellular pH (pHi) in catecholamine secretion and the regulation of pHi in bovine chromaffin cells, the pH-sensitive fluorescent indicator [2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein] was used to monitor the on-line changes in pHi. The pHi of chromaffin cells at resting state is approximately 7.2. The pHi was manipulated first by incubation of the cells with NH4+, and then the solution was replaced with a NH4(+)-free solution to induce acidification of the cytoplasm. The pHi returned toward the basal pH value after acidification within 5-10 min in the presence of Na+ or Li+, but the pHi stayed acidic when Na(+)-free buffers were used or in the presence of amiloride and its analogues. These results suggest that the pH recovery process after an acid load is due to the Na+/H+ exchange activity in the plasma membrane of the chromaffin cells. The catecholamine secretion evoked by carbachol and Na+ removal was enhanced after the cytoplasm had been made more acidic. It appears that acidic pH favors the occurrence of exocytosis.     

Distinct effects of various amino acids on 45Ca2+ fluxes in rat pancreatic islets.

The effects of three types of amino acids on 45Ca2+ fluxes in rat pancreatic islets have been compared. Alanine, a non-insulinotropic neutral amino acid, transported with Na+, increased 45Ca2+ efflux in the presence or in the absence of extracellular Ca2+, but not in the absence of Na+. Its effects in Na+-solutions were practically abolished by 7 mM-glucose. Alanine slightly stimulated 45Ca2+ influx (5 min uptake) only when Na+ was present. Two insulinotropic cationic amino acids (arginine and lysine) triggered similar changes in 45Ca2+ efflux. They accelerated the efflux in the presence of Ca2+ and inhibited the efflux in a Ca2+-free medium, whether glucose was present or not. In an Na+-free Ca2+-medium, arginine and lysine markedly accelerated 45Ca2+ efflux, but this effect was suppressed by 7 mM-glucose. Arginine stimulated 45Ca2+ influx irrespective of the presence or absence of glucose and Na+. Leucine, a neutral insulinotropic amino acid well metabolized by islet cells, inhibited 45Ca2+ efflux from the islets in a Ca2+-free medium; this effect was potentiated by glutamine. In the presence of Ca2+ and Na+, leucine was ineffective alone, but triggered a marked increase in 45Ca2+ efflux when combined with glutamine. In an Na+-free Ca2+-medium, leucine accelerated 45Ca2+ efflux to the same extent with or without glutamine. Leucine also stimulated 45Ca2+ influx in the presence or in the absence of Na+, but its effects were potentiated by glutamine only in the presence of Na+. The results show that amino acids of various types cause distinct changes in 45Ca2+ fluxes in pancreatic islets. Certain of these changes involve an Na+-mediated mobilization of cellular Ca2+ from sequestering sites where glucose appears to exert an opposite effect.     

Nutrient-induced changes in the pH of pancreatic islet cells

Fluorescein rapidly accumulates in rat pancreatic islets exposed to fluorescein diacetate. The influence of environmental agents upon cellular pH was examined in fluorescein-labelled islets by recording their fluorescence intensity at 520 nm after excitation at 490 and 435 nm, respectively. Glucose caused a rapid, sustained and dose-related increase in cellular pH. Another nutrient secretagogue, 2-ketoisocaproic acid, also increased cellular pH. The stimulation of islet cells by non-nutrient secretagogues, e.g. by glibenclamide or in response to an increase in extracellular K+ concentration, decreased cellular pH, indicating that the nutrient-induced increase in cellular pH is not merely a consequence of stimulated Ca2+ inflow and/or insulin release. In either the presence of amiloride or absence of bicarbonate, glucose decreased cellular pH. These results strongly suggest that the acidification of islet cells which can be expected from the increased metabolism of glucose in glucose-stimulated islets is normally masked and overcome by stimulation of such processes as Na+/H+ and HCO3-/Cl- exchange.     

Roles of K+ and Na+ in pH homeostasis and growth of the marine bacterium Vibrio alginolyticus.

The marine bacterium Vibrio alginolyticus, containing 470 mM-K+ and 70 mM-Na+ inside its cells, was able to regulate the cytoplasmic pH (pH(in)) in the narrow range 7.6-7.8 over the external pH (pH(out)) range 6.0-9.0 in the presence of 400 mM-Na+ and 10 mM-K+. In the absence of external K+, however, pHin was regulated only at alkaline pH(out) values above 7.6. When the cells were incubated in the presence of unusually high K+ (400 mM) and 4 mM Na+, the pH(in) was regulated only at acidic pH(out) values below 7.6. These results could be explained by postulating a K+/H+ antiporter as the regulator of pH(in) over the pH(out) range 6.0-9.0. When Na(+)-loaded/K(+)-depleted cells were incubated in 400 mM-Na+ in the absence of K+, an inside acidic delta pH was generated at pH(out) values above 7.0. After addition of diethanolamine the inside acidic delta pH collapsed transiently and then returned to the original value concomitant with the extrusion of Na+, suggesting the participation of a Na+/H+ antiporter for the generation of an inside acidic delta pH. In the presence of 400 mM-K+, at least 5 mM-Na+ was required to support cell growth at pH(out) below 7.5. An increase in Na+ concentration allowed the cells to grow at a more alkaline pH(out). Furthermore, cells containing more Na+ inside could more easily adapt to grow at alkaline pH(out). These results indicated the importance of Na+ in acidification of the cell interior via a Na+/H+ antiporter in order to support cell growth at alkaline pH(out) under conditions where the activity of a K+/H+ antiporter is marginal.     

