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.     

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.     

Effect of calcitonin on the regulation of intracellular pH in primary cultures of rabbit early distal tubule

To examine the intracellular pH (pH_i) regulation in primary cultures of rabbit distal convoluted tubules (DCTb) we used the pH-sensitive dye 2,7-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF/AM) and a video-microscopy technique. DCTb segments were microdissected from rabbit kidney cortex and cultured in a hormonally defined medium. The culture epithelia were grown on semi-transparent permeable supports. Before pH_i measurement, DCTb primary cultures were maintained for 48–96 h in growth-factor-free medium to obtain quiescent cells. We had previously shown that two mechanisms are involved in the regulation of intracellular pH: a basolateral Na⁺/H⁺ exchanger and an apical Cl^–/HCO ₃ ^– exchanger [1]. The pH_i of DCTb cells was significantly decreased by the addition of 60 nM human calcitonin (from 7.30±0.04 to 7.08±0.04). This response to calcitonin was dose-dependent and mimicked by both forskolin and permeant cyclic AMP derivatives. An initial acidification (of 0.25 pH unit in 7–8 min) was observed after the addition of basolateral amiloride (1 mM). The persistence of the effect induced by human calcitonin in these conditions, suggests that the Na⁺/H⁺ exchanger is not involved in the response. However, the acidification response was blocked in both the absence of chloride at the apical side and by the apical addition of 0.1 mM 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS). These experiments suggest that the target for the human calcitonin effect on pH_i is the Cl^–/HCO ₃ ^– exchanger. This study confirms the importance of this transporter in pH_i regulation within the physiological pH_i range and the influence of calcitonin in the regulation of DCTb cell function.     

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     

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     

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]     

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.     

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.     

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.     

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.     

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.     

Mechanism of uphill chloride transport of the mouse lacrimal acinar cells: Studies with Cl−-sensitive microelectrode

The mechanism of uphill Cl^– accumulation by mouse lacrimal acinar cells was studied using double-barrelled Cl^–-selective microelectrodes. When measured in standard tris-buffered saline solution, the membrane potential (V _m) was –39.2±0.4 mV and intracellular Cl^– activity (A _Cl ⁱ ) was 34.6±0.7 mmol/l which was 1.4 times higher than the equilibrium level. In Na⁺-free solution,A _Cl ⁱ decreased from 34 mmol/l to 19 mmol/l in 100 min, a level that was close to the equilibrium activity. Return to the standard solution restored the normal level ofA _Cl ⁱ in 5 min. In the presence of furosemide (1 mmol/l), Cl^– uptake induced by Na⁺-readmission was inhibited by 44%. Superfusion with a K⁺-free solution gradually decreasedA _Cl ⁱ until it was close to the equilibrium level after 75 min; superfusion with a high-K⁺ (29.5 mmol/l) solution increasedA _Cl ⁱ significantly. In the presence of ouabain (1 mmol/l), switching the superfusing solutions from K⁺-free to high-K⁺ and from high-K⁺ to K⁺-free at timed intervals of 15 min caused, respectively, an increase (+9 mmol/l) and a decrease (–7 mmol/l) inA _Cl ⁱ . These changes inA _Cl ⁱ were inhibited by furosemide respectively by 61% and 24%. In the presence of furosemide, DIDS (1 mmol/l) or furosemide plus DIDS, the initial rate of Cl^– uptake after cessation of acetylcholine (ACh 1 mol/l) stimulation was inhibited by 47%, 37% or 74%, respectively. Present results show that the characteristics of the uphill chloride uptake by the mouse lacrimal acinar cells are consistent with those of Na⁺–K⁺–Cl^– cotransport. The additional inhibitory effect of DIDS to furosemide inhibition suggests an involvement of anion exchange transport, in parallel with the cotransport, in uphill Cl^– uptake into the cells.     

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.     

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.     

Uncoupling of Na+H+ from Cl−HCO 3 − exchange under some steady state conditions in rabbit gallbladder

The transapical Cl^– influx and transepithelial Na⁺ transport were measured in rabbit gallbladder. Only 11.7% of the transported Na⁺ was found to be accompanied by HCO ₃ ^– . 10^–4 M SITS eliminated the HCO ₃ ^– dependent fraction of Cl^– influx (50%) but did not significantly alter intracellular Na⁺ activity and Na⁺ transport. Exposure to HCO₃-free salines or to 10^–4 M acetazolamide about halved Cl^– influx and Na⁺ transport. 25 mM SCN^– reduced Cl^– influx to zero, decreased intracellular Na⁺ activity, but only halved Na⁺ transport which under these conditions was abolished only in the absence of HCO ₃ ^– . Exposure to a Cl^–-free saline produced effects similar to those caused by SCN^–. These resuits suggest that when Cl^–/HCO ₃ ^– exchange is inhibited at the apical membrane, Na⁺/H⁺ exchange and transepithelial Na⁺ transfer are unmodified if HCO ₃ ^– is available for transport. The permanent uncoupling of the exchangers and the elevated transepithelial transport of Na⁺ are not due to an increased activity of the parallel Na⁺–Cl^– cotransport but to a redirection of HCO ₃ ^– flux toward the basolateral side.     

