Basolateral regulation of pHi in proximal tubules of avian loopless and long-looped nephrons in bicarbonate.

In isolated, nonperfused chicken proximal tubules from both loopless reptilian-type and long-looped mammalian-type nephrons, resting intracellular pH (pHi), measured with pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), was approximately 7.1 under control HCO3- conditions [20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)/5 mM HCO3(-)-buffered medium with pH 7.4 at 37 degrees C] and was reduced to approximately 6.8 in response to NH4Cl pulse. The rate of recovery of pHi (dpHi/dt) from this level to the resting level in proximal tubules from both nephron types was (1) significantly reduced by the removal of Na+ or both Na+ and Cl- from the bath, and (2) unaffected by the removal of Cl- from the bath or the presence of a high K+ concentration or Ba2+ in the bath. In proximal tubules from long-looped mammalian-type, but not loopless reptilian-type, nephrons, dpHi/dt was significantly reduced by the addition of either 5-(N-ethyl-N-isopropyl) amiloride (EIPA) or 4,4'-diisothiocyanostilbene-2,2'disulfonate (DIDS) to the bath. These data suggest that a Na+/H+ exchanger and most likely a Na(+)-dependent Cl-/HCO3- exchanger are involved in basolateral regulation of pHi in mammalian-type nephrons whereas none of the commonly identified basolateral acid-base transporters appear to be involved in regulation of pHi in reptilian-type nephrons.     

Intracellular pH regulation in cultured embryonic chick heart cells. Na(+)-dependent Cl-/HCO3- exchange

The contribution of Cl-/HCO3- exchange to intracellular pH (pHi) regulation in cultured chick heart cells was evaluated using ion-selective microelectrodes to monitor pHi, Na+ (aiNa), and Cl- (aiCl) activity. In (HCO3- + CO2)-buffered solution steady-state pHi was 7.12. Removing (HCO3- + CO2) buffer caused a SITS (0.1 mM)-sensitive alkalinization and countergradient increase in aiCl along with a transient DIDS-sensitive countergradient decrease in aiNa. SITS had no effect on the rate of pHi recovery from alkalinization. When (HCO3- + CO2) was reintroduced the cells rapidly acidified, aiNa increased, aiCl decreased, and pHi recovered. The decrease in aiCl and the pHi recovery were SITS sensitive. Cells exposed to 10 mM NH4Cl became transiently alkaline concomitant with an increase in aiCl and a decrease in aiNa. The intracellular acidification induced by NH4Cl removal was accompanied by a decrease in aiCl and an increase in aiNa that led to the recovery of pHi. In the presence of (HCO3- + CO2), addition of either amiloride (1 mM) or DIDS (1 mM) partially reduced pHi recovery, whereas application of amiloride plus DIDS completely inhibited the pHi recovery and the decrease in aiCl. Therefore, after an acid load pHi recovery is HCO3o- and Nao- dependent and DIDS sensitive (but not Ca2+o dependent). Furthermore, SITS inhibition of Na(+)-dependent Cl-/HCO3- exchange caused an increase in aiCl and a decrease in the 36Cl efflux rate constant and pHi. In (HCO3- + CO2)-free solution, amiloride completely blocked the pHi recovery from acidification that was induced by removal of NH4Cl. Thus, both Na+/H+ and Na(+)-dependent Cl-/HCO3- exchange are involved in pHi regulation from acidification. When the cells became alkaline upon removal of (HCO3- + CO2), a SITS-sensitive increase in pHi and aiCl was accompanied by a decrease of aiNa, suggesting that the HCO3- efflux, which can attenuate initial alkalinization, is via a Na(+)-dependent Cl-/HCO3- exchange. However, the mechanism involved in pHi regulation from alkalinization is yet to be established. In conclusion, in cultured chick heart cells the Na(+)-dependent Cl-/HCO3- exchange regulates pHi response to acidification and is involved in the steady-state maintenance of pHi.     

