Cell membrane potential: a signal to control intracellular pH and transepithelial hydrogen ion secretion in frog kidney

The dependence of intracellular pH (pH_i) and transepithelial H⁺ secretion on the cell membrane potential (V _m) was tested applying pH-sensitive and conventional microelectrodes in giant cells fused from single epithelial cells of the diluting segment and in intact tubules of the frog kidney. An increase of extracellular K⁺ concentration from 3 to 15 mmol/l decreasedV _m from –49±4 to –29±1 mV while pH_i increased from 7.44±0.04 to 7.61±0.06. Addition of 1 mmol/l Ba²⁺ depolarizedV _m from –45±3 to –32±2 mV, paralleled by an increase of pH_i from 7.46±0.04 to 7.58±0.03. Application of 0.05 mmol/l furosemide hyperpolarizedV _m from –48±3 to –53±3 mV and decreased pH_i from 7.47±0.05 to 7.42±0.05. In the intact diluting segment of the isolated-perfused frog kidney an increase of peritubular K⁺ concentration from 3 to 15 mmol/l increased the luminal pH from 7.23±0.08 to 7.41±0.08. Addition of Ba²⁺ to the peritubular perfusate also increased luminal pH from 7.35±0.07 to 7.46±0.07. Addition of furosemide decreased luminal pH from 7.32±0.03 to 7.24±0.05. We conclude: cell depolarization reduces the driving force for the rheogenic HCO ₃ ^– exit step across the basolateral cell membrane. HCO ₃ ^– accumulates in the cytoplasm and pH_i increases. An alkaline pH_i inactivates the luminal Na⁺/H⁺ exchanger. This diminishes transepithelial H⁺ secretion. Cell hyperpolarization leads to the opposite phenomenon. Thus, pH_i serves as signal transducer between cell voltage and Na⁺/H⁺ exchange.     

Effects of intra- and extracellular H+ and Na+ concentrations on Na+-H+ antiport activity in the lacrimal gland acinar cells

Kinetic properties of the Na⁺-H⁺ antiport in the acinar cells of the isolated, superfused mouse lacrimal gland were studied by measuring intracellular pH (pH_i) and Na⁺ activity (aNa_i) with the aid of double-barreled H⁺- and Na⁺-selective microelectrodes, respectively. Bicarbonate-free solutions were used throughout. Under untreated control conditions, pH_i was 7.12±0.01 and aNa_i was 6.7±0.6 mmol/l. The cells were acid-loaded by exposure to an NH ₄ ⁺ solution followed by an Na⁺-free N-methyl-d-glucamine (NMDG⁺) solution. Intracellular Na⁺ and H⁺ concentrations were manipulated by changing the duration of exposure to the above solutions. Subsequent addition of the standard Na⁺ solution rapidly increased pH_i. This Na⁺-induced increase in pH_i was almost completely inhibited by 0.5 mmol/l amiloride and was associated with a rapid, amiloride-sensitive increase in aNa_i. The rate of pH_i recovery induced by the standard Na⁺ solution increased in a saturable manner as pH_i decreased, and was negligible at pH_i 7.2–7.3, indicating an inactivation of the Na⁺-H⁺ antiport. The apparent K _m for intracellular H⁺ concentration was 105 nmol/l (pH 6.98). The rate of acid extrusion from the acid-loaded cells increased proportionally to the increase in extracellular pH. Depletion of aNa_i to less than 1 mmol/l by prolonged exposure to NMDG⁺ solution significantly increased the rate of Na⁺-dependent acid extrusion. The rate of acid extrusion increased as the extracellular Na⁺ concentration increased following Michaelis-Menten kinetics (V _max was 0.55 pH/min and the apparent K _m was 75 mmol/l at pH_i 6.88). The results clearly showed that the Na⁺-H⁺ antiport activity is dependent on the chemical potential gradient of both Na⁺ and H⁺ ions across the basolateral membrane, and that the antiporter is asymmetric with respect to the substrate affinity of the transport site. The data agree with the current model of activation and inactivation of the antiporter by an intracellular site through changes in the intracellular Na⁺ and H⁺ concentrations.     

Activation of luminal Na+/H+ exchange in distal nephron of frog kidney

Increased chronic intake of K⁺ induced H⁺ and K⁺ secretion in amphibian distal tubule, paralleled by an elevation of plasma aldosterone. The present experiments test whether the mineralocorticoid hormone is responsible for the alteration of ion transport. The blood capillaries of the isolated kidneys of NaCl-adapted (i.e. aldosterone-suppressed)Rana pipiens were perfused with HEPES-buffered amphibian Ringer solution (pH 7.8). Limiting intraluminal pH (pH_1u) was measured continuously with pH-sensitive microelectrodes while aldosterone (3·10^–7 to 3·10^–6 mol/l) was applied in the peritubular perfusate. Concomitant with a decrease of the lumen-positive transepithelial potential (V _te) from 8.5±1.1 mV to 4.0±0.6 mV pH_1u dropped from 7.73±0.02 to a new steady-state value of 7.17±0.05 within 60 to 180 min of aldosterone administration. Significant luminal acidification occurred already 20 min after application of aldosterone. Luminal addition of 10^–3 mol/l amiloride reversed luminal acidification to a pH_1u of 7.68±0.04; at the same timeV _te recovered partially. Pretreatment of the distal tubules with spironolactone prevented the aldosterone-induced acidification of the tubule fluid. We conclude that in early distal tubule of the amphibian kidney aldosterone — after interaction with cytoplasmic receptors — activates the luminal, amiloride-inhibitable Na⁺/H⁺ exchanger. This mechanism could explain enhanced H⁺ secretion found in the K⁺ adapted animal.     

