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     

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.     

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.     

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]     

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.     

Intracellular pH regulation of human colonic crypt cells

In order to investigate the regulation of intracellular pH (pH_i) in freshly isolated human colonocytes, we have used a newly developed technique for the rapid isolation and covalent attachment of these cells to glass surfaces and microspectrofluorimetric measurement of the pH-sensitive fluorescence of 2,7-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded specimens in a perfusion chamber (37°C). In N-2-hydroxyethylpiperazine — N — 2 — ethanesulphonic — acid — (HEPES)-buffered Ringer solution (HBS) a baseline pH_i of 7.35±0.03 (mean ± SD; n=42) was found for human colonocytes and in HBS, NH₄Cl-prepulse-induced intracellular acidification in colonocytes is reversed rapidly by the ubiquitous amiloride-sensitive (1 mmol/l) Na⁺/H⁺ exchanger. Switching from HBS to HCO ₃ ^– buffered solution (BBS) led to a transient intracellular acidification (7.29±0.09), followed by a recovery to a final resting pH_i of 7.43±0.03. One-third of the acid extrusion in BBS is amiloridesensitive; the remaining two-thirds are caused by the dihydroderivative of 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (H₂DIDS)-sensitive HCO ₃ ^– -dependent mechanisms. The functional activity of an acid-extruding Na⁺/HCO ₃ ^– cotransporter in human colonocytes was observed in response to the reintroduction of Na⁺ into amiloride-containing Na⁺/Cl^–-free BBS. In addition, the mechanism leading to alkalinization (7.56±0.05) in Cl^–-free BBS was identified as Na⁺-dependent Cl^–/HCO ₃ ^– exchange, by its H₂DIDS sensitivity and the specific requirement for Cl^– and Na⁺. The intrinsic buffering capacity ( _i) of the human colonocytes was calculated from pH changes induced by sequential NH₄Cl-loading steps during blockage of acid/base transporters. With _i=80 mmol · l^–1 · pH unit^–1 for the pH interval ranging from 6.9 to 7.1 (n=8) the colonocytes exhibited a relatively high intrinsic buffering in comparison with other cell types. In conclusion, the freshly isolated human colonocytes express a Na⁺/H⁺ exchanger, a Na⁺/HCO ₃ ^– cotransporter and a Na⁺-dependent Cl^–/HCO ₃ ^– exchanger, all of them likely to be involved in the regulation of pH homeostasis in vivo in the presence of widely varying extracellular conditions. Maintenance of a stable pH_i of human colonocytes seems to be facilitated by a comparatively high _i at physiological pH values.     

Intracellular pH and buffer power of type 1 and 2 fibres from skeletal muscle ofXenopus laevis

Intracellular pH (pH_i) and buffering power of type 1 and type 2 fibres from the iliofibularis muscle of the clawed frog,Xenopus laevis, have been measured using pH-sensitive microelectrodes. In phosphate buffered Ringer's solution (extracellular pH 7.25, 20–22°C), mean pH_i and its variance were similar in the two fibre types (6.86±SD 0.15±SEM 0.03,n=24, type 1, and 6.86±SD 0.12±SEM 0.03,n=15, type 2). On changing to Ringer's solution containing CO₂ and HCO ₃ ^– (extracellular pH 7.25, 20–22°C), pH_i became more acid in both fibre types. Although H⁺ ions were not at electrochemical equilibrium across the surface membrane, active transport did not return pH_i to its original value during exposure to CO₂. The buffering powers calculated from the changes in pH_i were not significantly different, 41.6 mmol·l^–1 per pH unit (±SEM 4.0,n=17) for type 1 and 49.3 mmol·l per pH unit (±SEM 7.2,n=11) for type 2 fibres. Thus differences in the mechanical properties of these fibre types are not due simply to a difference of the intracellular pH or buffering of resting fibres. Other possible explanations are discussed for the changes in some contractile properties that occur when pH_i is acidified.     

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.     

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     

Bicarbonate-dependent changes of intracellular sodium and pH in identified leech glial cells

A new triple-barrelled ion-sensitive microelectrode was used to investigate the importance of bicarbonate for the regulation of intracellular Na⁺ and pH (Na_i and pH_i, respectively) of neuropile glial cells in the central nervous system of the leech Hirudo medicinalis. Addition of CO²/HCO ₃ ^– produced an increase of the Na_i activity and an intracellular alkalinization, indicating bicarbonate accumulation in the glial cells. Changes of external pH (from 7.4 to 7.0 and 7.8) produced large and rapid shifts of pH_i and Na_i and of the membrane potential in the presence, but not in the absence, of bicarbonate. Thus, acid/base transport and Na⁺ movements across the glial membrane into and out of the cells were accelerated severalfold in CO²/HCO ₃ ^– -buffered saline as compared to a CO²/HCO ₃ ^– -free, HEPES-buffered saline. The results suggest that the electrogenic, reversible, cotransport of Na⁺ and HCO ₃ ^– in the glial cell membrane [3, 9] can produce significant changes in intraglial pH and Na activity, and can carry a significant fraction of the total Na⁺ flux across the cell membrane.     

