Inhibited pHi Recovery Research Articles

IRBIT plays an important role in NHE3-mediated pHi regulation in HSG cells

Expression of inositol-1,4,5-trisphosphate (IP3) receptor-binding protein (IRBIT) has been reported in epithelial cells. However, its role in pHi regulation is not well understood. In this study, we investigated the role of IRBIT in pHi regulation, mediated by Na+/H+ exchangers (NHEs), in salivary glands. We measured pHi recovery from cell acidification in BCECF-loaded salivary HSG cells. Western blot and co-immunoprecipitation (CO-IP) assays were also performed, showing that NHE1, 2 and 3 are expressed, and IRBIT binds to NHE3. HOE642, a specific NHE1 blocker, inhibited pHi recovery, but 40% pHi recovery was still observed even at the highest concentration of HOE642. Furthermore, pretreatment of the cells with siIRBIT significantly inhibited pHi recovery, indicating that NHE3 potentially plays a role in pHi recovery as well. The amount of membrane-localized NHE3 and its interaction with IRBIT are also significantly increased by cell acidification. In addition, we found that Ste20p-related proline alanine-rich kinase (SPAK) reverses the effect of IRBIT on membrane NHE3 translocation. Taken together, we conclude that IRBIT plays an important role in pHi regulation, mediated by NHE3, and further regulated by SPAK.

Effect of Sodium Nitroprusside on pHi Recovery in Porcine Non‐pigmented Ciliary Epithelium

ObjectiveAtriopeptin, acting through cGMP, inhibits NHE1 in the bovine ocular non‐pigmented ciliary epithelium (NPE)1. Here we examined NHE isoforms and tested whether SNP, a NO donor and a stimulator of cGMP, alters intracellular pH (pHi) responses.MethodsNHE isoforms in native and cultured NPE were detected by immunohistochemistry and immunoblot. BCECF fluorescence imaging was used to study the effect of drugs on pHi recovery after an acid load.ResultsNative and cultured NPE expressed NHE1 and NHE4 isoforms. pHi recovery in NPE was Na‐dependent and had DIDS and amiloride sensitive components. SNP inhibited pHi recovery concentration‐dependently. L‐arginine, the NOS substrate, also slowed pHi recovery. Dimethyl amiloride (DMA), a potent inhibitor of NHE, caused substantial inhibition of pHi recovery at 100μM. In cells exposed to both SNP and DMA, the degree of inhibition of pHi recovery was similar to that elicited by SNP alone. The cyclic GMP analog 8‐pCPT‐cGMP (10μM) mimicked the SNP effect. The inhibitory effect of SNP on pHi recovery was suppressed by 1μM H‐8, an inhibitor of cGMP‐dependent protein kinase.ConclusionPrimary cultures of porcine NPE express NHE isoforms found in native cells. NO donors slow pHi recovery through inhibition of NHE. The effect of NO donors on NHE involves a cGMP‐PKG signal transduction pathway.Funding: NIH‐EY06915

NHE2 is the main apical NHE in mouse colonic crypts but an alternative Na+-dependent acid extrusion mechanism is upregulated in NHE2-null mice

The mechanism of apical Na(+)-dependent H(+) extrusion in colonic crypts is controversial. With the use of confocal microscopy of the living mouse distal colon loaded with BCECF or SNARF-5F (fluorescent pH sensors), measurements of intracellular pH (pH(i)) in epithelial cells at either the crypt base or colonic surface were reported. After cellular acidification, the addition of luminal Na(+) stimulated similar rates of pH(i) recovery in cells at the base of distal colonic crypts of wild-type or Na(+)/H(+) exchanger isoform 2 (NHE2)-null mice. In wild-type crypts, 20 microM HOE694 (NHE2 inhibitor) blocked 68-75% of the pH(i) recovery rate, whereas NHE2-null crypts were insensitive to HOE694, the NHE3-specific inhibitor S-1611 (20 microM), or the bicarbonate transport inhibitor 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS; 1 mM). A general NHE inhibitor, 5-(N-ethyl-N-isopropyl)amiloride (EIPA; 20 microM), inhibited pH(i) recovery in NHE2-null mice (46%) but less strongly than in wild-type mice (74%), suggesting both EIPA-sensitive and -insensitive compensatory mechanisms. Transepithelial Na(+) leakage followed by activation of basolateral NHE1 could confound the outcomes; however, the rates of Na(+)-dependent pH(i) recovery were independent of transepithelial leakiness to lucifer yellow and were unchanged in NHE1-null mice. NHE2 was immunolocalized on apical membranes of wild-type crypts but not NHE2-null tissue. NHE3 immunoreactivity was near the colonic surface but not at the crypt base in NHE2-null mice. Colonic surface cells from wild-type mice demonstrated S1611- and HOE694-sensitive pH(i) recovery in response to luminal sodium, confirming a functional role for both NHE3 and NHE2 at this site. We conclude that constitutive absence of NHE2 results in a compensatory increase in a Na(+)-dependent, EIPA-sensitive acid extruder distinct from NHE1, NHE3, or SITS-sensitive transporters.