pH-regulatory mechanisms in in vitro perfused rectal gland tubules of Squalus acanthias

 Isolated in vitro perfused rectal gland tubules (RGT) were preincubated with the pH-sensitive dye 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and pH-regulatory mechanisms were studied. A reduction of bath Cl^– concentration from 269 to 6 mmol/l increased the fluorescence ratio 488/436 [corresponding to cytosolic pH (pH_i)] slightly but significantly (n=10). Depolarization by Ba²⁺ (1 mmol/l) or a bath solution containing 30 mmol/l K⁺ (n=4–6) increased the fluorescence ratio (pH_i). These data suggest that HCO₃ ^– uptake and/or H⁺ extrusion is dependent on Cl^– and/or voltage. A reduction of bath Na⁺ from 278 to 5 mmol/l reduced the ratio significantly (n=3). Addition of trimethylamine (Trima⁺, 20 mmol/l) alkalinized cytosolic pH (n=7). Similarly, addition of NH₄ ⁺ (20 mmol/l) led to an initial alkalinization and a strong acidification when NH₄ ⁺ was removed (n=59). The initial pH_i-recovery rates after NH₄ ⁺ removal were quantified and the responsible H⁺ extrusion and/or HCO₃ ^– import systems were examined. The recovery was almost completely abolished when the extracellular Na⁺ concentration was reduced to 5 mmol/l. In the presence of normal Na⁺, recovery was slower in the absence as compared to the presence of HCO₃ ^– (n=5). It was inhibited by 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) (0.5 mmol/l, n=11) in the presence of HCO₃ ^– and in the absence of HCO₃ ^– by the Na⁺/H⁺-exchange blocker HOE694 (0.5 mmol/l, n=6). These data suggest that acid extrusion probably occurs by basolateral Na⁺-2HCO₃ ^–/Cl^– exchange in the presence of HCO₃ ^– and by basolateral Na⁺/H⁺ exchange in the absence of HCO₃ ^–. Luminal perfusion with a solution containing a low Cl^– concentration (6 mmol/l) increased the fluorescence ratio (pH_i) (n=5). The ratio (pH_i) was further increased and pH recovery further delayed by basolateral addition of Trima⁺ (20 mmol/l, n=3). These data suggest that the HCO₃ ^–/Cl^– exchanger is present in the luminal membrane. Luminal HCO₃ ^–/Cl^– exchange and basolateral Na⁺-2HCO₃ ^–/Cl^– exchange may work in tandem to secrete HCO₃ ^– and exchange it for luminal Cl^–. Received: 7 January 1998 / Received after revision and accepted: 5 March 1998     

Regulation of intracellular sodium and pH by the electrogenic H+ pump in frog skin

We have investigated the role of the electrogenic hydrogen ion pump in the regulation of intracellular sodium ion activity (a _Na ⁱ ) and intracellular pH (pH_i) in frog skin epithelial cells using double-barreled ion sensitive microelectrodes. WhenRana esculenta skin is mounted in an Ussing chamber and bathed in 1 mM Na₂SO₄ buffered to pH 7.34 with imidazole on the apical side and in normal Ringer on the serosal side, the apical addition of the carbonic anhydrase inhibitor, ethoxzolamide (10^–4M) blocks net H⁺ ion excretion and Na absorption, producing a depolarization of 25–30 mV of the apical membrane, potential (mc). We demonstrate the these changes are accompanied by a fall ina _Na ⁱ from 6.2±0.5 mmol/l to 3.4±0.6 mmol/l and an increase in pH_i from 7.20±0.03 to 7.38±0.08 (n=12 skins). Voltage clamping mc to its control value in the presence of ethoxzolamide restoreda _Na ⁱ but the pH_i remained alkaline. Furthermore, the fall ina _Na ⁱ produced by ethoxzolamide could be mimicked by voltage clamping mc towards the value of the Nernst potential for Na at the apical membrane. These results indicate that the maintenance of the cellular Na⁺ transport pool is dependent on a favourable electrical driving force and counter-current generated by an electrogenic H⁺ pump at the apical membrane.Addition of amiloride (10^–5 mol/l) or substitution of external Na⁺ by Mg²⁺ or K⁺ caused a hyperpolarization of mc and a fall ina _Na ⁱ . These effects were accompanied by an inhibition of H⁺ excretion and a fall in pH_i of 0.14 ±0.08 units (n=6 skins). However, when the effect, of Na⁺ transport inhibition on mc was prevented by imposing a voltage clamp no effects of amiloride on H⁺ excretion or pH_i were observed. Moreover, the effect of amiloride on pH_i could be reproduced in control skins by voltage clamping mc to –100 mV. The metabolic inhibitors vanadate (1 mmol/l) and di-cyclo hexyl carbodiimide (5×10^–5 mol/l) inhibited H⁺ excretion and decreased pH_i from 7.28±0.08 to 7.02±0.06 and from 7.30±0.06 to 7.12±0.05 (n=6 skins), respectively.These results indicate that an apical membrane H⁺ ATPase plays a role in regulating pH_i and the mechanism is sensitive to membrane potential.     