Intracellular pH regulation in the S3 segment of the rabbit proximal tubule in HCO3- -free solutions

We used the absorbance spectrum of 4',5'-dimethyl-5-(and 6) carboxyfluorescein to measure intracellular pH (pHi) in the isolated, perfused S3 segment of the rabbit proximal tubule. Experiments were conducted in HCO3- -free solutions. pHi recovered from an acid load imposed by an NH4+ prepulse, indicating the presence of one or more active acid-extrusion mechanisms. Removal of Na+ from bath and lumen caused pHi to decrease by approximately 0.6, whereas Na+ readdition caused complete pHi recovery. Removal of Na+ from the bath caused only a slow pHi decrease that was enhanced about fourfold when Na+ was subsequently removed from the lumen also. Similarly, the pHi recovery produced by the readdition of Na+ to the bath and lumen was about ninefold faster than when Na+ was returned to the bath only. Amiloride (1-2 mM) inhibited the pHi recovery that was elicited by returning 15 or 29 mM Na+ to lumen by only approximately 30%. However, in the absence of external acetate (Ac-), 1 mM amiloride inhibited approximately 66% of the pHi recovery induced by the readdition of 29 mM Na+ to the lumen only. The removal of external Ac- reduced the pHi recovery rate from an NH4+-induced acid load by approximately 47%, and that elicited by Na+ readdition, by approximately 67%. Finally, when bilateral removal of Na+ was maintained for several minutes, pHi recovered from the initial acidification, slowly at first, and then more rapidly, eventually reaching a pHi approximately 0.1 higher than the initial one. This Na+-independent pHi recovery was not significantly affected by lowering [HEPES]o from 32 to 3 mM or by adding N'N'-dicyclohexylcarbodiimide (10(-4) M) to the lumen, but it was reduced approximately 57% by iodoacetate (0.5 mM) plus cyanide (1 mM). We conclude that in the nominal absence of HCO3-, three transport systems contribute to acid extrusion by S3 cells: (a) a Na+-independent mechanism, possibly an H+ pump; (b) a Na-H exchanger, confined primarily to the luminal membrane; and (c) an Ac- and luminal Na+-dependent mechanism. The contribution of these three mechanisms to total acid extrusion, assessed by the rapid readdition of Na+, was approximately 13, approximately 30, and approximately 57%, respectively.     

Effects of ammonium on intracellular pH in rat medullary thick ascending limb: mechanisms of apical membrane NH4+ transport

The renal medullary thick ascending limb (MTAL) actively reabsorbs ammonium ions. To examine the effects of NH4+ transport on intracellular pH (pHi) and the mechanisms of apical membrane NH4+ transport, MTALs from rats were isolated and perfused in vitro with 25 mM HCO3(-)-buffered solutions (pH 7.4). pHi was monitored using the fluorescent dye BCECF. In the absence of NH4+, the mean pHi was 7.16. Luminal addition of 20 mM NH4+ caused a rapid intracellular acidification (dpHi/dt = 11.1 U/min) and reduced the steady state pHi to 6.67 (delta pHi = 0.5 U), indicating that apical NH4+ entry was more rapid than entry of NH3. Luminal furosemide (10(-4) M) reduced the initial rate of cell acidification by 70% and the fall in steady state pHi by 35%. The residual acidification observed with furosemide was inhibited by luminal barium (12 mM), indicating that apical NH4+ entry occurred via both furosemide (Na(+)-NH4(+)-2Cl- cotransport) and barium- sensitive pathways. The role of these pathways in NH4+ absorption was assessed under symmetric ammonium conditions. With 4 mM NH4+ in perfusate and bath, mean steady state pHi was 6.61 and net ammonium absorption was 12 pmol/min/mm. Addition of furosemide to the lumen abolished net ammonium absorption and caused pHi to increase abruptly (dpHi/dt = 0.8 U/min) to 7.0. Increasing luminal [K+] from 4 to 25 mM caused a similar, rapid cell alkalinization. The pronounced cell alkalinization observed with furosemide or increasing [K+] was not observed in the absence of NH4+. In symmetric 4 mM NH4+ solutions, addition of barium to the lumen caused a slow intracellular alkalinization and reduced net ammonium absorption only by 14%. Conclusions: (a) ammonium transport is a critical determinant of pHi in the MTAL, with NH4+ absorption markedly acidifying the cells and maneuvers that inhibit apical NH4+ uptake (furosemide or elevation of luminal [K+]) causing intracellular alkalinization; (b) most or all of transcellular ammonium absorption is mediated by apical membrane Na(+)- NH4(+)-2Cl- cotransport; (c) NH4+ also permeates a barium-sensitive apical membrane transport pathway (presumably apical membrane K+ channels) but this pathway does not contribute significantly to ammonium absorption under physiologic (symmetric ammonium) conditions.     