pH dependence of K+ conductances of rat cortical collecting duct principal cells

The K⁺ channels of the principal cells of rat cortical collecting duct (CCD) are pH sensitive in excised membranes. K⁺ secretion is decreased with increased H⁺ secretion during acidosis. We examined whether the pH sensitivity of these K⁺ channels is present also in the intact cell and thus could explain the coupling between K⁺ and H⁺ secretion. Membrane voltages (V _m), whole-cell conductances (g _c), and single-channel currents of K⁺ channels were recorded from freshly isolated CCD cells or isolated CCD segments with the patch-clamp method. Intracellular pH (pH_i) was measured using the pH-sensitive fluorescent dye 2-7-bis(carboxyethyl)-5-6-carboxyfluorescein (BCECF). Acetate (20 mmol/l) had no effect on V _m, g _c, or the activity of the K⁺ channels in these cells. Acetate, however, acidified pH_i slightly by 0.17±0.04 pH units (n=19). V _m depolarized by 12±3 mV (n=26) and by 23±2 mV (n=66) and g _c decreased by 26±5% (n=13) and by 55±5% (n=12) with 3–5 or 8–10% CO₂, respectively. The same CO₂ concentrations decreased pH_i by 0.49±0.07 (n=15) and 0.73±0.11 pH units (n=12), respectively. Open probability (P _o) of all four K⁺ channels in the intact rat CCD cells was reversibly inhibited by 8–10% CO₂. pH_i increased with the addition of 20 mmol/l NH₄ ⁺/NH₃ by a maximum of 0.64±0.08 pH units (n=33) and acidified transiently by 0.37±0.05 pH units (n=33) upon NH₄ ⁺/NH₃ removal. In the presence of NH₄ ⁺/NH₃ V _m depolarized by 16±2 mV (n=66) and g _c decreased by 26±7% (n=16). The activity of all four K⁺ channels was also strongly inhibited in the presence of NH₄ ⁺/NH₃. The effect of NH₄ ⁺/NH₃ on V _m and g _c was markedly increased when the pH of the NH₄ ⁺/NH₃-containing solution was set to 8.5 or 9.2. From these data we conclude that cellular acidification in rat CCD principal cells down-regulates K⁺ conductances, thus reduces K⁺ secretion by direct inhibition of K⁺ channel activity. This pH dependence is present in all four K⁺ channels of the rat CCD. The inhibition of K⁺ channels by NH₄ ⁺/NH₃ is independent of changes in pH_i and rather involves an effect of NH₃.     

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.     

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.     

Simultaneous measurement of intracellular and extracellular carbonic anhydrase activity in intact muscle fibres

The presence and properties of membrane-bound carbonic anhydrases have been difficult to establish with conventional enzymological and immunohistochemical techniques. We have therefore studied carbonic anhydrase (CA) activity in single intact crayfish muscle fibres by superfusing them alternately with a 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES)-buffered and a 5% CO₂/HCO ₃ ^– -buffered solution (pH of both solutions 7.4) while recording the intracellular pH (pH_i) and extracellular surface pH (pH_s) with H⁺-selective microelectrodes. In order to prevent regulation of pH_i, Na⁺ ions were replaced with N-methyl-D-glucamine. Application of the CO₂-containing solution produced a fast fall in pH_i coupled with a marked (0.5–0.8 pH units) transient increase in pH_s. Submicromolar concentrations of acetazolamide (AA) and benzolamide (BA) immediately blocked the pH_s transients. A concentration of 8×10^–8 M (both compounds) reduced the response by 50%. A more prolonged application of BA and AA at concentrations of 10^–7 M and higher slowed the CO₂-induced fall in pH_i, which attained a rate corresponding to uncatalysed intracellular CO₂ hydration at an AA concentration of 10^–4 M. The effect of BA and AA on the pH_i changes developed with a time constant of 25±4 min and 7.6±1.5 min respectively, indicating that BA is less permeant than AA. CNO^– ions (5×10_–4 M) had little effect on the CO₂-induced pH_s and pH_i changes. The results are consistent with the view that the muscle fibres are equipped with an intracellular CA and with a sarcolemmal CA which has its active site at the extracellular surface. The pharmacological inhibition pattern of the latter is not fully compatible with any CA isozyme identified so far.     

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     

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]     

Histamine reduces Cl− activity in surface epithelial cells of frog gastric mucosa

Intracellular chloride activity (a _c ^Cl ) and serosal as well as mucosal membrane potentials (V _cs andV _cm) were recorded in surface epithelial cells (SEC) of frog gastric mucosa during the resting state (cimetidine, 10^–4 mol/l) or during stimulation with histamine (10^–4 mol/l). Stimulation leads to a fall ina _c ^Cl from 18.7 SD±5.9 mmol/l (n=26) to 13.3 SD±4.9 mmol/l (n=33). Simultaneously both cell membranes hyperpolarize,V _cs from –56.0 SD±4.8 (n=42) to –62.8±7.6 (n=43) andV _cm from –39.6 SD±5.8 (n=42) to –47.9±7.6 (n=43), so that intracellular chloride remains elevated above electrochemical equilibrium at both cell membranes. Reduction or omission of chloride in the lumen perfusate does not affecta _c ^Cl , suggesting that the luminal cell membrane is virtually tight for chloride ions. Current induced hyperpolarization of the serosal cell membrane potential which simulates the electrical effects of stimulation, does not affecta _c ^Cl either; however, inhibition of gastric acid secretion by a benzimidazol derivative which is known to block the H⁺/K⁺ ATPase prevents the fall ina _c ^Cl in response to histamine. The same holds if the experimental solutions are gassed with 25% CO₂ which does not interfere with acid secretion but may block cell to cell communication via gap junctions. From these results we conclude that 1. the SEC are not involved in non-acidic Cl^– secretion, neither in resting nor in stimulated state, and 2. that the fall ina _c ^Cl of the surface epithelial cells under stimulation does not seem to reflect a direct action of histamine on the SEC but seems to reflect a fall ina _c ^Cl of the oxyntic cells which may be coupled to the surface epithelial cells by permeable cell-to-cell junctions.Part of this work has been presented at the SIBS-SIF-SINU-Congress, Abano-Terme, 1984This study was supported by CNR grant number 84.01734.04 and by Ministero della Pubblica Istruzione, Roma, Italy     

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.     

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.     

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.