Role of Na+/H+ exchange and HCO3- transport in pHi recovery from intracellular acid load in cultured epithelial cells of sheep rumen.

This study sought to investigate effects of short-chain fatty acids and CO2 on intracellular pH (pHi) and mechanisms that mediate pHi recovery from intracellular acidification in cultured ruminal epithelial cells of sheep. pHi was studied by spectrofluorometry using the pH-sensitive fluorescent indicator 2',7'-bis (carboxyethyl)-5(6')-carboxyfluorescein acetoxymethyl ester (BCECF/AM). The resting pHi in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution was 7.37 +/- 0.03. In HEPES-buffered solution, a NH4+/NH3-prepulse (20 mM) or addition of butyrate (20 mM) led to a rapid intracellular acidification (P < 0.05). Addition of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA: 10 microM) or HOE-694 (200 microM) inhibited pHi recovery from an NH4+/NH3-induced acid load by 58% and 70%, respectively. pHi recovery from acidification by butyrate was reduced by 62% and 69% in the presence of EIPA (10 microM) and HOE-694 (200 microM), respectively. Changing from HEPES-(20 mM) to CO2/HCO3(-)-buffered (5%/20 mM) solution caused a rapid decrease of pHi (P < 0.01), followed by an effective counter-regulation. 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS; 100 microM) blocked the pHi recovery by 88%. The results indicate that intracellular acidification by butyrate and CO2 is effectively counter-regulated by an Na+/H+ exchanger and by DIDS-sensitive, HCO3(-)-dependent mechanism(s). Considering the large amount of intraruminal weak acids in vivo, both mechanisms are of major importance for maintaining the pHi homeostasis of ruminal epithelial cells.

Characterization of ion transport mechanisms regulating intracellular pH in hepatic stellate cells.

The aim of this study was to evaluate intracellular pH (pHi) regulation in nonactivated and activated rat hepatic stellate cells (HSC). The fluorescent pHi indicator 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein was used to measure pHi in the presence and absence of HCO3-. In the absence of HCO3-, baseline pHi was significantly higher (P < 0.001) in activated than in nonactivated HSC (7.1 +/- 0.1 vs. 6.9 +/- 0.2) and decreased, in both groups, after amiloride administration and after Na+ removal. After an acid-loading maneuver, pHi recovery was significantly higher (P < 0.03) in activated than in nonactivated HSC (H+ flux = 11.0 +/- 3.8 vs. 7.7 +/- 2.9 mM/min at pHi 6.6) and was inhibited by amiloride and Na+ removal. In the presence of HCO3-, baseline pHi was higher in both groups and decreased after amiloride administration. Amiloride and Na+ removal inhibited pHi recovery after an intracellular acid load by 77 and 93%, respectively, in nonactivated and by 82 and 92%, respectively, in activated HSC, whereas 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibited pHi recovery by only 27%. Acute Cl- removal increased pHi by 0.07 +/- 0.01 pH unit/min in the absence but not in the presence of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid in nonactivated and activated HSC in an Na(+)-independent manner. In activated HSC, 24 h of incubation with 25 ng/ml platelet-derived growth factor (PDGF)-BB (in 0.5% serum) did not modify baseline pHi (7.07 +/- 0.1 vs. 7.08 +/- 0.1 in HSC cultured in 0.5% serum only) but significantly (P < 0.02) increased, with respect to controls, pHi recovery after an acute acid load. Incubation with PDGF for 24 h induced a fivefold increase in HSC proliferation expressed as percentage of bromodeoxyuridine-positive cells (30.8 +/- 6.7 vs. 6.1 +/- 1.9% in controls). When amiloride (0.1 mM) was present, PDGF-induced HSC proliferation was significantly inhibited (8.1 +/- 0.4%, P < 0.001). Our results show that 1) the Na+/H+ exchanger is the main pHi regulator in rat HSC, 2) activation of HSC is associated with an increase in pHi and in the activity of the Na+/H+ exchanger, 3) PDGF increases the activity of this exchanger, and 4) amiloride is able to inhibit HSC proliferation induced by PDGF.

Na(+)-H+ exchange in frog early distal tubule: effect of aldosterone on the set-point.