The effect of ouabain on intracellular activities of K+, Na+, Cl−, H+ and Ca2+ in proximal tubules of frog kidneys

Using conventional and ion selective microelectrodes, the effect of ouabain (10^–4 mol/l) on peritubular cell membrane potential (PD_pt), on intracellular pH (pH_i) as well as on the intracellular ion activities of Cl^– (Cl _i ^– ), K⁺ (K _i ⁺ ), Na⁺ (Na _i ⁺ ) and Ca²⁺ (Ca _i ²⁺ ) was studied in proximal tubules of the isolated perfused frog kidney. In the absence of ouabain (PD_pt=–57.0±1.9 mV), the electrochemical potential difference of chloride (apparent {ie6-1} and of potassium {ie6-2} is directed from cell to bath, of H⁺ {ie6-3}, of Na⁺ {ie6-4} and of Ca²⁺ {ie6-5} from bath to cell. Ouabain leads to a gradual decline of PD_pt, which is reduced to half (PD_{pt, 1/2}) within 31±4.6 min (in presence of luminal glucose and phenylalanine), and to a decline of the absolute values of apparent {ie6-6}, of {ie6-7}, {ie6-8} and {ie6-9}. In contrast, an increase of {ei6-10} is observed. At PD_{pt, 1/2} apparent Cl _i ^– increases by 6.2±1.0 mmol/l, pH_i by 0.13±0.03, Ca _i ²⁺ by 185±21 nmol/l, and Na _i ⁺ by 34.2±4.6 mmol/l, whereas K _i ⁺ decreases by 37.7±2.2 mmol/l. The results suggest that the application of ouabain is followed by a decrease of peritubular cell membrane permeability to K⁺, by an accumulation of Ca²⁺, Na⁺ and HCO ₃ ^- in the cell and by a dissipation of the electrochemical Cl^– gradient.Supported by Österr. Forschungsrat, Proj. No. 4366     

The colon carcinoma cell line Caco-2 contains an H+/K+-ATPase that contributes to intracellular pH regulation

The presence of an H⁺/K⁺-ATPase and its contribution to the regulation of intracellular pH (pH_i) was investigated in Caco-2 cells. The H⁺/K⁺-ATPase was detected immunologically using the monoclonal antibody 5-B6, which was raised against hog gastric H⁺/K⁺-ATPase. Cell pH was determined using the pH-sensitive dye 2,7-bis(carboxyethyl)-carboxyfruorescein. Control pH_i, measured in HCO ₃ ^– -free medium, was 7.62±0.03 (n=27) when cells were cultured for 14 days and decreased to 7.40±0.03 (n=18) after 35 days in culture. Recovery of pH_i following a NH ₄ ⁺ /NH₃ pulse could be reduced by either 100 M SCH 28080 or 1 mM amiloride, or by removing extracellular Na⁺. The inhibitory effects of SCH 28080 and amiloride were additive, demonstrating the involvement of a gastric-like H⁺/K⁺-ATPase and a Na⁺/H⁺ exchanger in regulating pH_i. Recovery rates at pH_i 6.8 were not significantly different in cells cultured for up to 21 days, but were significantly lower in cells cultured for 28 and 35 days. This decrease in recovery rate was due to a decrease in the SCH-28080-insensitive recovery, indicating a reduction of the relative importance of Na⁺/H⁺ exchange to the recovery. Recovery of pH_i was also inhibited by 1 mM N-ethylmaleimide. However, it is unlikely that N-ethylmaleimide inhibited a vacuolar type of H⁺-ATPase, since bafilomycin A₁ had no effect on pH_i recovery. In conclusion, Caco-2 cells contain a SCH-28080-sensitive mechanism for regulating pH_i, which is most conveniently studied after 28 days in culture, when the relative contribution of a Na⁺/H⁺ exchanger to pH_i regulation is decreased.     

