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.     

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.     

Phasic changes in intracellular pH during action potentials of sheep Purkinje fibres

Regulation of intracellular pH (pH_i) and the relationship between H⁺ and Ca²⁺ may vary during activity. Ion-selective microelectrodes were used to record pH_i during action potentials of sheep Purkinje fibres prolonged by low temperature (21°C) and elevated CO₂ content. Intracellular pH also was measured during changes in extracellular calcium concentration, [Ca²⁺]_o. Cytosolic alkalinization (peak pH_i change, 0.03–0.05) was observed during the long action-potential plateau and transient acidification (0.01–0.02 units) upon repolarization. Potassium-induced depolarization to plateau potentials (i.e. to –15±2 mV) simulated the peak magnitude of the alkalinization. However, compensation for the alkalinization occurred at a faster rate during the action potential (8.9±4.3 nM/min) than during K⁺ depolarization (1.2±0.5 nM/min). In comparison, the cytoplasm acidified in resting fibres (0.06–0.07 log units) during changes of [Ca²⁺]_o thought to increase intracellular calcium concentration. Alterations of pH_i were translated into changes of proton concentration ([H⁺]_i). Ten-to twenty-fold elevation of [Ca²⁺]_o evoked a comparable change in [H⁺]_i (mean increase, 5.7 nM) but oppositely directed from that during the plateau (mean decrease, 8.8 nM). The findings in resting fibres seem consistent with displacement of bound protons by Ca²⁺. In contrast, the initial change in pH_i during the plateau is proposed to be consequent to Ca²⁺-release from sarcoplasmic reticulum and/or phosphocreatine hydrolysis coupled to ATP regeneration.     

Intracellular pH in diluting segment of frog kidney

Chronic exposure to high potassium (K⁺ adaptation) stimulates H⁺ net secretion in the diluting segment of the frog kidney. In order to investigate the cellular mechanism of the H⁺ secretory process intracellular pH (pH_i) measurements were performed in cells of the diluting segment of the isolated doubly-perfused kidney of K⁺ adaptedRana esculenta. pH_i changes were monitored by pH-sensitive microelectrodes while the tubule lumen was rapidly perfused with various solutions. With control solutions (extracellular pH=7.80) pH_i averaged 7.60±0.05. Luminal application of furosemide (5 · 10^–5 mol/l) or reduction of luminal Cl^– (from 104 mmol/l to 9 mmol/l) hyperpolarized the cell membrane potentials but pH_i was not altered. Reduction of luminal Na⁺ (from 98 mmol/l to 3 mmol/l) depolarized the cell membrane potentials but pH_i remained constant. Complete removal of luminal Na⁺, however, led to a significant decrease of pH_i from 7.61±0.08 to 7.18±0.08. Luminal application of amiloride (1 · 10^–3 mol/l) also decreased pH_i significantly (pH_i=0.15±0.02).The results indicate that an amiloride-sensitive H⁺ extrusion mechanism exists in the luminal cell membrane of the K⁺ adapted frog diluting segment. The data are consistent with Na⁺/H⁺ exchange which maintains a constant pH_i even at extreme experimental conditions.Parts of the data were presented at the 16th Ann. Meeting of the Am. Soc. Nephrol., Washington (1983)     

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.     

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.     

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.     

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

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

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

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

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

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

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.     

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     

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.     

Effects of procaine on intracellular pH and its regulation: measurements with pH-selective micro-electrodes in Retzius neurones of the leech

The experiments were performed with double-barelled micro-electrodes in Retzius neurones of the leech in order to see if the tertiary amine local anesthetic procaine affects intracellular pH (pH_i) and its regulation. Lasting about 20 min, exposures to procaine (3 mM) saline caused an intracellular alkalosis of about 0.2 pH units which gradually degradated; its removal initiated a fast decrease of pH_i resulting in an acidosis, from which the cells recovered. Detailed studies, involving CO₂ or NH ₄ ⁺ acid loaded cells as well as inhibition of the pH_i regulating exchange mechanism by various drugs or exposures to Na-free salines suggest, that neither the Na⁺–H⁺, nor a probably Na⁺-dependent HCO ₃ ^– –Cl^– exchange are affected by procaine. We propose a model for procaine action on pH_i assuming that neutral procaine passes the membrane easily through lipophilic pathways. The degradation of alkalinity during procaine exposure hints on an entry of loaded procaine, some possible transport paths of which are discussed.     

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.     

Roles of pH in control of cell proliferation

Precise spatiotemporal regulation of intracellular pH (pH_i) is a prerequisite for normal cell function, and changes in pH_i or pericellular pH (pH_e) exert important signalling functions. It is well established that proliferation of mammalian cells is dependent on a permissive pH_i in the slightly alkaline range (7.0‐7.2). It is also clear that mitogen signalling in nominal absence of is associated with an intracellular alkalinization (~0.3 pH unit above steady‐state pH_i), which is secondary to activation of Na⁺/H⁺ exchange. However, it remains controversial whether this increase in pH_i is part of the mitogenic signal cascade leading to cell cycle entry and progression, and whether it is relevant under physiological conditions. Furthermore, essentially all studies of pH_i in mammalian cell proliferation have focused on the mitogen‐induced G0‐G1 transition, and the regulation and roles of pH_i during the cell cycle remain poorly understood. The aim of this review is to summarize and critically discuss the possible roles of pH_i and pH_e in cell cycle progression. While the focus is on the mammalian cell cycle, important insights from studies in lower eukaryotes are also discussed. We summarize current evidence of links between cell cycle progression and pH_i and discuss possible pH_i‐ and pH_e sensors and signalling pathways relevant to mammalian proliferation control. The possibility that changes in pH_i during cell cycle progression may be an integral part of the checkpoint control machinery is explored. Finally, we discuss the relevance of links between pH and proliferation in the context of the perturbed pH homoeostasis and acidic microenvironment of solid tumours.     

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.     

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.     

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.     

Evidence for the presence of a Na+-H+ exchanger in the endolymphatic sac epithelium of guinea-pigs

 The intracellular pH (pH_i) of epithelial cells from the endolymphatic sac (ES) of the guinea-pig was measured microfluorometrically with the pH-sensitive fluorescent dye, 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to examine the presence of a Na⁺-H⁺ exchanger (NHE) in the ES epithelial cells. pH_i recovery from acid loading with an NH₄ ⁺-prepulse in a nominally HCO₃-free solution was dependent on extracellular Na⁺ ([Na⁺]_o) and was inhibited by amiloride and its analogue ethylisopropylamiloride (EIPA), suggesting that a decreased pH_i induced by an acute acid load may be equilibrated by a NHE. In the steady-state, amiloride had no effect on pH_i, indicating that the NHE activity is low at the resting pH_i. However, the intracellular acidification induced by the removal of [Na⁺]_o was inhibited by the simultaneous application of amiloride. H⁺-efflux rate (J _H, mean activity of NHE), which was calculated as the product of the recovery rate (dpH_i/dt) from the acid loading and the intrinsic buffering capacity (β_i) at the corresponding pH_i, was decreased as pH_i was increased. The concentration/response curve for the inhibition of initial J _H by EIPA revealed an apparent 50% inhibitory constant (K _i ) of 0.85 μM. Kinetic analysis of initial J _H as a function of [Na⁺]_o revealed a Michaelis-Menten constant (K _m) of 24.14 mM for Na⁺-dependent H⁺ efflux. The results indicate that NHE in the ES epithelium belongs to an amiloride-sensitive subtype. Received: 5 August 1997 / Received after revision and accepted: 26 February 1998