1. Intracellular pH (pHi) regulation was investigated in frog early distal tubule. Single tubules were dissected and perfused, such that the compositions of apical and basolateral solutions could be varied independently. pHi was measured using the fluorescent probe 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). 2. Brief exposure to NH4+ on the basolateral aspect of the tubules elicited an intracellular acidification, followed by an active recovery. The recovery was inhibited by amiloride and its analogue 5-(N-ethyl-N-isopropyl) amiloride (EIPA) when added to the basolateral, but not the apical, solution. Omission of Na+ from the basolateral solution alone completely inhibited pHi recovery. Thus the Na(+)-H+ exchangers appear to be located on the basolateral membrane. 3. Neither amiloride nor EIPA had any effect on pHi under control conditions, suggesting that the activity of the Na(+)-H+ exchangers at the resting pHi is low. However, removal of basolateral Na+ caused an acidification that was blocked by amiloride, indicating that the Na(+)-H+ exchangers can be activated from the resting state. 4. Intrinsic buffering power (beta i) was determined by stepwise removal of ammonium from the cells in Na(+)-free conditions, to prevent pH regulation, and in the presence of Ba2+ and furosemide (frusemide), to inhibit ammonium transport. beta i was a function of pHi, increasing as pHi decreased. 5. Proton efflux was calculated during the recovery from an acid load in tubules from normal and K(+)-loaded frogs and in tubules which had been incubated for 30 min with aldosterone. Potassium loading produces a chronic increase in plasma aldosterone. Both acute and chronic aldosterone treatment caused an intracellular alkalinization. This was due to an alkaline shift in the set-point of the basolateral Na(+)-H+ exchanger, with no change in the density and/or turnover rate.

Acid‐base transport systems in a polarized human intestinal cell monolayer: Caco‐2

Acid-base transport systems have been incompletely characterized in intact intestinal epithelial cells. We therefore studied the human cell line Caco-2, cultured on Teflon membranes to form confluent monolayers with apical microvilli on transmission electron microscopy and progressive enrichment in microvillar hydrolases. Monolayers (16- to 25-day-old), loaded with the pH-sensitive dye BCECF-AM (2',7'-bis (carboxyethyl)-5-carboxyfluorescein), were mounted in a spectrofluorometer cuvette to allow selective superfusion of apical and basolateral surfaces with Hepes- or HCO(3-)-buffered media. Intracellular pH (pHi) was measured by dual-excitation spectrofluorimetry; calibration was with standards containing nigericin and 110 mM K+ corresponding to measured intracellular [K+] in Caco-2 cell monolayers. In HCO(3-)-free (Hepes-buffered) media, bilateral superfusion with 1 mM amiloride or with Na(+)-free media reversibly inhibited pHi recovery from an intracellular acid load (NH4Cl pulse) by 86 and 98% respectively. Selective readdition of Na+ to the apical or basolateral superfusate also induced a pHi recovery, which was inhibited by ipsilateral but not by contralateral amiloride (1 mM). The pHi recovery induced by apical Na+ readdition had a Michaelis constant (Km) for Na+ of 30 mM and a relatively high inhibitor constant (Ki) for amiloride of 45.5 microM. Initial pHi in HCO(3-)-buffered media was lower than in the absence of HCO3- (7.35 vs. 7.80). pHi recovery from an acid load in HCO3- was Na- dependent but was inhibited only 18% by 1 mM amiloride. The amiloride-independent pHi recovery was inhibited 49% by pre-incubation of cells in 5 mM DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid). These data suggest that Caco-2 cells possess: (a) both apical and basolateral membrane Na(+)-H+ exchange mechanisms, the apical exchanger being relatively resistant to amiloride, similar to apical Na(+)-H+ exchangers in several normal epithelia; and (b) a Na(-)-dependent HCO3- transport system, either Na(+)-HCO3- cotransport or Na(-)-dependent Cl(-)-HCO3- exchange.

Intracellular pH regulation by the plasma membrane V-ATPase in Malpighian tubules of Drosophila larvae.

The functional significance of the apical vacuolar-type proton pump (V-ATPase) in Drosophila Malpighian tubules was studied by measuring the intracellular pH (pHi) and luminal pH (pHlu) with double-barrelled pH-microelectrodes in proximal segments of the larval anterior tubule immersed in nominally bicarbonate-free solutions (pHo 6.9). In proximal segments both pHi (7.43 +/- 0.20) and pHlu (7.10 +/- 0.24)) were significantly lower than in distal segments (pHi 7.70 +/- 0.29, pHlu 8.09 +/- 0.15). Steady-state pHi of proximal segments was much less sensitive to changes in pHo than pH of the luminal fluid (delta pHlu/delta pHo was 0.49 while delta pHi/delta pHo was 0.18; pHo 6.50-7.20). Re-alkaliniziation from an NH4Cl-induced intracellular acid load (initial pHi recovery rate 0.55 +/- 0.34 pH.min-1) was nearly totally inhibited by 1 mmol.l-1 KCN (96% inhibition) and to a large degree (79%) by 1 mumol.l-1 bafilomycin A1. In contrast, both vanadate (1 mmol.l-1) and amiloride (1 mmol.l-1) inhibited pHi recovery by 38% and 33%, respectively. Unlike amiloride, removal of Na+ from the bathing saline had no effect on pHi recovery, indicating that a Na+/H+ exchange is not significantly involved in pHi regulation. Instead pHi regulation apparently depended largely on the availability of ATP and on the activity of the bafilomycin-sensitive proton pump.