Evidence for a Na+/H+ antiport in stomach smooth muscle cells

Single smooth muscle cells were isolated from circular muscle of the canine gastric corpus by collagenase incubation. Cytoplasmic pH (pHi) of these cells was measured fluorometrically using the trapped dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. Cells were examined for their Na+/H+ exchange activity after intracellular acidification. Cells acid-loaded by propionate exposure, the NH4+ prepulse technique or suspension in a Na+-depleted medium regained almost normal pHi upon exposure to a Na+ medium. The Na+-dependent alkalinization was amiloride sensitive. As well, addition of amiloride to cells suspended in a Na+ medium caused a concurrent decrease in pHi. The study indicates that a Na+/H+ antiport is present in these smooth muscle cells.     

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.     

Intracellular pH-regulatory mechanisms in pancreatic acinar cells. I. Characterization of H+ and HCO3- transporters

Rat pancreatic acini loaded with the pH sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein were used to characterize intracellular pH (pHi) regulatory mechanisms in these cells. The acini were attached to cover slips and continuously perfused. In 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered solutions recovery from acid load (H+ efflux) required extracellular Na+ (Na+out) and was blocked by amiloride. Likewise, H+ influx initiated by removal of Na+out was blocked by amiloride. Hence, in HEPES-buffered medium the major operative pHi regulatory mechanism is a Na+/H+ exchange. In HCO3(-)-buffered medium, amiloride only partially blocked recovery from acid load and acidification due to Na+out removal. The remaining fraction required Na+out, was inhibited by H2-4,4'-diisothiocyanostilbene-2,2'-disulfunic acid (H2DIDS) and was independent of C1-. Hence, a transporter with characteristics of a Na(+)-HCO3- cotransport exists in pancreatic acini. Measurement of pHi changes due to Na(+)-HCO3- cotransport, suggests that the transporter contributes to HCO3- efflux under physiological conditions. Changing the Cl- gradient across the plasma membrane of acini maintained in HCO3(-)-buffered solutions reveals the presence of an H2DIDS-sensitive, Na(+)-independent, Cl(-)-dependent, HCO3- transporter with characteristics of a Cl-/HCO3- exchanger. In pancreatic acini the exchanger transports HCO3- but not OH- and under physiological conditions functions to remove HCO3- from the cytosol. In summary, only the Na+/H+ exchanger is functional in HEPES-buffered medium to maintain pHi at 7.28 +/- 0.03. In the presence of 25 mM HCO3- at pHo of 7.4, all the transporters operate simultaneously to maintain a steady-state pHi of 7.13 +/- 0.04.     