Thrombin-induced cytosolic alkalinization in human platelets occurs without an apparent involvement of HCO 3 − /Cl− exchange

We have estimated the changes in cytosolic pH (pH_i) that occur when human platelets are stimulated by thrombin. Changes in pH_i were estimated (i) from the H⁺ efflux across the plasma membrane using an extracellular pH electrode and (ii) using an intracellular pH-sensitive fluorescent dye (BCECF). Stimulation of platelets with thrombin (0.5 unit/ml) resulted in an H⁺ efflux that averaged 7.7±1.6 mol/10¹¹ platelets (means±SD) leading to an increase in pH_i, from 7.05±0.04 to 7.45±0.05. Both H⁺ efflux and pH_i changes were unaffected by 0.1 mM 4,4-diisothiocyanostilbene-2,2 disulphonate (DIDS), 0.1 mM 4-acetamido 4-isothiostilbene-2,2-disulphonic acid (SITS), or 0.5 mM bumetanide, suggesting no involvement of anion transport systems, e.g. an HCO ₃ ^– /Cl^– exchange. Removal of HCO ₃ ^– or Cl^– from the suspending buffer had no effect on the extent of the rise in pH_i. After blockade of Na⁺/H⁺ exchange by 100 M ethylisopropylamiloride (EIPA), thrombin induced a decrease in pH_i the rate of which averaged 0.39 unit/min in HCO ₃ ^– -containing medium, and 0.57 unit/min in HCO ₃ ^– -free medium. The cytosolic buffer capacity for H⁺ was determined by the nigericin/ NH₄Cl technique in BCECF-loaded platelets and averaged 25.3 mmol/(1xpH) in buffer containing 8 mM HCO ₃ ^– , but only 17.2 mmol/(1xpH) in HCO ₃ ^– -free buffer. The total amount of H⁺ transferred by Na⁺/H⁺ exchange can be estimated from our measurements at 10 mmol/l platelet cytosol in the absence of HCO ₃ ^– and to 14 mmol/l platelet cytosol in the presence of HCO ₃ ^– , and is in good agreement with the estimated amount of Na⁺ uptake by ADP-stimulated platelets. We conclude that net extrusion of H⁺ from stimulated platelets is predominantly mediated by Na⁺/H⁺ exchange without an apparent contribution of HCO ₃ ^– /Cl^– exchange.     

Proton transport mechanism in the cell membrane of Xenopus laevis oocytes

Mechanisms of H⁺ transport across the plasma cell membrane of prophase-arrested oocytes of Xenopus laevis were investigated by testing the effect of ion substitutions and inhibitors on cytoplasmic pH (pH_i), membrane potential (V _m) and membrane resistance (R _m). During superfusion with control solution of pH=7.4, pH_i was 7.49±0.12 (n=15), V _m was –61.9±7.8 mV (n=34) (cytoplasm negative), and R _m was 2.9±1.5 M (n=19). These data confirm that H⁺ ions are not distributed at electrochemical equilibrium. By following pH_i during recovery of the oocytes from an acid load (20 mmol/l NH₄Cl) in the presence and absence of extracellular Na⁺ or amiloride (1 mmol/l), a Na/H exchanger was identified. On the basis of the known Na⁺ gradient across the cell membrane, this transporter could suffice to generate the observed H⁺ disequilibrium distribution. Utilizing blockers or ion-concentration-step experiments no evidence was obtained for an ATP-driven H⁺ pump or for passive acid/base transporters such as H⁺ conductances or Na⁺ (HCO ₃ ^– )₃ cotransport. The membrane depolarization observed in response to extracellular acidification appeared to result from a pH-dependent, Ba²⁺-inhibitable K⁺ conductance.     

Effect of parathyroid hormone on acid/base transport in rabbit renal proximal tubule S3 segment

The effect of parathyroid hormone (PTH) on acid/base transport in isolated rabbit renal proximal tubule S3 segment was investigated with double-barreled and conventional microelectrodes. PTH (10 nM) induced a small depolarization and enhanced the initial rates of cell pH (pH_i) increase and cell Cl^– ([Cl^–]_i) decrease in response to bath Cl^– removal by 28.0±2.1% and 31.0±6.4% respectively. The calculated initial HCO₃ ^– influx to bath Cl^– removal was also enhanced by 28%. On the other hand, PTH reduced the initial rate of pH_i decrease to luminal Na⁺ removal in the absence of HCO₃ ^–/CO₂ by 20.4±3.9%. The PTH-induced depolarization was not accompanied with changes in steadystate pH_i or [Cl^–]_i levels, but was greatly attenuated in the presence of ouabain (0.1 mM). Either dibutyrylcAMP (0.1 mM) plus theophylline (1 mM) or forskolin (10 M) alone could reproduce all the effects of PTH. These results indicate that (a) PTH inhibits the luminal Na⁺/H⁺ exchanger but stimulates the basolateral Cl^–/HCO₃ ^– exchanger in the S3 segment; (b) the PTH-induced depolarization largely results from inhibition of Na⁺/K⁺-ATPase and (c) all these effects are at least partly mediated by a cAMP-dependent mechanism.     