PH recovery from intracellular alkalinization in Retzius neurones of the leech central nervous system.

1. Neutral-carrier pH-sensitive microelectrodes were used to investigate intracellular pH (pHi) recovery from alkalinization in leech Retzius neurones in Hepes- and in CO2-HCO3(-)-buffered solution. The Retzius neurones were alkaline loaded by the addition and subsequent removal of 16 mM acetate, by changing from 5% CO2-27 mM HCO3- to 2% CO2-11 mM HCO3- or by changing from CO2-HCO3(-)- to Hepes-buffered solution. 2. In Hepes-buffered solution (pH 7.4) the mean pHi was 7.29 +/- 0.11 and the mean membrane potential -44.7 +/- 5.9 mV (mean +/- S.D.; n = 83). 3. The rate of pHi recovery from alkalinization increased with decreasing pH of the bathing medium (pHb). pHi changed about 0.30 pH units for a pHb unit change. 4. A decrease of extracellular buffer concentration (Hepes concentration lowered from 20 to 5 mM) caused an acidification of extracellular and intracellular pH and an acceleration of pHi recovery from alkalinization. 5. A depolarization of the Retzius cell membrane-induced by increasing the K+ concentration of the bathing medium from 4 to 20 mM (delta Em = 16.5 +/- 5.5 mV) or from 4 to 40 mM (delta Em = 24.8 +/- 3.5 mV)--evoked a decrease of pHi and an acceleration of pHi recovery from alkalinization. 6. The H+ current blocker Zn2+ (0.5 mM) inhibited pHi recovery from alkalinization at resting membrane potential as well as during depolarization. The inhibition was more pronounced during depolarization. 7. In Cl(-)-free, CO2-HCO3(-)-buffered solution pHi recovery from an alkaline load by changing from 5% CO2-27 mM HCO3- to 2% CO2-11 mM HCO3- was slowed by 48-71%. The rate of pHi recovery from an alkaline load induced by changing from CO2-HCO3- to Hepes buffer was reduced by 33-56% in Cl(-)-free solution. The removal of external Cl- did not affect pHi recovery in Hepes-buffered solution. 8. The pHi recovery from alkalinization was DIDS-insensitive in CO2-HCO3(-)- as in Hepes-buffered solutions and was not slowed in the absence of external Na+. 9. It is concluded that in Retzius neurones pHi recovery from alkalinization is mediated by a passive voltage-dependent H+ influx along the electrochemical proton gradient. In the presence of CO2-HCO3- buffer a DIDS-insensitive Cl(-)-HCO3- exchanger additionally regulates pHi after an intracellular alkaline load. It cannot be excluded that intracellular processes (e.g. H+ release from organelles, metabolic H+ production) are also involved in pHi recovery from alkalinization.

Hydrogen Ion Regulation in Rat Cerebellar Granule Cells Studied by Single-Cell Fluorescence Microscopy.

The internal pH (pHi) regulatory mechanisms of individual rat cerebellar granule cells maintained in tissue culture have been investigated using the fluorescent indicator BCECF (2,7' bis carboxyethyl 5,6 carboxy-fluorescein) and quantitative fluorescence microscopy. The steady-state pHi was estimated as 7.27 +/- 0.25 in bicarbonate-buffered media and 7.49 +/- 0.35 in HEPES-buffered media. Buffering power was estimated at about 8 mM/pH unit from the peak alkalinization and acidification transients seen on addition and removal of NH4Cl. Bicarbonate did not appear to contribute to the buffering power estimated in this way. Following an acid load imposed by the ammonium prepulse technique, pHi recovered to steady-state values with first-order kinetics. Recovery was absolutely dependent upon extracellular sodium and, in about half of the cells tested, bicarbonate ions. In cells that did not require bicarbonate for pHi recovery, amiloride (1 mM) inhibited pHi recovery. Removal of extracellular chloride produced a reversible alkalinization of pHi in a third of the cells studied. This alkalinization persisted even when extracellular sodium had been reduced to zero. Removal of extracellular chloride did not inhibit bicarbonate-dependent pHi recovery following an acid load. These results are best explained by the existence of three independent pHi regulatory mechanisms: Na+/H+ exchange, Na+/HCO3- cotransport and Cl-/HCO3- exchange.

Intracellular pH and its regulation in isolated type I carotid body cells of the neonatal rat.

