Video microscopy of intracellular pH in primary cultures of rabbit proximal and early distal tubules

The purpose of this study was to investigate intracytoplasmic pH (pH_i) regulation in primary cultures of proximal (PCT) and distal bright (DCTb) convoluted tubules. PCT and DCTb segments were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The cultured epithelia were grown on semi-transparent permeable supports. The pH_i was determined by video microscopy and digital image processing using 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and measuring the ratio of BCECF fluorescence excited by two successive wavelengths (490 nm and 450 nm). Resting pH_i values, determined in bicarbonatefree medium (extracellular pH: 7.40), were 7.25±0.02 (n=23) and 7.17±0.04 (n=30) for cultured PCT and DCTb respecitively. After the acid-loading procedure, cultured proximal cells recovered their pH_i by means of the classic Na⁺/H⁺ antiporter, sensitive to amiloride and located in the apical membrane only. In cultured DCTb part of the pH_i recovery was mediated by a Na⁺/H⁺ exchange present in the basolateral side. Moreover, at physiological initial pH_i values, chloride removal from the apical solution caused the pH_i to increase in the presence of bicarbonate. In acidified cultured DCTb cells, a partial pH_i recovery was induced in sodium-free media by 15 mM HCO ₃ ^– in the presence of an outward chloride gradient. This pH_i change was completely abolished by 4,4-diisothiocyanostilbene 2,2-disulfonic acid (1 mM). These data suggest that DCTb cells possess in apical anion/base exchanger that resembles the Na⁺-independent Cl^–/HCO ₃ ^– exchanger.     

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.     

pH regulation in HT29 colon carcinoma cells

The pH regulation in HT₂₉ colon carcinoma cells has been investigated using the pH-sensitive fluorescent indicator 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Under control conditions, intracellular pH (pH_i) was 7.21±0.07 (n=22) in HCO ₃ ^– -containing and 7.21±0.09 (n=12) in HCO ₃ ^– -free solution. HOE-694 (10 mol/l), a potent inhibitor of the Na⁺/H⁺ exchanger, did not affect control pH_i. As a means to acidify cells we used the NH ₄ ⁺ /NH₃ (20 mmol/l) prepulse technique. The mean peak acidification was 0.37±0.07 pH units (n=6). In HCC ₃ ^– -free solutions recovery from acid load was completely blocked by HOE-694 (1 mol/l), whereas in HCO3 ₃ ^– -containing solutions a combination of HOE-694 and 4,4-diisothiocyanatostilbene-2, 2-disulphonate (DIDS, 0.5 mmol/l) was necessary to show the same effect. Recovery from acid load was Na⁺-dependent in HCO ₃ ^– -containing and HCO ₃ ^– -free solutions. Removal of external Cl^– caused a rapid, DIDS-blockable alkalinization of 0.33±0.03 pH units (n=15) and of 0.20±0.006 pH units (n=5), when external Na⁺ was removed together with Cl^–. This alkalinization was faster in HCO ₃ ^– -containing than in HCO ₃ ^– -free solutions. The present observations demonstrate three distinct mechanisms of pH regulation in HT₂₉ cells: (a) a Na⁺/H⁺ exchanger, (b) a HCO ₃ ^– /Cl^– exchanger and (c) a Na⁺-dependent HCC ₃ ^– transporter, probably the Na⁺-HCO ₃ ^– /Cl^– antiporter. Under HCO ₃ ^– — free conditions the Na⁺/H⁺ exchanger fully accounts for recovery from acid load, whereas in HCO ₃ ^– -containing solutions this is accomplished by the Na⁺/H⁺ exchanger and a Na⁺-dependent mechanism, which imports HCO ₃ ^– . Recovery from alkaline load is caused by the HCO ₃ ^– /Cl^– exchanger.This study was supported by DFG Gr 480/10     

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.     