Evidence for Na+ dependent rheogenic HCO 3 − transport in fused cells of frog distal tubules

The mechanism of HCO ₃ ^– transport was studied applying microelectrodes in giant cells fused from single epithelial cells of the diluting segment of frog kidney. A sudeen increase of extracellular HCO ₃ ^– concentration from 10 to 20 mmol/l at constant pH hyperpolarized the cell membrane potential of the fused cell. This cell-voltage response was totally abolished by 10^–3 mol/l SITS and significantly reduced by 10^–4 mol/l acetazolamide or by omission of Na⁺ from the extracellular perfusate. Removal of Na⁺ from the perfusate caused a transient depolarization. Reapplication of Na⁺ induced a transient hyperpolarization. 10^–3 mol/l SITS abolished the cell-voltage response to removal and reapplication of Na⁺. In the intact diluting segment of the isolated perfused frog kidney peritubular perfusion of 10^–4 mol/l acetazolamide reduced the limiting transepithelial electrochemical gradient for H⁺ significantly from 30±4 mV to 14±3 mV. The results suggest: (i) In the diluting segment of the frog kidney a Na⁺-dependent rheogenic HCO ₃ ^– transport system exists across the peritubular cell membrane. (ii) This rheogenic peritubular Na⁺/HCO ₃ ^– cotransporter cooperates with a Na⁺/H⁺ exchanger in the luminal membrane, thus driving HCO ₃ ^– reabsorption. (iii) Reabsorption of HCO ₃ ^– and secretion of H⁺ depend upon the presence of carbonic anhydrase.     

The effect of phenylalanine on intracellular pH and sodium activity in proximal convoluted tubule cells of the frog kidney

The present study was performed to test the influence of sodium coupled transport of neutral substrates on intracellular pH and sodium activity in proximal tubules of the amphibian kidney. To this end, kidneys of rana esculenta have been isolated and perfused both through the portal vein (peritubular capillaries) and the aorta (luminal perfusate). The potential difference across the peritubular membrane of proximal tubule cells has been redorced with conventional (PD_pt) as well as with sodium (PD_na) and hydrogen ion (PD_h) selective microelectrodes continuously before during and after the luminal application of 10 mmol/l phenylalanine, replacing 10 mmol/l raffinose. PD_b and PD_na allowed the calculation of intracellular pH (pH_i) and sodium activity (Na_i), respectively. In the absence of phenylalanine in the tubule lumen, PD_pt approximates –57.5±2.3 mV (n=27), pH_i 7.73±0.04 (n=14, extracellular pH 7.77), and Na_i 13.3±0.9 mmol/l (n=13, extracellular sodium activity 74 mmol/l). Within 1 min the luminal application of phenylalanine leads to a depolarisation of PD_pt by +32±2 mV, as well as an increase of pH_i by 0.24±0.04 and of Na_i by 5.2±1.0 mmol/l. At 8 min from luminal application of phenylalanine, Na_i plateaus 5±1 mmol/l above control value, PD_pt increases again to a value of +12±2 mV below and pH_i decreases to a value 0.04±0.07 above their respective control values. All changes are fully reversed after removal of phenylalanine from the tubule lumen. The steady state of intracellular sodium activity might be explained by an extrusion of sodium via the sodium/potassium-ATPase, which approaches the entry across the luminal membrane, the intracellular alkalinisation is probably due to the reduced exit of bicarbonate across the peritubular cell membrane following the depolarisation of PD_pt.     

Mechanism of hydrogen ion transport in the diluting segment of frog kidney

Transepithelial H⁺ transport was studied in diluting segments of the isolated-perfused kidney ofrana esculenta. The experiments were performed in controls as well as in K⁺-adapted and Na⁺-adapted animals (exposed to 50 mmol/l KCl or NaCl, resp. for at least 3 days). Conventional and single-barreled, liquid ion-exchanger H⁺-sensitive microelectrodes were applied in the tubule lumen to evaluate transepithelial H⁺ net flux (J _te ^H ) as well as limiting transepithelial electrical and H⁺ electrochemical potential differences (PD _te ,E _te ^H ) and luminal pH at zero net flux conditions. The measurements were made in absence (control) and presence of furosemide (5·10^–5 mol/l) or amiloride (10^–3 mol/l). E _te ^H (lumen positive vs ground) was 19±3 mV in controls, 43±3 mV in K⁺ adapted but about zero in Na⁺ adapted animals. Using the correspondingPD _te -values, steady state luminal pH of 7.63±0.05, 7.13±0.05 and 8.02±0.02 was calculated for the respective groups of animals (peritubular pH 7.80). In parallel, significant secretoryJ _te ^H (from blood to lumen) was found in controls (14±2 pmol·cm^–2·S^–1) which was stimulated by K⁺ adaptation (61±8 pmol·cm^–2·s^–1) but reversed in direction by Na⁺-adaptation (–8±1 pmol·cm^–2·s^–1). Amiloride inhibited secretoryJ _te ^H . Elimination of the lumen positivePD _te by furosemide did not affect significantlyE _te ^H andJ _te ^H in control and K⁺ adapted animals but abolished reabsorptiveJ _te ^H in Na⁺ adapted animals.We conclude that in frog diluting segment H⁺ secretion is an active, amiloride-sensitive, furosemide-insensitive transport process. The data are consistent with luminal Na⁺/H⁺ exchange. The activity of this system depends critically on the metabolic state of the animal.Parts of the data were presented at the 16th Ann. Meeting of the Am. Soc. Nephrol., Washington (1983)This work was supported by österr. Forschungsrat, Proj. No.: 4366 and by Dr. Legerlotz Stiftung     