Inhibitory action of norepinephrine on sodium transport in vascular smooth muscle cells in culture

Cultured vascular smooth muscle cells from porcine aortas incubated in Na⁺-free medium rapidly release their intracellular Na⁺ contents (Na_i) (23±4% of baseline after 60 min incubation, mean ± SEM of 18 experiments). Total Na_i release was inhibited by 35–40% after addition of ouabain and by 60–70% after addition of ouabain + bumetanide. Norepinephrine inhibited ouabain and bumetanide-sensitives Na⁺ efflux with an IC₅₀ of about 10^–9–10^–8 M. Addition of the alpha-adrenergic agonist phenylephrine (10 M) to the cells mimicked the inhibitory action of norepinephrine on Na_i release. Conversely, the beta-adrenergic agonist isoproterenol was without effect on Na_i release. Simultaneous addition of 10 M norepinephrine and the alpha-adrenergic antagonist phentolamine prevented any effect of norepinephrine on the rate of Na_i decline. In A-10 cultured vascular smooth muscle cells, the alpha-adrenergic agonist phenylephrine (10 M) inhibited 40.0±8.1% of ouabain-sensitive Rb⁺ influx and 70.7±6.9% of bumetanide-sensitive Rb⁺ influx (mean ± SEM of three experiments). 50% inhibition of bumetanide-sensitive Rb⁺ influx was obtained with about 5×10^–7 M of phenylephrine. Our results show that in vascular smooth muscle cells a [Na⁺, K⁺, Cl^–]-cotransport system is able to catalyze outward Na⁺ movements (in Na⁺-free media) of a similar order of magnitude to those of the Na⁺, K⁺ pump and that alpha-adrenergic stimulation markedly inhibits Na⁺ efflux (and Rb⁺ influx) through these two transport systems.     

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     

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]     

Mechanism of NaCl secretion in the rectal gland of spiny dogfish (Squalus acanthias)

Rectal gland tubules (RGT) of spiny dogfish were dissected and perfused in vitro. Transepithelial PD (PD_te), resistance (R_te), the PD across the basolateral membrane (PD_bl) and intracellular chloride and potassium activities (a _Cl– ^cell ,a _K+ ^cell ) were measured. In a first series, 67 RGT segments were perfused with symmetric shark Ringers solution. The bath perfusate contained in addition db-cAMP 10^–4, forskolin 10^–6, and adenosine 10^–4 mol · l^–1. PD_te was –11±1 (n=67) mV lumen negative, R_te 27±2 (n=47) cm². PD_bl –75±0.4 (n=260) mV.a _K+ ^cell anda _Cl– ^cell were 109±22 (n=4) and 38±4 (n=36) mmol · l^–1 respectively. These data indicate that Cl^– secretion across the RGT must be an uphill transport process, whereas secretion of Na⁺ could be driven by the lumen negative PD_te. Intracellular K⁺ is 14 mV above equilibrium with respect to the basolateral membrane PD and Cl^– is 23 mV above equilibrium across the apical membrane. In series 2, the conductivity properties of the apical and basolateral membrane as well as that of the paracellular pathway were examined in concentration step experiments. Decrease of the basolateral K⁺ concentration led to a rapid hyperpolarization of PD_bt with a mean slope of 19 mV per decade of K⁺ concentration change. Addition of 0.5 mmol · l^–1 Ba²⁺ to the bath solution lead to a marked depolarization and abolished the response to K⁺ concentration steps. In the lumen a Cl^– concentration downward step led to a depolarization of the lumen membrane; resulting in a mean slope of 18 mV per decade of Cl^– concentration change. When dilution potentials were generated across the epithelium, the polarity indicated that the paracellular pathway is cation selective. In series 3 the equivalent short circuit current (I_sc=PD_te/R_te) was determined as a function of symmetrical changes in Na⁺ concentration, with Cl^– held at 276 mmol · l^–1, and as a function of symmetrical changes in Cl^– concentration, with Na⁺ held at 278 mmol · l^–1 I_sc was a saturable function of Na⁺ concentration (Hill coefficient 0.9±0.1,K _1/2 4.4 mmol · l^–1,n=7) and also a saturable function of Cl^– concentration (Hill coefficient 2.0±0.1,K _1/2 75 mmol · l^–1,n=11). These data are compatible with the assumption that the carrier responsible for NaCl uptake has a 1 Na⁺ per 2 Cl^– stoichiometry. In series 4, the effect of a K⁺ concentration downward step on PD_bl anda _Cl– ^cell transients was followed with high time resolution in the presence and absence of basolateral furosemide (5 · 10^–5 to 10^–4 mol · l^–1) in an attempt to examine whether K⁺ reduction on the bath side inhibits Na⁺Cl^– uptake by the carrier system as does e.g. furosemide. The data indicate that removal of K⁺ from the bath side exerts an effect comparable to that of furosemide, i.e. it inhibits the carrier. We conclude that NaCl secretion in the RGT cell comprises at the least the following components: In the basolateral membrane, the (Na⁺+K⁺)-ATPase, probably the Na⁺ 2 Cl^–K⁺ carrier, and a K⁺ conductance. In the apical membrane a Cl^– conductance; and a Na⁺ conductive paracellular pathway.Supported by Deutsche Forschungsgemeinschaft DFG-Gr 480/8-1. Parts of this study have been presented at the 3rd International Symposium on Ion Selective Electrodes, Burg Rabenstein 1983, 16th Annual Meeting American Society of Nephrology, Washington DC 1983, 49th Tagung der Deutschen Physiologischen Gesellschaft, Dortmund 1984. A summary of the present study was published in Bulletin Mount Desert Island Biological Laboratory (Vol. 83)