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+.     

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.     

Two-cell stage mouse embryos appear to lack mechanisms for alleviating intracellular acid loads.

Mouse embryos at the two-cell stage, like other cells, can recover from an intracellular acid-load. Our previous work has shown, surprisingly, that there is no contribution to this recovery by Na+/H+ antiport activity. Here we show that the recovery similarly is not affected by inhibition of other known intracellular pH (pHi) regulatory mechanisms. Specifically, the recovery is unaffected by lack of external Na+, inhibition of anion exchange, or lack of bicarbonate, which eliminates the Na(+)-dependent HCO3-/Cl- exchanger as a possible mechanisms. These conditions also eliminate any possible Na+,HCO3- cotransporter operating to relieve acid-loading. Recovery is unaffected similarly by nonspecific inhibitors of H(+)-ATPase activity. These observations lead to the conclusion that recovery from acid-load is a passive process in the two-cell mouse embryo. Similarly, the mean base-line pHi (6.84) is not dependent on known pHi regulatory mechanisms. The embryos exhibit a marked intracellular alkalinization when exposed to Cl(-)-free medium in the presence of bicarbonate. This response is eliminated by an inhibitor of anion exchange and by lack of bicarbonate, but is independent of Na+. These results indicate that there is probably a Na(+)-independent HCO3-/Cl- exchanger active in these cells, presumably functioning to alleviate alkaline loads.     

Effects of intracellular signals on Na+/K(+)-ATPase pump activity in the frog skin epithelium.

The effects of intracellular signals (pHi, Na+i, Ca2+i, and the electrical membrane potential), on Na+ transport mediated by the Na+/K+ pump were investigated in the isolated Rana esculenta frog skin. In particular we focussed on pHi sensitivity since protons act as an intrinsic regulator of transepithelial Na+ transport (JNa) by a simultaneous control of the apical membrane Na+ conductance (gNa) and the basolateral membrane K+ conductance (gK). pHi changes which modify JNa, gNa and gK, do not affect the Na+ transport mediated by the pump as shown by kinetic and electrophysiological studies. In addition, no changes were observed in the number of 3H-ouabain binding sites in acid-loaded epithelia. Our attempts to modify cellular Ca2+ (by using Ca(2+)-free/EGTA Ringer solution or A23187 addition) also failed to produce any significant effects in the Na+ pump turnover rate or the number of 3H-ouabain binding sites. The Na+ pump current was found to be sensitive to the basolateral membrane potential, saturating for very positive (cell) potentials and a reversal potential of -160 mV was calculated from I-V relationships of the pump. Changes in Na+i considerably affected the Na+ pump rate. A saturating relationship was found between pump rate and Nai+ with maximal activation at Nai+ greater than 40 mmol/l; a high dependence of the pump rate and of the number of 3H-ouabain binding sites was observed in the physiological range of Nai+. We conclude that protons (in the physiological pH range) which act directly and simultaneously on the passive transport pathways (gNa and gK), have no direct effect on the Na+/K+ pump rate. After an acid load, the inhibition of JNa is primarily due to the reduction of gNa. This results in a reduction of Nai and the pump turnover rate then becomes dependent on other pathways of Na+ entry such as the basolateral membrane Na+/H+ exchanger.     

Chemotactic factor-induced activation of Na+/H+ exchange in human neutrophils. II. Intracellular pH changes

The intracellular pH (pHi) changes resulting from chemotactic factor-induced activation of Na+/H+ exchange in isolated human neutrophils were characterized. Intracellular pH was measured from the equilibrium distribution of [14C]-5,5-dimethyloxazolidine-2,4-dione and from the fluorescence of 6-carboxyfluorescein. Exposure of cells to 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (FMLP) in 140 mM Na+ medium at extracellular pH (pHo) 7.40 led to a rise in pHi along an exponential time course (rate coefficient approximately 0.55 min-1). By 10 min, a new steady-state pHi was reached (7.75-7.80) that was 0.55-0.60 units higher than the resting pHi of control cells (7.20-7.25). The initial rate of H+ efflux from the cells (approximately 15 meq/liter X min), calculated from the intrinsic intracellular buffering power of approximately 50 mM/pH, was comparable to the rate of net Na+ influx (approximately 17 meq/liter X min), an observation consistent with a 1:1 stoichiometry for Na+/H+ exchange. This counter-transport could be inhibited by amiloride (apparent Ki approximately 75 microM). When either the external ([Na+]o) or internal Na ([Na+]i) concentrations, pHo, or pHi were varied independently, the new steady-state [Na+]i and pHi values in FMLP-stimulated cells were those corresponding to a chemical equilibrium distribution of Na+ and H+ across the cell membrane. By analogy to other activated cells, these results indicate that an alkalinization of pHi in human neutrophils is mediated by a chemotactic factor-induced exchange of internal H+ for external Na+.     