Basolateral Na-H exchange in the rabbit cortical collecting tubule

We used the intracellular absorbance spectrum of the dye 4',5'-dimethyl-5- (and -6-) carboxyfluorescein (Me2CF) to measure intracellular pH (pHi) in the isolated, perfused cortical collecting tubule (CCT) of the rabbit nephron. The incident spot of light was generally 10 micron in diameter, large enough to illuminate from two to six cells. No attempt was made to distinguish principal from intercalated cells. All experiments were carried out in HCO3- -free Ringer to minimize HCO3- transport. When cells were acid-loaded by briefly exposing them to Ringer containing NH+4 and then withdrawing the NH+4, pHi spontaneously recovered from the acid load. The pHi recovery was best fit by the sum of two exponentials. When the acid loading was performed in the absence of Na+, the more rapid of the two phases of pHi recovery was absent. The remaining slow phase never returned pHi to normal and was sometimes absent. Returning Na+ to the lumen had only a slight effect on the pHi recovery. However, when Na+ was returned to the basolateral (i.e., blood-side) solution, pHi recovered rapidly and completely. The apparent Km for basolateral Na+ was 27.3 +/- 4.5 mM. The basolateral Na-dependent pHi recovery was reversibly inhibited by amiloride. We conclude that the mechanism responsible for the rapid phase of pHi recovery is an Na-H exchanger confined primarily, if not exclusively, to the basolateral membrane of the CCT.     

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+].     

Regulation of intracellular pH in cultured hippocampal neurons by an amiloride-insensitive Na+/H+ exchanger.

Regulation of intracellular pH (pHi) in single cultured rat hippocampal neurons was investigated using the fluorescent pHi indicator dye bis-carboxyethylcarboxyfluorescein. Resting pHi was dependent on the presence of bicarbonate and external Na+ but was not altered significantly by removal of Cl- or treatment with the anion exchange inhibitor diisothiocyanatostilbene-2,2'-disulfonate. Recovery of pHi from acute acid loading was due, in large part, to a pharmacologically distinct variant of the Na+/H+ antiporter. In nominally HCO3(-)-free solutions, this recovery exhibited a saturable dose dependence on extracellular Na+ (Km = 23-26 mM) or Li+. The antiporter was activated by decreasing pHi and was unaffected by collapse of the membrane potential with valinomycin. Like the Na+/H+ antiporter described in other cell systems, the hippocampal activity was inhibited by harmaline, but in sharp contrast, neither amiloride nor its more potent 5-amino-substituted analogues were able to prevent the recovery from an acid load. These data indicate that Na(+)-dependent mechanisms dominate pHi regulation in hippocampal neurons and suggest a role for a novel variant of the Na+/H+ antiporter.     

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.     

Direct measurement of intracellular pH in identified glial cells and neurones of the leech central nervous system

Neutral carrier pH-sensitive double-barrelled microelectrodes were used to investigate intracellular pH (pHi) in leech neuropile glial cells and in Retzius neurones. The mean pHi of the glial cells was 6.87 +/- 0.13 (+/- SD, n = 27) in HEPES-buffered saline (pHo 7.4) and 7.18 +/- 0.19 (n = 13) in solutions buffered with 2% CO2- 11 mM HCO3-. The distribution of H+ ions in both the glia and neurones was found not to be in electrochemical equilibrium. To investigate pHi regulation, the pHi was decreased by exposure to CO2 or by adding and then removing NH4Cl. Acidification by any method was followed by a recovery to normal pHi values within minutes. The pHi recovery from acidification in neuropile glial cells in HEPES-buffered saline and CO2-HCO3- buffered saline was, however, blocked by removing external Na. In HCO3(-)-free solutions the diuretic amiloride (2 mM) reduced the rate of pHi recovery. In the presence of HCO3-, the rate of acid efflux was stimulated; the stilbene 4-acetamido-4'-isothiocyanatostilbene-2,3'-disulfonic acid (SITS; 0.5 mM) slowed pHi recovery. In HEPES buffered and CO2-HCO3- buffered solutions pHi regulation in neurones was inhibited by removing external Na. In HCO3(-)-free solutions amiloride reduced the rate of pHi recovery considerably. In the presence of HCO3-, SITS or amiloride slowed but did not completely block pHi recovery. We conclude that leech glial cells and neurones have two mechanisms of pHi regulation, one being Na+-H+ exchange and the other Na+ and HCO3- dependent.     