1. The dual-emission pH-sensitive fluoroprobe carboxy-SNARF-1 (carboxy-seminaptharhodofluor) was used to measure pHi in type I cells enzymically dispersed from the neonatal rat carotid body. 2. Steady-state pHi in cells bathed in a HEPES-buffered Tyrode solution (pH 7.4) was found to be remarkably alkaline (pHi = 7.77) whereas cells bathed in a CO2-HCO3(-)-buffered Tyrode solution (pH 7.4) had a more 'normal' pHi (pHi = 7.28). These observations were further substantiated by using an independent nullpoint test method to determine pHi. 3. Intracellular intrinsic buffering (beta, determined by acidifying the cell using an NH4Cl pre-pulse) was in the range 7-20 mM per pH unit and appeared to be dependent upon pHi with beta increasing as pHi decreased. 4. In cells bathed in a HEPES-buffered Tyrode solution, pHi recovery from an induced intracellular acid load (10 mM-NH4Cl pre-pulse) was inhibited by the Na(+)-H+ exchange inhibitor ethyl isopropyl amiloride (EIPA; 150 microM) or substitution of Nao+ with N-methyl-D-glucamine (NMG). Both EIPA and Nao+ removal also caused a rapid intracellular acidification, which in the case of Nao+ removal, was readily reversible. The rate of this acidification was similar for both Nao+ removal and EIPA addition. 5. Transferring cells from a HEPES-buffered Tyrode solution to one buffered with 5% CO2-HCO3- resulted in an intracellular acidification which was partially, or wholly, sustained. The rate of acidification upon transfer to CO2-HCO3- was considerably slowed by the membrane permeant carbonic anhydrase inhibitor, acetazolamide, thus indicating the presence of the enzyme in these cells. 6. In CO2-HCO3(-)-buffered Tyrode solution, pHi recovery from an intracellular acidosis (NH4+ pre-pulse) was only partially inhibited by EIPA or amiloride whereas Nao+ removal completely inhibited the recovery. The stilbene DIDS (4,4-diisothiocyanatostilbenedisulphonic acid, 200 microM) also partially inhibited pHi recovery following an induced intracellular acidosis. Furthermore, the pre-treatment with 200 microM-DIDS of a pre-acidified cell in Na(+)-free Tyrode solution completely inhibited pHi recovery when Nao+ was reintroduced together with concomitant addition of 150 microM-EIPA. We conclude, that in the presence of CO2-HCO3-, a Na(+)- and HCO3-dependent (DIDS inhibitable) mechanism aids acid extrusion.(ABSTRACT TRUNCATED AT 400 WORDS)

Intracellular pH regulation in isolated rat bile duct epithelial cells.

To evaluate ion transport mechanisms in bile duct epithelium (BDE), BDE cells were isolated from bile duct-ligated rats. After short-term culture pHi was measured with a single cell microfluorimetric set-up using the fluorescent pHi indicator BCECF, and calibrated with nigericin in high K+ concentration buffer. Major contaminants were identified using vital markers. In HCO3(-)-free media, baseline pHi (7.03 +/- 0.12) decreased by 0.45 +/- 0.18 pH units after Na+ removal and by 0.12 +/- .04 after amiloride administration (1 mM). After acid loading (20 mM NH4Cl) pHi recovery was inhibited by both Na+ removal and amiloride (JH+ = 0.74 +/- 1.1, and JH+ = 2.28 +/- 0.8, respectively, vs. 5.47 +/- 1.97 and 5.97 +/- 1.76 mM/min, in controls, respectively). In HCO3- containing media baseline pHi was higher (7.16 +/- 0.1, n = 36, P less than 0.05) and was decreased by Na+ substitution but not by amiloride. Na+ removal inhibited pHi recovery after an intracellular acid load (0.27 +/- 0.26, vs. 7.7 +/- 4.1 mM/min, in controls), whereas amiloride reduced JH+ only by 27%. pH recovery was inhibited by DIDS (0.5-1 mM), but not by Cl- depletion. Finally, acute Cl- removal increased pHi by 0.18 pH units in the absence but not presence of DIDS. These data indicate that BDE cells possess mechanisms for Na+/H+ exchange, Na+:HCO3- symport and Cl-/HCO3 exchange. Therefore BDE may be capable of transepithelial H+/HCO3- transport.

Intracellular pH regulation in the embryonic chicken lens epithelium.