Acetazolamide inhibition of basolateral Cl−/HCO 3 − exchange in rabbit renal proximal tubule S3 segment

Cell pH (pH_i) and cell membrane potential (V _b) were measured in isolated S3 segments of rabbit renal proximal tubule with double-barrelled microelectrodes to search for a possible effect of the carbonic anhydrase inhibitor, acetazolamide (ACZ), on Cl^–/HCO ₃ ^– exchange in the basolateral cell membrane. ACZ was found to retard and reduce the pH_i response to bath Cl^– removal reversibly with half-maximal inhibition at 0.42 mmol/l and a rather flat concentration dependence (Hill coefficient 0.36). To determine whether the retardation resulted from inhibition of cytoplasmic carbonic anhydrase, which might have delayed the attainment of HCO ₃ ^– /CO₂ equilibrium, we have measured the response of pH_i to step changes in PCO₂ in the presence and absence of ACZ. ACZ greatly retarded the pH_i response to CO₂ steps; however, the concentration dependence differed (half-maximal inhibition at 18 mol/l) and even at maximal ACZ concentrations the response to CO₂ steps was more than twice as fast as the response to Cl^– replacement. Since, in addition, the ACZ inhibition of Cl^–/HCO ₃ ^– exchange could not be overcome by increasing PCO₂ we conclude that the ACZ effect on Cl^–/HCO ₃ ^– exchange in rabbit proximal tubule S3 segments does not result from inhibition of cytosolic or membrane-bound carbonic anhydrase, but from a direct interaction with the exchanger molecule.     

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.     

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]     

Intracellular pH during secretion in the perfused rabbit mandibular salivary gland measured by31P NMR spectroscopy

Intracellular pH (pH_i) was measured in the isolated, perfused rabbit mandibular salivary gland by³¹P NMR spectroscopy. In the unstimulated gland perfused with HCO ₃ ^– /CO₂-buffered Ringer's solution, pH_i was 7.27±0.01. Continuous stimulation with acetylcholine elicited dose- and time-dependent changes in pH_i. 10^–6 mol/l acetylcholine caused a brief intracellular acidosis (–0.19±0.06 pH units) followed by an increase in pH_i to a more alkaline steady-state value (7.33±0.02). In the absence of perfusate HCO ₃ ^– or in the presence of 10^–4 mol/l DIDS (4,4-diisothiocyanatostilbene-2,2-disulphonic acid), the transient acidosis was abolished and pH_i increased rapidly to give a sustained alkalosis (7.49±0.03 and 7.44±0.03 respectively). In the presence of 10^–3 mol/l amiloride, the response to acetylcholine was a rapid decrease in pH_i to 7.02±0.02. The data suggest that, during perfusion with HCO ₃ ^– /CO₂-buffered solutions, stimulation with acetylcholine results in a transient loss of HCO ₃ ^– from the acinar cells (causing a transient acidosis), and, independently, the activation of Na⁺–H⁺ exchange (causing a sustained alkalosis). In the unstimulated gland, DIDS and the HCO ₃ ^– -free perfusate caused decreases in pH_i to 7.12±0.02 and 7.04±0.01 respectively. In contrast, amiloride had little effect. The relatively high value of pH_i maintained by the unstimulated gland is therefore probably not due to Na⁺–H⁺ exchange.     