Role of monocarboxylic acid transport in intracellular pH regulation of isolated proximal cells

Isolated proximal cells were prepared from rabbit kidney cortex by mechanical dissociation. The intracytoplasmic pH (pHi) was measured in HCO3(-)-free media (external pH (pHe), 7.3) using the fluorescent dye 2,7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF). Cells were acid-loaded by the nigericin technique. Addition of 70 mM Na+ to the cells caused a rapid pHi recovery, which was blocked by 0.5 mM amiloride. When the cells were exposed to 5 mM sodium butyrate in the presence of 1 mM amiloride, the H+ efflux was significantly increased and followed Michaelis-Menten kinetics. Increasing pHe from 6.4 to 7.6 at a constant pHi of 6.4 enhanced the butyrate activation of the H+ efflux. Increasing pHi from 6.5 to 7.2 at a constant pHe of 7.2 reduced the butyrate effect. 22Na uptake experiments in the presence of 1 mM amiloride showed that 1.5 mM butyrate increased the Na+ flux in the proximal cells (pHi 7.10). The efficiency of monocarboxylic anions in promoting a pHi recovery increased with the length of their straight chain (acetate less than propionate less than butyrate less than valerate). The data show that when the Na+/H+ antiporter is blocked, the proximal cells can regulate their pHi by a Na+-coupled absorption of butyrate followed by non-ionic diffusion of butyric acid out of the cell and probably also by OH- influx by means of the OH-/anion exchanger.     

Effects of intracellular pH on hypoxic vasoconstriction in rat lungs

Isolated rat lungs perfused with physiological salt-Ficoll solutions were studied to test whether hypoxic pulmonary vasoconstriction was potentiated by increases in intracellular pH (pHi) and blunted by decreases in pHi. Whereas addition to perfusate of 5 nM phorbol myristate acetate (PMA), a stimulator of exchange of intracellular H+ for extracellular Na+, potentiated hypoxic vasoconstriction, 1 mM amiloride, an inhibitor of Na+-H+ exchange, blunted the hypoxic response. Hypoxic vasoconstriction was also potentiated by the weak bases NH4Cl (20 mM), methylamine (10 mM), and imidazole (5 mM) and was inhibited by the weak acid sodium acetate (40 mM). NH4Cl, imidazole, and acetate had the same effects on KCl-induced vasoconstriction and on the hypoxic response. Hypoxic vasoconstriction was greater in lungs perfused with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution than in those perfused with CO2/HCO3--buffered solution. Similarly, lungs perfused with CO2/HCO3--buffered solution containing 1.8 mM Cl- (NaNO3 and KNO3 substituted for NaCl and KCl) had larger hypoxic and angiotensin II pressor responses than those perfused with 122.5 mM Cl-. Because PMA, NH4Cl, methylamine, imidazole, HEPES-buffered solutions, and low-Cl- solutions can cause increases in pHi and amiloride and acetate can cause decreases in pHi, these results suggest that intracellular alkalosis and acidosis, respectively, potentiate and blunt vasoconstrictor responses to hypoxia and other stimuli in isolated rat lungs. These effects could be related to pHi-dependent changes in either the sensitivity of the arterial smooth muscle contractile machinery to Ca2+ or the release of a vasoactive mediator or modulator by some other lung cell.     