1. The intracellular pH (pHi) of embryonic lens epithelia was measured by the emission ratio technique using the fluorescent pH probe carboxy-seminaphthorhodafluor-1 (Snarf-1). 2. In artificial aqueous humour solutions (AAH) containing HCO3-, pHi was 7.45, a value more alkaline than that of the bathing medium (pH = 7.3). In HCO3- -free AAH, pHi was 7.29. 3. Acetazolamide, an inhibitor of carbonic anhydrase, had no effect on resting pHi. 4. The pHi could be manipulated experimentally by changing the external pH (pHo) of HEPES-buffered AAH, the addition or withdrawal of CO2-HCO3-, or by perfusion with the weak bases NH4Cl and procaine. 5. The pHi change induced by withdrawal of 5 mM-procaine was used to calculate a value for the intrinsic cytoplasmic buffering capacity (beta i) of 16.5 mM. 6. The addition of amiloride (1 mM) or treatment with low-Na+ AAH solutions led to a decrease in pHi of 0.23 over the 10 min exposure. In addition, these treatments inhibited pHi recovery from NH4(+)-induced acidosis. These observations are consistent with the presence of amiloride-sensitive N(+)-H+ antiport. 7. Addition of exogenous antiport activity in the form of 50 microM-monensin caused an increase in pHi of 0.24. 8. In HCO3(-)-containing media, replacing Cl- by gluconate or isothionate led to an immediate, reversible increase in pHi which could be completely inhibited by 2 mM-4-acetamido-4'-isothiocyanato-stillbene-2,2'-disulphonic acid (SITS). This indicates the presence of Cl(-)-HCO3- exchange in this tissue. 9. Under HCO3(-)-free conditions, replacement of Cl- by gluconate or isothionate caused a small transient acidification followed, 5 min later, by a large sustained alkalinization. The delayed increase in pHi could be completely blocked by 1 mM-amiloride and may reflect volume-sensitive stimulation of the Na(+)-H+ antiporter as cell volume (estimated by cell height measurements) was shown to decrease significantly during this period.

ATP-dependent pHi recovery in lung macrophages: evidence for a plasma membrane H(+)-ATPase

We have previously shown that cytoplasmic pH (pHi) recovery in pulmonary macrophages, under nominally HCO3(-)-free conditions, after acute intracellular acidification is Na+ and amiloride insensitive and is blocked by nonspecific proton adenosinetriphosphatase (ATPase) inhibitors N-ethyl-maleimide and N,N'-dicyclohexylcarbodiimide [Am. J. Physiol. 257 (Cell. Physiol. 26): C65-C76, 1989]. To further delineate the mechanism of H+ extrusion across plasma membranes of pulmonary macrophages, we investigated the effects of metabolic inhibitors of oxidative phosphorylation and glycolysis on cellular ATP content and pHi recovery from an intracellular acid load under nominally HCO3(-)-free conditions. Dose-dependent reductions in ATP levels and in the rate of pHi recovery were obtained in the presence of KCN (50% inhibition, 10(-4) M). Parallel reductions in ATP content and the rate of pHi recovery were noted in the presence of antimycin A, rotenone, oligomycin, and iodoacetate. However, inhibition by iodoacetate was reduced in the presence of pyruvate. The more specific vacuolar H(+)-ATPase inhibitors, bafilomycin A1 and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, resulted in no decrement in cellular ATP levels but significantly inhibited pHi recovery. These studies demonstrate that recovery from an acid load is ATP dependent and provide support for a plasmalemmal proton ATPase, perhaps of the vacuolar type, that participates in regulation of pHi in pulmonary macrophages.

Na(+)-H+ and Na(+)-dependent Cl(-)-HCO3- exchange control pHi in vascular smooth muscle

The mechanisms that control intracellular pH (pHi) in vascular smooth muscle are not fully understood. These studies were performed to determine the identity and relative importance of the sarcolemmal transport systems that mediate net acid efflux in primary cultured vascular smooth muscle cells from canine femoral artery. In HEPES- or HCO3(-)-buffered physiological salt solution (HEPES-PSS, HCO3(-)-PSS), recovery from an acute acid load was totally dependent on external Na+. 5-[N-ethyl-N-isopropyl]amiloride (EIPA, 50 microM) inhibited pHi recovery 100 and 68% in HEPES-PSS and HCO3(-)-PSS, respectively. EIPA-insensitive pHi recovery in HCO3(-)-PSS was inhibited 48% by 4,4'-diisothyocyanostilbene-2,2'-disulfonic acid (DIDS). An outwardly directed H+ gradient stimulated amiloride-sensitive 22Na+ uptake, and an inwardly directed HCO3- gradient stimulated amiloride-insensitive 22Na+ uptake. The latter was inhibited by DIDS or prior depletion of cell Cl-. In HEPES-PSS, resting pHi was 7.17 +/- 0.03, was not affected by DIDS, but was lowered by EIPA or by removing extracellular Na+. In HCO3(-)-PSS, resting pHi was 7.25 +/- 0.02 (P less than 0.05) and was not affected by EIPA. Removing extracellular Na+ in the presence of EIPA decreased pHi in HCO3(-)-PSS but not in HEPES-PSS. DIDS lowered resting pHi in HCO3(-)-PSS, after which EIPA further lowered pHi. We conclude that acid efflux from these cells is mediated by a Na(+)-H+ exchanger and a Na(+)-dependent Cl(-)-HCO3- exchanger. In HEPES-PSS, acid efflux via the Na(+)-H+ exchanger maintains resting pHi. In HCO3(-)-PSS, additional acid efflux via the Na(+)-dependent Cl(-)-HCO3- exchanger results in a higher pHi. Although the Na(+)-H+ exchanger is primarily responsible for acid efflux after an acute acid load, the Na(+)-dependent Cl(-)-HCO3- exchanger is responsible for acid efflux under physiological conditions.