Mechanisms of intracellular pH regulation in the hamster inner medullary collecting duct perfused in vitro

To examine the mechanisms of H⁺ transport in the mid-inner medullary collecting duct of hamsters, we measured the intracellular pH (pH_i) in the in vitro perfused tubules by microscopic fluorometry using 2,7-bis(carboxyethyl)-carboxyfluorescein (BCECF) as a fluorescent probe. In the basal condition, pH_i was 6.74±0.04 (n=45) in HCO ₃ ^– -free modified Ringer solution. Either elimination of Na⁺ from the bath or addition of amiloride (1 mM) to the bath produced a reversible fall in pH_i After acid loading with 25 mM NH₄Cl, pH_i spontaneously recovered with an initial recovery rate of 0.096±0.012 (n=23) pH unit/min. In the absence of ambient Na⁺, after removal of NH ₄ ⁺ , the pH_i remained low (5.95±0.10, n=8) and showed no signs of recovery. Subsequent restoration of Na⁺ only in the lumen had no effect on pH_i. However, when Na⁺ in the bath was returned to the control level, pH_i recovered completely. Amiloride (1 mM) in the bath completely inhibited the Na⁺-dependent pH_i recovery. Furthermore, elimination of Na⁺ from the bath, but not from the lumen, decreased pH_i from 6.97±0.07 to 6.44±0.05 (n=12) in the HCO ₃ ^– /Ringer solution or 6.70±0.03 to 6.02±0.05 (n=8) in the HCO ₃ ^– free solution. pH_i spontaneously returned to 6.76±0.08 with a recovery rate of 0.017±0.5 pH unit/min in the presence of CO₂/HCO ₃ ^– , whereas it did not recover in the absence of CO₂/HCO ₃ ^– . Although elimination of ambient Na⁺ depolarized the basolateral membrane voltage (V _B) from –78±1.2 to –72 ±0.6 mV (n=5, P<0.01), the level of V _B was not sufficient to explain the pH_i recovery solely by HCO ₃ ^– entry driven by the voltage. These results indicate that (a) pH_i of the inner medullary collecting duct is regulated mainly by a Na⁺/H⁺ exchanger in the basolateral membranes, (b) no apparent Na⁺-dependent H⁺ transport system exists in the luminal membranes and (c) Na⁺-independent H⁺ transport may also operate in the presence of CO₂/HCO ₃ ^– Preliminary data were reported at the Conference on Bicarbonate, Chloride, and Proton Transport Systems, New York, USA, in January 1989     

Ba2+ and amiloride uncover or induce a pH-sensitive and a Na+ or non-selective cation conductance in transitional cells of the inner ear

The membrane potential V _m the cytosolic pH (pH_i), the transference numbers (t) for K⁺, Cl^– and Na⁺/ non-selective cation (NSC) and the pH-sensitivity of V _m were investigated in transitional cells from the vestibular labyrinth of the gerbil. V _m, pH_i, , and the pH_i sensitivity of V _m were under control conditions were –92±1 mV (n=89 cells), pH_i 7.13±0.07 (n=11 epithelia), 0.87±0.02 (n=22), 0.02±0.01 (n=19), 0.01±0.01 (n=24) and –5 mV/pH unit (n=13 cells/n=11 epithelia), respectively. In the presence of 100 mol/l Ba²⁺ the corresponding values were: –70±1 mV (n=32), pH_i 7.16±0.08 (n=6), 0.31±0.05 (n=4), 0.06±0.01 (n=6), 0.20±0.03 (n=10) and -16 mV/pH-unit (n=15/n=6). In the presence of 500 mol/l amiloride the corresponding values were: –72±2mV (n=34), pH_i 7.00±0.07 (n=5), 0.50±0.04 (n=6), 0.04±0.01 (n=11), 0.28±0.04 (n=9) and –26 mV/pH-unit (n=20/n=5). In the presence of 20 mmol/l propionate plus amiloride the corresponding values were: –61±2 mV (n=27), pH_i 6.72±0.06 (n=5), 0.30±0.02 (n=6), 0.06±0.01 (n=5) and 0.40±0.02 (n=8), respectively. V _m was depolarized and and pH_i decreased due to (a) addition of 1 mmol/l amiloride in 150 mmol/l Na⁺ by 38±1 mV (n=8), from 0.82±0.02 to 0.17±0.02 (n=8) and by 0.13±0.01 pH unit (n=6), respectively; (b) reduction of [Na⁺] from 150 to 1.5 mmol/l by 3.3±0.5 mV (n=30), from 0.83±0.02 to 0.75±0.04 (n=9) and by 0.33±0.07 pH unit (n=4), respectively and (c) addition of 1 mmol/l amiloride in 1.5 mmol/l Na⁺ by 20±1 mV (n=11) and from 0.83±0.03 to 0.53±0.02 (n=5), respectively. These data suggest that the K⁺ conductance is directly inhibited by amiloride and Ba²⁺ and that Ba²⁺ and amiloride uncover or induce a pH-sensitive and a Na⁺/NSC conductance which may or may not be the same entity.Some of the data have been presented at various meetings and appear in abstract form in [31, 35, 37]     