H+ extrusion by an apical vacuolar-type H+-ATPase in rat renal proximal tubules

The activity of Na+/H(+)-exchange and H(+)-ATPase was measured in the absence of CO2/HCO3 by microfluorometry at the single cell level in rat proximal tubules (superficial S1/S2 segments) loaded with BCECF [2'7'-bis(carboxyethyl)5-6-carboxyfluorescein- acetoxymethylester]. Intracellular pH (pHi) was lowered by a NH4Cl-prepulse technique. In the absence of Na+ in the superfusion solutions, pHi recovered from the acid load by a mechanism inhibited by 0.1 microM bafilomycin A1, a specific inhibitor of a vacuolar-type H(+)-ATPase. Readdition of Na+ in the presence of bafilomycin A1 produced an immediate recovery of pHi by a mechanism sensitive to the addition of 10 microM EIPA (ethylisopropylamiloride), a specific inhibitor of Na+/H+ exchange. The transport rate of the H(+)-ATPase is about 40% of Na+/H(+)-exchange activity at a similar pHi (0.218 +/- 0.028 vs. 0.507 +/- 0.056 pH unit/min. Pre-exposure of the tubules to 30 mM fructose, 0.5 mM iodoacetate and 1 mM KCN (to deplete intracellular ATP) prevented a pHi recovery in Na(+)-free media; readdition of Na+ led to an immediate pHi recovery. Tubules pre-exposed to Cl(-)-free media for 2 hr also reduced the rate of Na(+)-independent pHi recovery. In free-flow electrophoretic separations of brush border membranes and basolateral membranes, a bafilomycin A1-sensitive ATPase activity was found to be associated with the brush border membrane fraction; half maximal inhibition is at 6 x 10(-10) M bafilomycin A1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glucose-induced increase in cytoplasmic pH in pancreatic beta-cells is mediated by Na+/H+ exchange, an effect not dependent on protein kinase C.

Glucose-induced changes in cytoplasmic pH (pHi) were investigated using pancreatic beta-cells isolated from obese hyperglycemic mice. Glucose, at concentrations above 3-5 mM, depolarized the beta-cell and increased pHi, cytoplasmic free Ca2+ ([Ca2+]i), and insulin release. This increase in pHi was dependent on the presence of extracellular Na+ and was inhibited by 5-(N-ethyl-N-isopropyl) amiloride, a blocker of Na+/H+ exchange. Stimulation of protein kinase C with phorbol ester also induced an alkalinization. However, when protein kinase C activity was down-regulated, glucose stimulation still induced alkalinization. At 20 mM glucose, 10 mM NH4Cl induced a marked rise in pHi, paralleled by repolarization, inhibition of electrical activity, and decreases in both [Ca2+]i and insulin release. Reduction in [Ca2+]i was prevented by 200 microM tolbutamide, but not by 10 mM tetraethylammonium. At 4 mM glucose, NH4Cl induced a transient increase in insulin release, without changing [Ca2+]i. Exposure of beta-cells to 10 mM sodium acetate caused a persistent decrease in pHi, an effect paralleled by a small transient increase in [Ca2+]i. Acidification per se did not change the beta-cell sensitivity to glucose, not excluding that the activity of the ATP-regulated K+ channels may be modulated by changes in pHi.     

Loss-of-function and gain-of-function phenotypes of stomatocytosis mutant RhAG F65S

Four patients with overhydrated cation leak stomatocytosis (OHSt) exhibited the heterozygous RhAG missense mutation F65S. OHSt erythrocytes were osmotically fragile, with elevated Na and decreased K contents and increased cation channel-like activity. Xenopus oocytes expressing wild-type RhAG and RhAG F65S exhibited increased ouabain and bumetanide-resistant uptake of Li(+) and (86)Rb(+), with secondarily increased (86)Rb(+) influx sensitive to ouabain and to bumetanide. Increased RhAG-associated (14)C-methylammonium (MA) influx was severely reduced in RhAG F65S-expressing oocytes. RhAG-associated influxes of Li(+), (86)Rb(+), and (14)C-MA were pharmacologically distinct, and Li(+) uptakes associated with RhAG and RhAG F65S were differentially inhibited by NH(4)(+) and Gd(3+). RhAG-expressing oocytes were acidified and depolarized by 5 mM bath NH(3)/NH(4)(+), but alkalinized and depolarized by subsequent bath exposure to 5 mM methylammonium chloride (MA/MA(+)). RhAG F65S-expressing oocytes exhibited near-wild-type responses to NH(4)Cl, but MA/MA(+) elicited attenuated alkalinization and strong hyperpolarization. Expression of RhAG or RhAG F65S increased steady-state cation currents unaltered by bath Li(+) substitution or bath addition of 5 mM NH(4)Cl or MA/MA(+). These oocyte studies suggest that 1) RhAG expression increases oocyte transport of NH(3)/NH(4)(+) and MA/MA(+); 2) RhAG F65S exhibits gain-of-function phenotypes of increased cation conductance/permeability, and loss-of-function phenotypes of decreased and modified MA/MA(+) transport, and decreased NH(3)/NH(4)(+)-associated depolarization; and 3) RhAG transports NH(3)/NH(4)(+) and MA/MA(+) by distinct mechanisms, and/or the substrates elicit distinct cellular responses. Thus, RhAG F65S is a loss-of-function mutation for amine transport. The altered oocyte intracellular pH, membrane potential, and currents associated with RhAG or RhAG F65S expression may reflect distinct transport mechanisms.     