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.     

The Na+/H+ exchange system in glial cell lines. Properties and activation by an hyperosmotic shock

The properties of the Na+/H+ exchange system in the glial cell lines C6 and NN were studied from 22Na+ uptake experiments and measurements of the internal pH (pHi) using intracellularly trapped biscarboxyethyl-carboxyfluorescein. In both cell types, the Na+/H+ exchanger is the major mechanism by which cells recover their pHi after an intracellular acidification. The exchanger is inhibited by amiloride and its derivatives. The pharmacological profile (ethylisopropylamiloride greater than amiloride greater than benzamil) is identical for the two cell lines. Both Na+ and Li+ can be exchanged for H+. Increasing the external pH increases the activity of the exchanger in the two cell lines. In NN cells the external pH dependence of the exchanger is independent of the pHi. In contrast, in C6 cells, changing the pHi value from 7.0 to 6.5 produces a pH shift of 0.6 pH units in the external pH dependence of the exchanger in the acidic range. Decreasing pHi activates the Na+/H+ exchanger in both cell lines. Increasing the osmolarity of the external medium with mannitol produces an activation of the exchanger in C6 cells, which leads to a cell alkalinization. Mannitol action on 22Na+ uptake and the pHi were not observed in the presence of amiloride derivatives. Mannitol produces a modification of the properties of interaction of the antiport with both internal and external H+. It shifts the pHi dependence of the system to the alkaline range and the external pH (pHo) dependence to the acidic range. It also suppresses the interdependence of pHi and pHo controls of the exchanger's activity. NN cells that possess an Na+/H+ exchange system with different properties do not respond to mannitol by an increased activity of the Na+/H+ exchanger. The action of mannitol on C6 cells is unlikely to be mediated by an activation of protein kinase C.     

Phosphate transport in Ehrlich ascites tumor cells: inhibition by H+

The effect of changes in extracellular pH (pHo) and intracellular pH (pHi) on Na+-dependent and Na+-independent inorganic phosphate (Pi) transport in Ehrlich cells was investigated. In the presence of Na+, acutely reducing pHo from 7.30 to 5.50 results first in a transient (approximately 7 min) stimulation of Pi transport. The enhanced rate of transport is a saturable function of the extracellular [H+]; the Ks equals 2.3 X 10(-6) M (pHo 6.68). However, Pi transport is progressively inhibited as pHi falls below 6.50. The effect of pHi on Pi transport measured at various intracellular [Na+] suggests that inhibition develops as a consequence of H+ interaction with an intracellular Na+ site(s) on the Na+-dependent carrier. At pHo 7.4, about 15% of the steady state Pi flux persists in the absence of Na+. However, when pHo is reduced, transport is stimulated to the same extent and with the same time course and kinetic characteristics as in the presence of Na+. Thus, H+ stimulated Pi transport does not require Na+, raising the possibility that the Na+-independent component is mediated by the anion (Cl-) exchanger.     