Dual regulatory mechanisms of proton transport in rat papillary collecting duct cells in culture.

The regulation of proton transport and cytosolic pH was studied in rat papillary collecting duct (PCD) cells in culture using a pH-sensitive fluorescence probe, 2,7-bis-carboxyethyl-5,6-carboxyfluorescein (BCECF). Data were obtained from confluent monolayers grown on glass coverslips and dipped in a HCO3- -free medium, pH 7.40. The resting intracellular pH (pHi) was 7.16 +/- 0.03 (n = 20). When PCD cells had been acidified by pretreatment with NH4Cl, pHi immediately recovered toward the resting value. Two mechanisms participated in this recovery: a Na+-dependent mechanism which could be inhibited by amiloride (indicative of Na+-H+ exchanger) and a Na+-independent process (a proton ATPase). The pHi recovery from acid loading was inhibited by amiloride to about 55% of the control recovery (half-maximal effect at 100 microM). The rate of pHi recovery after the readdition of Na+ to a sodium-free medium exhibited saturation kinetics (half maximal rate at 28 mM). Dicyclohexylcarbodiimide (DCCD), an inhibitor of a plasma membrane proton ATPase, and the depletion of cellular ATP induced by 2 mM potassium cyanide (KCN) also partially inhibited the rate of pHi recovery after cell acidification with a NH4Cl load. When PCD cells were treated with 1 mM DCCD, amiloride almost completely inhibited pHi recovery. Amiloride and the removal of external Na+ had induced a gradual fall in pHi to a new resting value and rapidly recovered when Na+ was added. We conclude that PCD cells grown in culture have at least two proton transport mechanisms: a Na+-H+ exchanger and a plasma membrane proton ATPase. The kinetics of these processes can be reliably assessed by the pH-sensitive fluorescent probe, BCECF. Both the Na+-H+ exchanger and the plasma membrane proton ATPase may contribute to urinary acidification.

Evidence for Na+/H+ exchange and pH sensitive membrane voltage in cultured bovine corneal epithelial cells.

Two methods were used to investigate cellular ion transport processes in confluent monolayers of cultured bovine corneal epithelial cells: measurements of membrane voltage (V) using conventional microelectrodes, and intracellular pH (pHi) measurements using the pH sensitive absorbance of intracellularly trapped 5 (and 6)-carboxy-4', 5'dimethyl-fluorescein. (1) V averaged -39.2 +/- 0.9 mV (mean +/- SEM, n = 71) with a range of -30 to -59 mV. Increasing extracellular potassium depolarized the cell membrane with a K+-slope of 43.3 mV/decade [K+] (for [K+] between 20 and 80 mM). Intracellular as well as extracellular acidification reversibly depolarized the cell membrane. Depolarization induced by 40 mM K+-pulses was smaller at extracellular pH (pHo) of 6.9 as compared to pHo = 7.9. These findings are compatible with a pH-sensitive K+ conductance. (2) During steady state pHi was 6.96 +/- 0.05 (mean +/- SEM, n = 7). After intracellular acidification, induced by NH4Cl-prepulse technique, pHi was regulated back towards normal steady state pHi. Application of 1 mM amiloride reversibly inhibited pHi recovery. Furthermore, pHi backregulation was inhibited by removing sodium from the extracellular solution. The effect was reversible after readdition of sodium. These findings suggest that a Na+/H+ exchange is present in corneal epithelial cells and participates in pHi backregulation after an intracellular acid load.

Intracellular pH regulation and proton transport by rabbit renal medullary collecting duct cells. Role of plasma membrane proton adenosine triphosphatase.

Proton secretion in the renal medullary collecting duct is thought to occur via a luminal proton-ATPase. In order to determine what mechanism(s) participate in proton transport across medullary collecting duct (MCD) cells membranes, intracellular pH (pHi) regulation and proton extrusion rates were measured in freshly prepared suspensions of rabbit outer MCD cells. Cells were separated by protease digestion and purified by Ficoll gradient centrifugation. pHi was estimated fluorometrically using the entrapped intracytoplasmic pH indicator, 6-carboxyfluorescein. Proton extrusion rates were measured using a pH stat. The resting pHi of MCD cells was 7.19 +/- 0.05 (SE) in a nonbicarbonate medium of pH 7.30. When cells were acidified by exposure to acetate salts or by abrupt withdrawal of ammonium chloride, they exhibited pHi recovery to the resting pHi over a 5-min time-course. Depletion of greater than 95% of cellular ATP content by poisoning with KCN in the absence of glucose inhibited pHi recovery. ATP depletion inhibited proton extrusion from MCD cells. Treatment with N-ethylmaleimide also inhibited pHi recovery. In addition, cellular ATP content was dependent on transmembrane pH gradients, suggesting that proton extrusion stimulated ATP hydrolysis. Neither removal of extracellular sodium nor addition of amiloride inhibited pHi recovery. These results provide direct evidence that a plasma membrane proton-ATPase, but not a Na+/H+ exchanger, plays a role in proton transport and pHi regulation in rabbit MCD.