Regulation of cytoplasmic pH in resident and activated peritoneal macrophages

Cytoplasmic pH (pHi) has been shown to be an important determinant of the activity of the NADPH oxidase in phagocytic cells. We hypothesized that a difference in pHi and/or its regulation existed between activated and resident macrophages (RES MOs) which might explain the increased NADPH oxidase activity observed in the former. The pHi of RES and lipopolysaccharide (LPS)-elicited MOs was examined using the fluorescent dye BCECF. Resting pHi did not differ between resident (RES) and elicited (ELI) MOs (7.16 +/- 0.05 and 7.20 +/- 0.05, respectively). pHi recovery after intracellular acid loading was partially dependent on the presence of Na+ in the extracellular medium, and was partially inhibited by the Na+/H+ antiport inhibitor, amiloride. At comparable pHi, the rate of acid extrusion during recovery was not different in RES and ELI MOs (1.48 +/- 0.12 and 1.53 +/- 0.06 mM/min, respectively). In both RES and ELI MOs, approx. 40% of total pHi recovery was insensitive to amiloride and independent of extracellular Na+. In both RES and ELI MOs, stimulation with TPA resulted in a biphasic pHi response: an initial acidification followed by a sustained alkalinization to a new steady-state pHi. This alkalinization was Na(+)-dependent and amiloride-sensitive, consistent with a TPA-induced increase in Na+/H+ antiport activity. The new steady-state pHi attained after TPA stimulation was equivalent in RES and ELI MOs (7.28 +/- 0.04 and 7.31 +/- 0.06, respectively), indicating comparable stimulated Na+/H+ antiport activity. However, the initial acidification induced by TPA was greater in ELI than in RES MOs (0.18 +/- 0.02 vs. 0.06 +/- 0.02 pH unit, respectively, P less than 0.05). The specific NADPH oxidase inhibitor diphenylene iodonium (DPI) completely inhibited the respiratory burst but reduced the magnitude of this pHi reduction by only about 50%. This suggested that the TPA-induced pHi reduction was due in part to acid produced via the respiratory burst, and in part to other acid-generating pathways stimulated by TPA.     

An Na(+)-independent short-chain fatty acid transporter contributes to intracellular pH regulation in murine colonocytes