Regulation of pH in individual pancreatic beta-cells as evaluated by fluorescence ratio microscopy

Pancreatic beta-cells are known to maintain intracellular pH (pHi) at a value well above that predicted from the electrochemical gradient. The mechanisms for the active extrusion of protons were examined by continuously monitoring pHi in individual beta-cells from ob/ob mice using the fluorescent indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In a medium nominally devoid of bicarbonate, the steady-state pHi was 6.82 +/- 0.02 and the intracellular buffering capacity was equivalent to 79 +/- 3 mM/pH unit. pHi remained unaffected after raising the glucose concentration from 3 to 20 mM, it was lowered when depolarizing the beta-cells with tolbutamide and it increased in the presence of carbachol. After removal of Na+ there was a significant drop of pHi and blockage of the pHi recovery following acid loading with the NH4+ prepulse technique. Whereas addition of amiloride had a similar, but less pronounced effect, omission of Cl- resulted in moderate alkalinisation. After switching to a medium containing bicarbonate, minor acidification was followed by adjustment of pHi to a steady state higher than the initial one. The results indicate that the acid load arising from glucose metabolism in the beta-cells is effectively buffered and the protons extruded both by Na+-H+ and Cl- -HCO3- exchangers.     

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

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

Intracellular pH regulation in ventral horn neurones cultured from embryonic rat spinal cord

Intracellular pH was measured with the pH-sensitive fluorescent probe BCECF in spinal cord neurones cultured from rat embryos. At an external pH of 7.3, the average steady-state pHi was 7.18 +/- 0.03 (SEM, n = 97) and 7.02 +/- 0.01 (n = 221) in HEPES-buffered and in bicarbonate-buffered medium, respectively. In both external media, pHi was strongly dependent on external pH (pHe). In HEPES-buffered medium, pHi recovery following an acid load induced by transient application of ammonium required external Na+ and was inhibited by amiloride, indicating the presence of a Na+/H+ exchange. Na(+)- and HCO3(-)-dependent, DIDS-sensitive alkalinizing mechanisms also contributed to pHi regulation in CO2/bicarbonate-buffered medium. The presence of an electrogenic Na(+)-HCO3- cotransporter was confirmed by the alkalinizing effect of KCl application. The fact that pHi is lower in CO2/bicarbonate- than in HEPES-buffered medium and the alkalinization observed upon suppression of external Cl- suggest that the acidifying Cl-/HCO3- transporter plays an important role in defining pHi.     

Control of cytoplasmic pH by Na+/H+ exchange in rat peritoneal macrophages activated with phorbol ester

The mechanisms underlying cytoplasmic pH (pHi) regulation in elicited rat peritoneal macrophages were investigated by electronic sizing and fluorescence determinations. Acid-loaded cells rapidly regained normal pHi by means of an amiloride-sensitive Na+/H+ exchange. When stimulated by 12-O-tetradecanoyl phorbol 13-acetate, macrophages displayed a biphasic pHi change: a marginal acidification followed by an alkalinization. The latter results from activation of Na+/H+ exchange, since it is Na+-dependent and prevented by amiloride. When the antiport is inhibited, the full magnitude of the initial acidification can be appreciated. This acidification is independent of the nature of the ionic composition of the medium and probably reflects accumulation of protons generated during the metabolic burst. Under physiological conditions, these protons are rapidly extruded by the Na+/H+ antiport.     

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.     

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+.     

Cl-/HCO3- exchange modulates intracellular pH in rat type II alveolar epithelial cells

The role of an anion exchange pathway in modulating intracellular pH (pHi) under steady-state and alkaline load conditions was investigated in confluent monolayers of rat type II alveolar epithelial cells using the pH-sensitive fluorescent probe 2'-7'-biscarboxy-ethyl-5,6-carboxylfluorescein. Under steady-state conditions in the presence of 25 mM HCO3-, 5% CO2 at pHo 7.4, pHi was 7.32 in a Na+-replete medium and 7.33 in the absence of Na+. Steady-state pHi was 7.19 in a nominally HCO3(-)-free medium at pHo 7.4, and 7.52 in a Cl(-)-free medium, with both values significantly different from that obtained in the presence of both HCO3- and Cl-. Monolayers in which pHi was rapidly elevated by removal of HCO3-/CO2 from the bathing medium demonstrated an absolute requirement for Cl- to recover toward base-line pHi. The Km of Cl- for the external site of the exchange pathway was 11 +/- 1 mM. Recovery of pHi from the alkaline load in the presence of Cl- was inhibited 60% by the stilbene derivative 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Removal of Cl- from the medium of cells bathed in HCO3-/CO2 resulted in a rapid increment in pHi which returned to base line when Cl- was reintroduced into the bathing medium. In contrast, pHi was not perturbed by removal or addition of Cl- to monolayers bathed in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered medium, indicating that HCO3- was the preferred species for transport. Recovery of pHi from an alkaline load was not affected by the presence or absence of Na+. These findings define the transport pathway as Na+-independent Cl-/HCO3- exchange. This pathway contributes importantly to determining resting pHi of pneumocytes and enables the cell to recover from an alkaline load.     