HCO 3 − -dependent ACh-activated Na+ influx in sheep parotid secretory endpieces

In the present study we used the Na⁺-sensitive fluorescent dye SBFI and optical measurement of endpiece volume to investigate the transport of Na⁺ in sheep parotid secretory cells. Sheep parotid endpiece cells bathed in a HCO ₃ ^– -free Cl^–-rich solution had a resting intracellular Na⁺ concentration ([Na⁺]_i) of 17±2 mmol/l (n=39). Exposure of the cells to a 2-min pulse of acetylcholine (ACh) (3×10^–7 mol/l) in a HCO ₃ ^– -free bathing solution produced no change in [Na⁺]_i or in cell volume. Changing from a Cl^–-containing HCO ₃ ^– -free bath solution to a Cl^– solution containing 25 mmol/l HCO ₃ ^– caused the endpieces to swell by 8±2 % (n=11) and the [Na⁺]_i to increase by 10±2 mmol/l (n=14). Subsequent exposure of the cells to ACh led to shrinkage of the cells by 12±2 % from the volume in the HCO ₃ ^– -containing solution prior to ACh exposure, with the maximum decrease occurring after 29±7 s (n=9). This shrinkage was accompanied by a rapid and transient increase in [Na⁺]_i, the [Na⁺]_i reaching a peak at 70±5 mmol/l above the unstimulated level (n=9). Substitution of gluconate for Cl^– did not significantly alter the effects of HCO ₃ ^– on unstimulated [Na⁺]_i or endpiece volume, nor did it significantly inhibit the effects of ACh on these two parameters when HCO ₃ ^– was present. Addition of 200 mol/l dihydrogen-4,4-diisothiocyanatostilbene-2,2-disulfonic acid (H₂-DIDS) to the gluconate/HCO ₃ ^– solution significantly reduced the peak of the ACh-induced increase in [Na⁺]_i to 34±10 mmol/l (n=4), but did not have any significant effect on the magnitude of the ACh-induced shrinkage. At 500 mol/l, H₂-DIDS abolished the ACh-induced increase in [Na⁺]_i and also significantly reduced the shrinkage due to ACh. Finally, we found that the rate of endpiece shrinkage following ACh stimulation did not depend on the presence of Cl^–.We interpret these results as indicating that sheep parotid secretory cells do not contain significant Na⁺-K⁺-2Cl^– co-transport activity and do not actively accumulate Cl^–. Rather, the mechanism of spontaneous basal secretion by these cells, in the presence of extracellular HCO ₃ ^– , is based on the accumulation of HCO ₃ ^– by the Na⁺-H⁺ exchanger. During ACh stimulation, the concentration of HCO ₃ ^– in the cytosol is also maintained by the operation of a H₂-DIDS-sensitive Na⁺-HCO ₃ ^– co-transporter. HCO ₃ ^– efflux across the apical membrane occurs via a HCO ₃ ^– conductance pathway rather than by the coupled operation of a Cl^– channel and a Cl^–-HCO ₃ ^– exchanger.     

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     

Basolateral mechanisms of intracellular pH regulation in the colonic epithelial cell line HT29 clone 19A

The intracellular pH (pH_i) of the colonic tumour cell line HT29 cl.19A was studied by microspectrofluorometry using the pH-sensitive dye BCECF. Single cells within a confluent monolayer, grown in a polarized manner on permeable supports, were examined. An amiloride-sensitive Na⁺/H⁺ exchange and a stilbene-insensitive Cl^– /HCO₃ ^– exchange mechanism have been identified in the basolateral membrane. Removal of Na⁺ from the basolateral solution caused a decrease of pH_i by 0.50±0.09 unit (n=4). Amiloride or Na⁺-free solution at the apical side had no effect on pH_i. Cl^– removal at the basolateral side led to an increase of pH_i by 0.20±0.03 unit (n=4) whereas apical removal had no influence on pH_i. This effect was independent of Na⁺ and was insensitive to 0.2 mM 4,4-diisothiocyanatodihydrostilbene-2, 2-disulphonic acid. A basolateral Cl^–/ HCO₃ ^– exchanger is the most likely explanation for this observation. The Na⁺/H⁺ exchange mechanism in the basolateral membrane is an acid extruder, whereas the C1^–/HCO₃ ^– exchanger is an acid loader. Both of these mechanisms are important for the maintenance of intracellular pH in HT29 cl.19A cells.     

Acetazolamide inhibition of basolateral base exit in rabbit renal proximal tubule S2 segment

The influence of the carbonic anhydrase inhibitor acetazolamide (ACZ) was investigated on HCO ₃ ^– transport mechanisms in the basolateral cell membrane of rabbit renal proximal tubule. Experiments were performed on isolated S2 segments using double-barrelled microelectrodes to measure cell membrane potential (V _b) and cell pH (pH_i) during step changes in bath perfusate ion concentrations. Peritubular application of ACZ (1 mmol/l) reduced the initial V _b response to 101 reduction of bath HCO ₃ ^– concentration only slightly, from +53.8±4.2 mV to+49.1±0.3 mV (n=5), but caused an intermittent overshooting repolarization in the secondary V _b response. In conjunction with these effects it left the initial pH_i response virtually unchanged but induced a secondary slow acidification. These observation indicate that — under the present experimental conditions — ACZ does not block the Na⁺-HCO ₃ ^– cotransporter but acts via inhibition of cytosolic carbonic anhydrase. This was confirmed by studying the effect of elevated intracellular HCO ₃ ^– concentrations under reduced flux conditions and by comparing the concentration dependence of the V _b response with the inhibition kinetics of cytosolic carbonic anhydrase. In contrast, peritubular ACZ inhibited Na⁺-independent Cl^–/HCO ₃ ^– exchange in the basolateral cell membrane of S2 segments directly in a similar way to that described in the preceding publication for S3 segments.     