Intracellular pH regulation in the sensory neurone of the stretch receptor of the crayfish (Astacus fluviatilis).

The ionic mechanisms of intracellular pH (pHi) regulation were studied in the slowly adapting sensory cell of the crayfish stretch receptor by using pH-, Na+- and Cl(-)-sensitive liquid ion exchanger electrodes. Under control conditions a mean pHi of 7.23 +/- 0.12 (S.D.) at a mean membrane potential of 68.3 +/- 4.1 mV S.D. was found in sixteen cells. Thus pHi is about 1 pH unit more alkaline than predicted from passive distribution, implying the presence of an acid extrusion mechanism. In order to acidify the cytoplasm, the cell was either acid-loaded by NH4Cl or exposed to CO2 and CO2/HCO3- solutions. During CO2 exposures pHi was regulated only if calculated amounts of HCO3- were added to keep external pH (pHo) constant. The pHo per se was found to be an important determinant of pHi and its regulation. Substitution of external Na+ by choline inhibited pHi recovery almost completely. As soon as Na+ was readmitted H+ extrusion occurred immediately at a rate similar to that of the control. The internal Na+ activity (aiNa) ranged between 6 and 13 mM with a mean of approximately 9.1 +/- 2.5 mM (S.D.; n = 8). The effects of various solutions on aiNa and the temporal relationship between aiNa and pHi in NH4Cl acid-loaded cells were investigated. The amount of aiNa increased during cell internal acidification and recovered in parallel with pHi recovery in NH4Cl acid-loaded cells. Experiments with 10(-4) M-ouabain and K+-free conditions suggest that neither the Na+-K+ pump nor external K+ are directly involved in pHi regulation. The internal chloride activity (aiCl), which was lower than predicted from a passive distribution, fell during exposure to HCO3-/CO2. Regulation of pHi was inhibited if the cell was completely depleted of Cl- by prolonged exposures to Cl(-)-free solution (isethionate and/or gluconate substituted). The pHi-regulating system of the sensory cell requires Na+ and Cl- which probably operate in a combined mechanism such as Na+ -H+-Cl(-)-HCO3- or an equivalent.

Sodium-dependent control of intracellular pH in Purkinje fibres of sheep heart.

Intracellular pH (pHi) of Purkinje fibres from sheep heart was recorded with pH-sensitive glass micro-electrodes. The cells were acidified by one of three methods: (1) exposure to and subsequent removal of NH4Cl, (2) exposure to solutions containing 5% CO2 or (3) exposure to an acidic Tyrode solution. The pHi recovery from these acidifications was studied. The time constant of recovery from an acidification induced by NH4Cl was almost twice as long as that from one induced by CO2 or acid extracellular pH. Following an acidification induced by exposure to CO2 the time constant of pHi recovery was not changed when the cell was depolarized to -40 mV (by replacement of some Na+ by K+). An intracellular acidification was produced when extracellular Na+ was removed and replaced by quaternary ammonium ions or K+. Such Na+-free solutions also inhibited pHi recovery from an acidification. A 50% inhibition of the rate of recovery was produced by lowering the [Na+]o to 8 mM. When used as a Na+ substitute, Li+ could permit recovery. Tris (22 mM) changed pHi in the alkaline direction. Amiloride (1 mM) or a decrease in temperature slowed the recovery from an acidification (Q10 = 2.65). There was no effect of SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid disodium salt; 100 microM) on the recovery. Na+-sensitive glass micro-electrodes were used to measure the intracellular Na+ activity when [Na+]o was lowered to levels used in our pHi recovery experiments. From these data we have calculated the apparent Na+ electrochemical gradient at different values of [Na+]o. If this gradient is responsible for H+ efflux from the cell then, by applying thermodynamic considerations, it can be shown that only low concentrations (1-2mM) of extracellular Na+ are required. Solutions containing a very low [Ca2+]o (less than 10(-8) M, buffered with EGTA) were used to prevent large rises of [Ca2+]i which may occur on removal of external Na+. Under these conditions pHi recovery is still dependent upon [Na+]o, and the apparent inhibition of pHi recovery by removal of Na+ is not simply due to rises in [Ca2+]i. The intracellular acidification which occurs on removal of Na+ does not occur when [Ca2+]o is very low (less than 10(-8) M).(ABSTRACT TRUNCATED AT 400 WORDS)