22Na+ fluxes in thymic lymphocytes. II. Amiloride-sensitive Na+/H+ exchange pathway; reversibility of transport and asymmetry of the modifier site

22Na+ flux and cytoplasmic pH (pHi) determinations were used to study the reversibility, symmetry, and mechanism of activation of the Na+/H+ exchange system in rat thymic lymphocytes. In acid-loaded cells, the antiport can be detected as an Na+-induced, amiloride-sensitive alkalinization. At pHi greater than or equal to 7.0, amiloride- sensitive net H+ fluxes are not detectable. To investigate whether at this pHi the transporter is operative in a different mode, e.g., Na+/Na+ exchange, 22Na+ uptake was measured as a function of pHi. The results indicate that the antiport is relatively inactive at pHi greater than or equal to 7.0. Comparison of the rates of H+ efflux (or equivalent OH- uptake) and Na+ uptake indicate that Na+/Na+ countertransport through this system is negligible at all values of pHi and that the Na+:H+ stoichiometry is 1:1. Measurements of pHi in Na+- loaded cells suspended in Na+-free medium revealed an amiloride- sensitive cytoplasmic acidification, which is indicative of exchange of internal Na+ for external H+. The symmetry of the system was analyzed by measuring the effect of extracellular pH (pHo) on Na+ efflux. Unlike cytoplasmic acidification, lowering pHo failed to activate the antiport. The results indicate that the amiloride-sensitive Na+/H+ exchanger is reversible but asymmetric. The system is virtually inactive at pHi greater than or equal to 7.0 but can be activated by protonation of a modifier site on the cytoplasmic surface. Activation can also occur by depletion of cellular Na+. It is proposed that Na+ may also interact with the modifier site, stabilizing the unprotonated (inactive) form.     

Activation of Na/H exchange by thrombin in peritoneal mast cells]

Using the fluorescent probe, BCECF, the changes in intracellular pH (pHi) in rat peritoneal mast cells were studied. alpha-Thrombin (0.1 nM) induced biphasic changes in pHi which consisted in a temporary decrease in pH with its subsequent steady increase due to the Na/H exchange activation which was inhibited by EIPA and controlled by extracellular Na+. The biphasic changes in pHi induced by DIP-alpha-thrombin (0.1 pM-1 nM), a catalytically inactive form with an intact recognition site, were similar to those of alpha-thrombin, whereas beta/gamma-thrombin (10-1000 pM), a catalytically active form characterized by structural disturbances in the recognition site, was able to induce only the initial phase of acidification. The thrombin recognition site modulators, alpha 1-thymosin and heparin, blocked the ability of the enzyme to induce the alkalinization of pHi. Nigericin stimulated the Na/H-exchange in mast cells. The rate of the Na/H-exchange activation determined with nigericin, decreased with an increase in the alpha-thrombin dose from 0.1 pM up to 10 nM. Activation of protein kinase C (PKC) in mast cells by PMA used at 1 nM and 10 nM led to the alkalinization of the cytoplasm as a result of the Na/H-exchange activation blocked by EIPA. The PKC inhibitor, H-7, suppressed the pHi increase induced by both PMA and alpha-thrombin. The alpha-thrombin-induced acidification of the cytoplasm was completely blocked by SITS in Ca(2+)-free media, whereas in media with Ca2+ SITS inhibited the pHi decline. Acidification of the cytoplasm by thrombin seems to be due to both Ca2+ influx and activation of Cl- fluxes. It is concluded that the observed activation of the Na/H-exchange by thrombin is induced by a cascade of intracellular reactions involving PKC.     