Intracellular pH regulation in cultured rat astrocytes in CO2/HCO 3 − -containing media

We studied the regulation of intracellular pH (pH_i) and the mechanisms of pH_i regulation in cultured rat astrocytes using microspectrofluorometry and the pH-sensitive fluorophore 2,7-bis(carboxyethyl-)-5,6-carboxyfluorescein. Control pH_i was 7.00±0.02 in HCO ₃ ^- containing solutions at an extracellular pH of 7.35. Addition of 4, 4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS) or amiloride decreased pH_i, as did removal of extracellular Na⁺, while removal of extracellular Cl^- was followed by an increase in pH_i. Following exposure to an acid transient induced by increasing the CO₂ content from 5 to 15%, pH_i rapidly returned to base line, with an average initial rate of recovery of 0.10 pH units min^-1 (corresponding to a mean acid extrusion rate of 6.3±0.36 mmolo 1^-1 min^-1). Regulation of pH_i was impaired when either amiloride or DIDS was added or Cl^- was removed. This inhibition was enhanced when both DIDS and amiloride were present, and pH_i regulation was completely blocked in the absence of extracellular Na⁺. The rapid regulation of pH_i normally seen following a transient alkalinisation was not inhibited by amiloride or removal of Na⁺, but was partially inhibited by DIDS and by the absence of extracellular Cl^-. The results are compatible with the presence of at least three different pH_i-regulating mechanisms: a Na⁺/H⁺ antiporter, a Na⁺-dependent HCO ₃ ^- /Cl^- exchanger (both regulating pH_i during a transient acidification), and a passive Cl^-/HCO ₃ ^- exchanger (regulating pH_i during transient alkalinisation). The results fail to provide firm evidence of the presence of an electrogenic Na⁺/HCO ₃ ^- symporter.     

Electrophysiological investigation of microdissected gastric glands of bullfrog II. Basolateral membrane properties in the presence of histamine

Following the technical approach described in the preceding publication we have investigated if, and how, stimulation of gastric HCl secretion affects the basolateral ion transport properties of oxyntopeptic cells of Rana catesbeiana stomach. To this end microdissected gastric glands were punctured with conventional or H⁺-sensitive glass microelectrodes and the effects of changing bath ion concentrations on the cell membrane potential (V _b) and cell pH (pH_i) were determined. Except for a transient alkalinization, histamine (0.5 mmol/l) did not significantly affect V _b or pH_i. The latter averaged 7.18±0.03 (mean±SEM, n=5) under resting conditions (0.1 mmol/l cimetidine) and 7.21±0.07 (n=5) in the presence of histamine. In addition, neither the initial velocity nor the final steady-state value of the cell alkalinization following a 101 reduction of bath Cl^– concentration changed in the presence of histamine, and the same holds true for the cell acidification following a 101 reduction of bath HCO₃ ^– concentration. These observations indicate that the basolateral Cl^–/HCO₃ ^– exchanger was not stimulated by histamine, and that no other base transporters were activated. By contrast, the V _b response to elevation of bath K ⁺ concentration decreased, and so did the initial depolarizing V _b response to bath Cl^– substitution, while the secondary hyperpolarizing response increased. The latter observations are compatible with the notion that stimulation by histamine reduced a pH-insensitive part of the basolateral K⁺ conductance and reduced also the basolateral Cl^– conductance.     

Intracellular pH regulation in papillary muscle cells from streptozotocin diabetic rats: an ion-sensitive microelectrode study

Intracellular pH regulation was studied in papillary muscle from STZ-induced diabetic rat hearts. In control bicarbonate solution there was no difference between the steady-state pH_i values recorded from diabetic or normal papillary muscle. The addition of insulin had no effect on the pH_i of either group. The amplitude of NH ₄ ⁺ -induced alkalinization and the time course of recovery from alkalinization were similar in both normal and diabetic muscles. In both preparations, the recovery from alkalinization was similarly delayed by the disulfonic stilbene DIDS. This suggests the participation of a Cl^–/HCO ₃ ^– exchange in the recovery from alkalosis in rat myocardial cells that is not changed by diabetes. On the other hand, the amplitude of the acidification induced by the withdrawal of NH ₄ ⁺ was markedly increased in diabetic papillary muscles as compared to normal muscles. Moreover, there was a marked slowing down of the recovery from acidosis in the diabetics. The amplitude of NH ₄ ⁺ withdrawal-induced acidification was increased equally by amiloride in both normal and diabetic muscles. These findings suggest that diabetes is associated with a change in the activity of the amiloride-sensitive Na⁺/H⁺ exchange.     