The regulation of intracellular pH in monkey kidney epithelial cells (BSC-1). Roles of Na+/H+ antiport, Na+-HCO3(-)-(NaCO3-) symport, and Cl-/HCO3- exchange

Using the pH-sensitive absorbance of 5 (and 6)-carboxy-4',5'- dimethylfluorescein, we investigated the regulation of cytoplasmic pH (pHi) in monkey kidney epithelial cells (BSC-1). In the absence of HCO3-, pHi is 7.15 +/- 0.1, which is not significantly different from pHi in 28 mM HCO3-, 5% CO2 (7.21 +/- 0.07). After an acid load, the cells regulate pHi in the absence of HCO3- by a Na+ (or Li+)-dependent, amiloride-inhibitable mechanism (indicative of Na+/H+ antiport). In 28 mM HCO3-, while still dependent on Na+, this regulation is only blocked in part by 1 mM amiloride. A partial block is also observed with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) (1 mM). With cells pretreated with DIDS, 1 mM amiloride nearly totally inhibits this regulation. Cl- had no effect on pHi regulation in the acidic range. In HCO3(-)-free saline, Na+ removal leads to an amiloride-insensitive acidification, which is dependent on Ca2+. In 28 mM HCO3-, Na+ (and Ca2+) removal led to a pronounced reversible and DIDS-sensitive acidification. When HCO3- was lowered from 46 to 10 mM at constant pCO2 (5%), pHi dropped by a DIDS-sensitive mechanism. Identical changes in pHo (7.6 to 6.9) in the nominal absence of HCO3- led to smaller changes of pHi. In the presence but not in the absence of HCO3-, removal of Cl- led to a DIDS-sensitive alkalinization. This was also observed in the nominal absence of Na+, which leads to a sustained acidification. It is concluded that in nominally bicarbonate-free saline, the amiloride-sensitive Na+/H+ antiport is the predominant mechanism of pHi regulation at acidic pHi, while being relatively inactive at physiological values of pHi. In bicarbonate saline, two other mechanisms effect pHi regulation: a DIDS-sensitive Na+-HCO3- symport, which contributes to cytoplasmic alkalinization, and a DIDS-sensitive Cl-/HCO3- exchange, which is apparently independent of Na+.     

Role of Na+ cycle in cell volume regulation of Mycoplasma gallisepticum.

The mechanism for the extrusion of Na+ from Mycoplasma gallisepticum cells was examined. Na+ efflux from cells was studied by diluting 22Na+-loaded cells into an isoosmotic NaCl solution and measuring the residual 22Na+ in the cells. Uphill 22Na+ efflux was found to be glucose dependent and linear with time over a 60-s period and showed almost the same rate in the pH range of 6.5 to 8.0. 22Na+ efflux was markedly inhibited by dicyclohexylcarbodiimide (DCCD, 10 microM), but not by the proton-conducting ionophores SF6847 (0.5 microM) or carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 microM) over the entire pH range tested. An ammonium diffusion potential and a pH gradient were created by diluting intact cells or sealed membrane vesicles of M. gallisepticum loaded with NH4Cl into a choline chloride solution. The imposed H+ gradient (inside acid) was not affected by the addition of either NaCl or KCl to the medium. Dissipation of the proton motive force by CCCP had no effect on the growth of M. gallisepticum in the pH range of 7.2 to 7.8 in an Na+-rich medium. Additionally, energized M. gallisepticum cells were stable in an isoosmotic NaCl solution, even in the presence of proton conductors, whereas nonenergized cells tended to swell and lyse. These results show that in M. gallisepticum Na+ movement was neither driven nor inhibited by the collapse of the electrochemical gradient of H+, suggesting that in this organism Na+ is extruded by an electrogenic primary Na+ pump rather than by an Na+-H+ exchange system energized by the proton motive force.     

Effect of internally loaded iodide, thiocyanate, and perchlorate on sodium-dependent iodide uptake by phospholipid vesicles reconstituted with thyroid plasma membranes: iodide counterflow mediated by the iodide transport carrier

Na+-dependent I- transport and I- counterflow were studied using phospholipid vesicles (P-vesicles) made of porcine thyroid plasma membranes and soybean phospholipid by sonication. 1) I- uptake by P-vesicles incubated in the presence of external Na+ was higher than that by P-vesicles incubated in choline+ instead of Na+. The vesicles exhibited Na+-dependent I- uptake. When P-vesicles were internally loaded with I- prior to incubation in Na+, a further increase in Na+-dependent I- uptake was observed, although the concentration of internal I- was very much higher than that outside. In the absence of external Na+, I- uptake by P-vesicles preloaded with I- was comparable to baseline uptake. 2) Na+-dependent I- uptake by P-vesicles not loaded with I- and enhanced Na+-dependent I- uptake by P-vesicles preloaded with I- were both inhibited by either of SCN- and ClO4- added outside the vesicles. 3) When P-vesicles were loaded with SCN- instead of I- and incubated in Na+, I- uptake by these vesicles was also higher than baseline Na+-dependent I- uptake. However, a ClO4- load did not result in an increase in I- uptake. These results indicate that Na+-dependent I- transport including Na+-dependent I- counterflow is specifically mediated by the thyroid I- carrier. SCN- - I- counterflow in addition to I- - I- counterflow occurs dependently on Na+, but ClO4- - I- counterflow does not.