Short-chain fatty acids (SCFAs) are the major anions in the colonic lumen. Experiments studied how intracellular pH (pHi) of isolated colonocytes was affected by exposure to SCFAs normally found in the colon. Isolated crypt fragments were loaded with SNARF-1 (a fluorescent dye with pH-sensitive excitation and emission spectra) and studied in a digital imaging microscope. Intracellular pH was measured in individual colonocytes as the ratio of fluorescence intensity in response to alternating excitation wavelengths (575/505 nm). After exposure to 65 mM acetate, propionate, n-butyrate, or iso-butyrate in isosmotic Na(+)- free media (substituted with tetramethylammonia), all colonocytes acidified rapidly and then > 90% demonstrated a pHi alkalinization (Na(+)-independent pHi recovery). Upon subsequent removal of the SCFA, pHi alkalinized beyond the starting pHi (a pHi overshoot). Using propionate as a test SCFA, experiments demonstrate that the acidification and pHi overshoot are explained by transmembrane influx and efflux of nonionized SCFA, respectively. The basis for the pHi overshoot is shown to be accumulation of propionate during pHi alkalinization. The Na(+)-independent pHi recovery (a) demonstrates saturable propionate activation kinetics; (b) demonstrates substrate specificity for unmodified aliphatic carbon chains; (c) occurs after exposure to SCFAs of widely different metabolic activity, (d) is electroneutral; and (e) is not inhibited by changes in the K+ gradient, Cl- gradient or addition of the anion transport inhibitors DIDS (1 mM), SITS (1 mM), alpha-cyano-4-hydroxycinnamate (4 mM), or probenicid (1 mM). Results suggest that most mouse colonocytes have a previously unreported SCFA transporter which mediates Na(+)-independent pHi recovery.     

Cellular NH4+/K+ transport pathways in mouse medullary thick limb of Henle. Regulation by intracellular pH

Fluorescence and electrophysiological methods were used to determine the effects of intracellular pH (pHi) on cellular NH4+/K+ transport pathways in the renal medullary thick ascending limb of Henle (MTAL) from CD1 mice. Studies were performed in suspensions of MTAL tubules (S-MTAL) and in isolated, perfused MTAL segments (IP-MTAL). Steady-state pHi measured using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) averaged 7.42 +/- 0.02 (mean +/- SE) in S-MTAL and 7.26 +/- 0.04 in IP-MTAL. The intrinsic cellular buffering power of MTAL cells was 29.7 +/- 2.4 mM/pHi unit at pHi values between 7.0 and 7.6, but below a pHi of 7.0 the intrinsic buffering power increased linearly to approximately 50 mM/pHi unit at pHi 6.5. In IP-MTAL, NH4+ entered cells across apical membranes via both Ba(2+)-sensitive pathway and furosemide-sensitive Na+:K+(NH4+):2Cl- cotransport mechanisms. The K0.5 and maximal rate for combined apical entry were 0.5 mM and 83.3 mM/min, respectively. The apical Ba(2+)-sensitive cell conductance in IP-MTAL (Gc), which reflects the apical K+ conductance, was sensitive to pHi over a pHi range of 6.0-7.4 with an apparent K0.5 at pHi approximately 6.7. The rate of cellular NH4+ influx in IP-MTAL due to the apical Ba(2+)-sensitive NH4+ transport pathway was sensitive to reduction in cytosolic pH whether pHi was changed by acidifying the basolateral medium or by inhibition of the apical Na+:H+ exchanger with amiloride at a constant pHo of 7.4. The pHi sensitivities of Gc and apical, Ba(2+)-sensitive NH4+ influx in IP-MTAL were virtually identical. The pHi sensitivity of the Ba(2+)-sensitive NH4+ influx in S-MTAL when exposed to (apical+basolateral) NH4Cl was greater than that observed in IP-MTAL where NH4Cl was added only to apical membranes, suggesting an additional effect of intracellular NH4+/NH3 on NH4+ influx. NH4+ entry via apical Na+:K+ (NH4+):2Cl- cotransport in IP-MTAL was somewhat more sensitive to reductions in pHi than the Ba(2+)-sensitive NH4+ influx pathway; NH4+ entry decreased by 52.9 +/- 13.4% on reducing pHi from 7.31 +/- 0.17 to 6.82 +/- 0.14. These results suggest that pHi may provide a negative feedback signal for regulating the rate of apical NH4+ entry, and hence transcellular NH4+ transport, in the MTAL. A model incorporating these results is proposed which illustrates the role of both pHi and basolateral/intracellular NH4+/NH3 in regulating the rate of transcellular N H4+ transport in the MTAL.