The chloride/base exchanger in the basolateral cell membrane of rabbit renal proximal tubule S3 segment requires bicarbonate to operate

Isolated microperfused S3 segments of rabbit renal proximal tubule were investigated with pH-sensitive double-barrelled intracellular microelectrodes to determine whether the Cl^–/base exchanger, which we have previously identified in the basolateral cell membrane of this segment requires HCO₃ ^– or can also work in CO₂/HCO₃ ^– free conditions. Cell pH (pH_i) was measured in response to sudden substitution of bath Cl^– by gluconate. In control solutions containing 25 mmol/l HCO₃ pH_i increased initially by 5.0±0.3 × 10^–3 unit/s but after perfusion with CO₂/HCO₃ ^–-free solutions pH_i of the same cells increased only by 1.3±0.2 × 10^–3 unit/s in response to Cl^– substitution. From measurements of the cellular buffering power it was calculated that the control base flux had fallen drastically from 3.7±0.3 to 0.3±0.1 × 10^–12 mols/s·cm tubule length. To test whether the remaining flux might have resulted from metabolic CO₂, oxidative metabolism was poisoned with cyanide (5 mmol/l). This abolished the pH change (pH_i) in CO₂/HCO₃ ^–-free solutions, but did not affect the pH shift in the presence of HCO₃ ^–. The data indicate that basolateral Cl^–/base exchange in S3 segment requires HCO₃ ^– to operate. A model in which HCO₃ ^– absorption proceeds in form of OH^– and CO₂ can be largely excluded.     

Intracellular pH in depolarized cardiac Purkinje strands

In isolated sheep cardiac Purkinje strands the effect of membrane depolarization on intracellular pH (pH_i) and on pH_i changes produced by addition and withdrawal of NH ₄ ⁺ and CO₂/HCO ₃ ^– was investigated. pH_i was continuously measured with double-barreled glass microelectrodes. Repetitive stimulation at high rate resulted in a moderate intracellular acidification (approximately 0.03 pH unit after a 3 Hz train of 2 min), whereafter pH_i returned toward its pre-stimulus level. Prolonged depolarization, evoked either by current injection or by superfusion with high K⁺ solutions, was accompanied by a small acid shift. In the depolarized cell, addition of NH ₄ ⁺ to the superfusate caused intracellular alkalinization followed by re-acidification which was slower than at normal membrane potential. Following intracellular acidification caused by withdrawal of NH ₄ ⁺ , pH_i recovery also was slightly slower than in the normally polarized cell. In the depolarized fiber, removal and readdition of CO₂/HCO ₃ ^– produced the expected intracellular alkalinization and acidification respectively. Recovery from CO₂-induced acidosis was slowed somewhat in high K⁺ (low Na⁺) superfused fibers, not in current depolarized fibers. In the depolarized cell, steady state pH_i in CO₂/HCO ₃ ^– containing and in CO₂/HCO ₃ ^– free solution tended to become identical. These experiments support the hypothesis that in the normally polarized Purkinje fiber passive shuttle movement of NH ₄ ⁺ /NH₃ and CO₂/HCO ₃ ^– occurs and could perhaps at least be partly responsible for the lower steady state pH_i as compared to that reached in NH ₄ ⁺ -free and CO₂/HCO ₃ ^– -free solutions respectively.     

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.     