Regulation of intracellular pH in cultured bovine retinal pigment epithelial cells

Regulation of intracellular pH (pH_i) in bovine retinal pigment epithelium (RPE) was investigated in cell culture. pH_i was measured using the pH-sensitive absorbance of intracellularly trapped 5 (and 6)-carboxy-dimethyl-fluorescein (CDMF). (1) Regulation of pH_i after induction of an acid load by removal of NH₄Cl could be blocked either totally by removal of extracellular sodium, or subtotally (about 90%) by application of amiloride (1 mmol/l). Additional flux measurements revealed a dose-dependent, amiloride-sensitive²²Na⁺-uptake into Na⁺-loaded cells. Both results suggest the presence of a Na⁺/H⁺ antiport.(2) When alkalinization of the cells was induced by preincubation with 50 mmol/l acetate in HCO ₃ ^– -Ringer's and subsequent removal of the weak acid, the following regulation was dependent on the presence of extracellular chloride. This process could be blocked with DIDS (1 mmol/l), suggesting the presence of a Cl^–/HCO ₃ ^– exchange mechanism.(3) We found no evidence for a Na⁺/HCO ₃ ^– -cotransport, which had been postulated to be present in RPE by others. We conclude that two processes are involved in regulation of pH_i in RPE: A Na⁺/H⁺ antiport responsible for recovery of pH_i from acid load, and a DIDS-sensitive Cl^–/HCO ₃ ^– exchange mechanism responsible for recovery of pH_i after alkalinization.Parts of this work jhave been published in abstract from [20, 21]     

Na+-HCO3 − cotransporter and intracellular pH regulation in chicken enterocytes

The current studies examine the presence of the Na⁺-HCO₃ ^– cotransporter in chicken enterocytes and its role in cytosolic pH (pH_i) regulation. The pH-sensitive dye 2,7-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF) was used to monitor pH_i. Under resting conditions, pH_i was 7.25 in solutions buffered with bis(2-hydroxyethyl)-1-piperazine ethanesulphonic acid (HEPES) and 7.17 in those buffered with HCO₃ ^–. Removal of external Na⁺ decreased pH_i and readdition of Na⁺ rapidly increased pH_i towards the control values. These Na⁺-dependent changes were greater in HCO ₃ ^– than in HEPES-buffered solutions. In HCO ₃ ^– - free solutions the Na⁺-dependent changes in pH_i were prevented by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and unaffected by 4,4-diisothiocyanatostilbene disulphonic acid (H₂-DIDS). In the presence of HCO ₃ ^– , the Na⁺-induced changes in pH_i were sensitive to both EIPA and H₂-DIDS. In the presence of EIPA, cells partially recovered from a moderate acid load only when both Na⁺ and HCO ₃ ^– were present. This pH_i recovery, which was EIPA resistant, and dependent on Na⁺ and HCO ₃ ^– , was inhibited by H₂-DIDS and occurred at equal rates in both Cl^–-containing and Cl^–-free solutions. Kinetic analysis of the rate of HCO ₃ ^– - and Na⁺- dependent pH_i recovery from an acid load as a function of the Na⁺ concentration revealed first-order kinetics with a Michaelis constant, K _m, of 11 mmol/l Na⁺. It is concluded that in HCO₃ ^/– buffered solutions both the Na⁺/H⁺ exchanger and the Na⁺-HCO₃ ^– cotransporter participate in setting the resting pH_i in isolated chicken enterocytes and help the recovery from acid loads.     

Na+/H+ exchange regulates intracellular pH of rat gastric surface cells in vivo

Intracellular pH (pH_i) and viability of gastric surface cells of the rat stomach in response to luminal acidification, and the role of Na⁺/H⁺ exchange in maintaining pH_i homeostasis were studied in vivo using a fluorescent microscopic technique. pH_i was measured during superfusion with buffers of pH 1.2–7.4. When the pH of the superfusate was 7.4, baseline pH_i was unchanged. Superfusion with pH 3 buffer rapidly decreased pH_i to 6.7, with subsequent recovery to baseline pH_i within 15 min despite continuing acid exposure. Superfusion with buffers of pH 1.7 and 1.2 decreased pH_i continuously to below 6.2 with no recovery observed. Despite the relentless decline in pH_i during superfusion with pH-1.2 and –1.7 solutions, over 75% of the surface cells were still viable, as measured by exclusion of the vital dye propidium iodide. We then examined the role of Na⁺/H⁺ exchange in the regulation of pH_i. Superfusion with amiloride did not affect recovery of pH_i from intracellular acidification induced by a NH₄Cl prepulse. Exposure to the potent, lipophilic Na⁺/H⁺ exchange inhibitor 5-(N,N-hexaniethylene)-amiloride (HMA), either in the superfusate or by close arterial perfusion, decreased baseline pH_i from 7.1 to 6.8. Close arterial perfusion of HMA additionally attenuated the recovery of pH_i to baseline during superfusion with pH 3 buffer. We conclude that luminal protons permeate into the cytoplasm of gastric surface cells, where they are eliminated by an Na⁺/H⁺ exchanger, most probably localized to the basolateral membrane.     