Regulation of cytoplasmic pH in rat sublingual mucous acini at rest and during muscarinic stimulation

The regulation of intracellular pH (pHi) in rat sublingual mucous acini was monitored using dual-wavelength microfluorometry of the pH-sensitive dye BCECF (2',7'-biscarboxyethyl-5(6)-carboxyfluorescein). Acini attached to coverslips and continuously superfused with HCO3(-)-containing medium (25 mM NaHCO3/5% CO2; pH 7.4) have a steady-state pHi of 7.25 +/- 0.02. Acid loading of acinar cells using the NH4+/NH3 prepulse technique resulted in a Na(+)-dependent, MIBA-inhibitable (5-(N-methyl-N-isobutyl) amiloride, Ki approximately 0.42 microM) pHi recovery, the kinetics of which were not influenced by the absence of extracellular Cl-. The rate and magnitude of the pHi recovery were dependent on the extracellular Na+ concentration, indicating that Na+/H+ exchange plays a critical role in maintaining pHi above the pH predicted for electrochemical equilibrium. When the NH4+/NH3 concentration was varied, the rate of pHi recovery was enhanced as the extent of the intracellular acidification increased, demonstrating that the activity of the Na+/H+ exchanger is regulated by the concentration of intracellular protons. Switching BCECF-loaded acini to a Cl(-)-free medium did not significantly alter resting pHi, suggesting the absence of Cl-/HCO3- exchange activity. Muscarinic stimulation resulted in a rapid and sustained cytosolic acidification (t 1/2 < 30 sec; 0.16 +/- 0.02 pH unit), the magnitude of which was amplified greater than two-fold in the presence of MIBA (0.37 +/- 0.05 pH unit) or in the absence of extracellular Na+ (0.34 +/- 0.03 pH unit). The agonist-induced intracellular acidification was blunted in HCO3(-)-free media and was inhibited by DPC (diphenylamine-2-carboxylate), an anion channel blocker. In contrast, the acidification was not influenced by removal of extracellular Cl-. The Ca2+ ionophore, ionomycin, mimicked the effects of stimulation, whereas preloading acini with BAPTA (bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid) to chelate intracellular Ca2+ blocked the agonist-induced cytoplasmic acidification. The above results indicate that during muscarinic stimulation an intracellular acidification occurs which: (i) is partially buffered by increased Na+/H+ exchange activity; (ii) is most likely mediated by HCO3- efflux via an anion channel; and (iii) requires an increase in cytosolic free [Ca2+].     

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+.     

Flow cytometric kinetic assay of the activity of Na+/H+ antiporter in mammalian cells.

BACKGROUND: The Na(+)/H(+) exchanger (NHE) of mammalian cells is an integral membrane protein that extrudes H(+) ion in exchange for extracellular Na(+) and plays a crucial role in the regulation of intracellular pH (pHi). Thus, when pHi is lowered, NHE extrudes protons at a rate depending of pHi that can be expressed as pH units/s. METHODS: To abolish the activity of other cellular pH-restoring systems, cells were incubated in bicarbonate-free Dulbecco's modified Eagle's medium buffered with HEPES. Flow cytometry was used to determine pHi with 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester or 5-(and-6)-carboxy SNARF-1 acetoxymethyl ester acetate, and the appropriate fluorescence ratios were measured. The calibration of fluorescence ratios versus pHi was established by using ionophore nigericin. The activity of NHE was calculated by a kinetic flow cytometric assay as the slope at time 0 of the best-fit curve of pHi recovery versus time after intracellular acidification with a pulse of exogenous sodium propionate. RESULTS: The kinetic method allowed determination of the pHi-dependent activity of NHE in cell lines and primary cell cultures. NHE activity values were demonstrated to be up to 0.016 pH units/s within the pHi range of 7.3 to 6.3. The inhibition of NHE activity by the specific inhibitor ethyl isopropyl amiloride was easily detected by this method. CONCLUSIONS: The assay conditions can be used to relate variations in pHi with the activity of NHE and provide a standardized method to compare between different cells, inhibitors, models of ischemia by acidification, and other relevant experimental or clinical situations.