Requirement of HCO 3 − for Cl−-absorption in seawater-adapted eel intestine

The role of HCO ₃ ^– /CO₂ buffer in Cl^– absorption was examined in the in vitro perfused eel intestine adapted to seawater. Cl^– absorption, expressed as short/circuit current (I _sc), was measured in either 20 mM HCO ₃ ^– /1% CO₂ Ringer or HEPES Ringer, pH 8.0. Unilateral (mucosal or serosal) substitution of HCO ₃ ^– /CO₂ with HEPES/O₂ was without effect on I _sc and transepithelial voltage (V _t), whereas bilateral removal of HCO ₃ ^– /CO₂ reduced I _sc and V _t by 50%, indicating that the presence of HCO ₃ ^– /CO₂ buffer at one side of the epithelium is sufficient to keep Cl^– absorption at the maximum rate. We examined in further detail the individual components of the HCO ₃ ^– /CO₂ system that stimulates Cl^– absorption. We found that, in tissues bathed with HEPES Ringer, addition of 1% CO₂ to the luminal or serosal solution (final pH=7.6 in the chamber) had no effect on I _sc and V _t, while both electrical parameters could be restored to control values by unilateral (luminal or serosal) substitution of HEPES Ringer with 20 mM HCO ₃ ^– /1% CO₂ Ringer or 20 mM HCO ₃ ^– alone. Stimulation of I _sc induced by unilateral (luminal or serosal) HCO ₃ ^– /CO₂ was inhibited by luminal or serosal 4-acetamido-4-isothiocyanostilbene-2,2-disulphonic acid (SITS) (0,25 mM) or by serosal Na⁺ removal, whereas amiloride (1 mM), luminal or serosal, had no effect. Acetazolamide (0.1 mM, both sides) inhibited stimulation of I _sc induced by luminal addition of HCO ₃ ^– /CO₂, whereas it was without effect when HCO ₃ ^– /CO₂ was added serosally or bilaterally. We reached the following conclusions, (a) Cl^– absorption is stimulated by HCO ₃ ^– /CO₂ buffer via an increase in intracellular HCO ₃ ^– concentration and/or pH_i changes consequent to the HCO ₃ ^– uptake mediated by HCO ₃ ^– transport systems operating on both cell membranes, (b) A Na⁺-dependent SITS-inhibitable HCO ₃ ^– transport mechanism operates at the basolateral membrane, (c) The transfer of HCO ₃ ^– through the luminal membrane is mediated by the carbonic anhydrase enzyme located on the brush-border membranes of the enterocyte: the movement of HCO ₃ ^– , via a SITS-sensitive transport system, occurs most likely in form of OH^–, which originates from the dehydration reaction of HCO ₃ ^– catalysed by the carbonic anhydrase. (d) There is no apparent amiloride-sensitive Na⁺/H⁺ antiporter on either cell membrane.This work was supported by a research grant from Ministero dell'Università e della Ricerca Scientifica e Tecnologica — Progetto di interesse nazionale e di rilevante interesse per lo sviluppo della Scienza     

Transport properties of the basolateral membrane of the oxyntic cells in frog fundic gastric mucosa

The conductive properties of the basolateral membrane of oxyntic cells (OC) of frog fundic gastric mucosa were investigated by utilizing the microelectrode technique. By examining the response of the basolateral cell membrane potential difference,V _cs, to sudden ion concentration changes in the serosal bath it was concluded that the basolateral membrane of OC has a high Ba²⁺-sensitive K⁺-conductance, and no Cl^–-conductance both in resting (cimetidine) and in stimulated (histamine) state. The response ofV _cs to serosal Cl^–-removal, consisting in a slight hyperpolarization (anomalous Nernst response), could not be explained by possible permeability changes to K⁺ and Na⁺ since the potential response to Cl^– was essentially preserved by blocking K⁺-permeability with Ba²⁺ and replacing all Na⁺ by choline. Conversely, hyperpolarization ofV _cs after Cl^–-free perfusion was abolished by exposure to HCO ₃ ^– -free solution, indicating that HCO ₃ ^– -ions are required at the serosal bath for Cl^– to get his effect. It was investigated wether the effect of Cl^– was due to an electrogenic Na⁺(HCO ₃ ^– ) _n /Cl^– exchange mechanism on the basolateral membrane. Experiments showed that the potential response to HCO ₃ ^– -removal and to Na⁺-removal, consisting in a depolarization ofV _cs, was similar both in presence and in absence of Cl^–. Furosemide (0.5 mmol/l) had no effect on steadyV _cs andV _t. The electrophysiological analysis of the data led to excluding the involvement of Na-Cl, Na-2Cl and NaK-2Cl cotransports, and to including the existence of an electrogenic Na⁺(HCO ₃ ^– ) _n /Cl^– exchange process, while suggests the presence of an electroneutral Cl^–/HCO ₃ ^– exchange mechanism to explain Cl^–-transport across the basolateral membrane of OC.This work was supported by a research grant from Ministero della Pubblica Istruzione, Rome, Italy