Effects of the Na+/H+-exchange inhibitor Hoe 642 on intracellular pH,calcium and sodium in isolated rat ventricular myocytes

 The inhibitors of the Na⁺/H⁺-exchange (NHE1) system Hoe 694 and Hoe 642 possess cardioprotective effects in ischaemia/reperfusion. It is assumed that these effects are due to the prevention of intracellular sodium (Na_i) and calcium (Ca_i) overload. The purpose of the present study was to investigate the effects of Hoe 642 on intracellular pH, Na⁺ and Ca²⁺ (pH_i, Na_i and Ca_i) in isolated rat ventricular myocytes under anoxic conditions or in cells in which oxidative phosphorylation had been inhibited by 1.5 mmol/l cyanide. In cells which were dually loaded with the fluorescent dyes 2,7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and Fura-2, anoxia caused acidification of the cells (from pH_i 7.2 to pH_i 6.8) and an increase in Ca_i from about 50 nmol/l to about 1 μmol/l. The decrease in pH_i began before the cells underwent hypoxic (rigor) contracture, whereas Ca_i only began to rise after rigor shortening had taken place. After reoxygenation, pH_i returned to its control value and Ca_i oscillated and then declined to resting levels. It was during this phase that the cells rounded up (hypercontracture). When 10 μmol/l Hoe 642 was present from the beginning of the experiment, pH_i and Ca_i were not significantly different from control experiments. At reoxygenation, pH_i did not recover, but Ca_i oscillated and returned to its resting level. To monitor Na_i, the cells were loaded with the dye SBFI. After adding 1.5 mmol/l cyanide or 100 μmol/l ouabain, Na_i increased from the initial 8 mmol/l to approximately 16 mmol/l. Hoe 642 or Hoe 694 (10 μmol/l) did not prevent the increase in Na_i. In contrast, the blocker of the persistent Na⁺ current R56865 (10 μmol/l) attenuated the CN^–-induced rise in Na_i. The substance ethylisopropylamiloride was not used because it augmented considerably the intensity of the 380 nm wavelength of the cell’s autofluorescence. In conclusion, the specific NHE1 inhibitor Hoe 642 did not attenuate anoxia-induced Ca_i overload, nor CN^–-induced Na_i and Ca_i overload. Hoe 642 prevented the recovery of pH_i from anoxic acidification. This low pH_i maintained after reoxygenation may be cardioprotective. Other possible mechanisms of NHE1 inhibitors, such as prevention of Ca²⁺ overload in mitochondria, cannot be ruled out. The increase in Na_i during anoxia is possibly due to an influx of Na⁺ via persistent Na⁺ channels. Received: 1 March 1996 / Received after revision: 30 April 1996 / Accepted: 12 July 1996     

Microelectrode determination of oxyntic cell pH in intact frog gastric mucosa. Effect of histamine

Intracellular pH (pH_i) of acid-secreting cells was measured in intact gastric fundus mucosa of Rana esculenta with double-barrelled pH microelectrodes. Tissues were mounted, serosal side up, between two half chambers and individual cells were impaled after microsurgical removal of the serosal muscle layer. Transepithelial potential difference (V _t) and resistance (R _t) as well as serosal cell membrane potential (V _s) and pH_i were continuously recorded at rest (0.1 mmol/l cimetidine) or during stimulation (0.5 mmol/l histamine). During chamber perfusion with HCO3 ₃ ^– /CO₂-buffered Ringer solution of pH_o=7.36, V _t and R _t were –21.7, SD±6.0 mV and 229±83 cm²(n=17) while V _s and pH_i averaged –7.3±6.9 mV and 7.4±0.11 (n=25). The latter value is considerably more alkaline than all recent pH_i measurements obtained with microspectrofluorometric techniques on isolated cells, glands or intact tissue. The difference may in part be explained by use of HCO ₃ ^– -free solutions in most of the previous studies because we observed that such solutions decrease pH_i to 6.89±0.18 (n=4). Again, in contrast to recent literature, application of histamine in HCO ₃ ^– /CO₂-buffered solution led to further transient alkalinization by 0.12±0.05 pH unit (n=8). Since in accidental punctures of the gastric gland lumen we noticed that H⁺ secretion only began approximately 5 min after histamine application, we conclude that the histamine-induced initial alkalinization does not reflect stimulation of the H⁺/K⁺ ATPase pump. Alternatively, it may result from histamine-induced activation or inactivation of other ion transporters, one possibility being activation of basolateral Na⁺/H⁺ and Cl^–/HCO ₃ ^– exchangers.