Microfluorimetric imaging study of the mechanism of activation of the Na+/H+ antiport by muscarinic agonist in rat mandibular acinar cells

The mechanism of regulation of intracellular pH (pH_i) in dispersed acini from the rat mandibular salivary gland has been studied with a microfluorimetric imaging method and the pH probe 2,7-bis(2-carboxyethyl)-5(and –6)-carboxyfluorescein. The pH_i in the TRIS/HEPES-buffered standard solution was 7.29±0.01. Addition of 1 mol/l acetylcholine (ACh) or ionomycin caused a sustained increase in the pH_i. These agents decreased pH_i in the absence of external Na⁺ or in the presence of amiloride. The rate of pH_i recovery from an acid load after NH ₄ ⁺ prepulse was a linear function of pH_i and increased as pH_i became more acidic. Addition of ACh shifted the relationship towards a more alkaline pH_i range. The increase in pH_i induced by ACh or ionomycin was not inhibited by the protein kinase C inhibitors staurosporine (10 nM) and 1-(5-isoquinolinesulfonyl)-1-methylpiperazine (50 mol/l). Addition of 0.1–1 mol/l phorbol 12-myristate 13-acetate (TPA) had little effect on pH_i within 10 min; however, exposure to TPA for 120 min resulted in a significant rise in pH_i. In Ca²⁺-free solution with 50 mol/l 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, the ACh-induced rise in both pH_i and cytosolic Ca²⁺ concentration was suppressed. ACh and ionomycin caused an increment of amiloride-sensitive acid output into the extracellular fluid, while 20 mol/l 1-oleoyl-2-acetylglycerol had little effect on it. It was concluded that (a) stimulation with ACh activated the Na⁺/H⁺ antiport in the plasma membrane, (b) ACh also stimulated the intracellular acid production but acid extrusion by the Na⁺/H⁺ antiport prevented the cell from intracellular acidification, and (c) the major route of signal transduction for the ACh-induced activation of the Na⁺/H⁺ antiport was independent of protein kinase C but was dependent on the rise in cytosolic Ca²⁺ concentration. The implication of the cytosolic acidification and cell volume change in pH_i regulation is discussed.     

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)     

Effect of intracellular pH on agonist-induced [Ca2+]i transients in HT29 cells

 In this study we examined the influence of intracellular pH (pH_i) on agonist-induced changes of intracellular Ca²⁺ activity ([Ca²⁺]_i) in HT₂₉ cells. pH_i and [Ca²⁺]_i were measured microspectrofluorimetrically using BCECF and fura-2, respectively. Buffers containing trimethylamine (TriMA), NH₃/NH₄ ⁺ and acetate were used to clamp pH_i to defined values. The magnitudes of the peak and plateau of [Ca²⁺]_i transients induced by carbachol (CCH, 10^–6 mol/l) were greatly enhanced by an acidic pH_i and nearly abolished by an alkaline pH_i. The relationship between pH_i and the [Ca²⁺]_i peak was nearly linear from pH_i 7.0 to 7.8. This effect of pH_i was also observed at higher CCH concentrations (10^–4 and 10^–5 mol/l), at which the inhibitory effect of an alkaline pH_i was more pronounced than the stimulatory effect of an acidic pH_i. An acidic pH_i shifted the CCH concentration/response curve to the left, whereas an alkaline pH_i led to a rightward shift. The influence of pH_i on [Ca²⁺]_i transients induced by neurotensin (10^–8 mol/l) or ATP (5 × 10^–7 mol/l) was similar to its influence on those induced by CCH, but generally not as pronounced. Measurements of cellular inositol 1,4,5-trisphosphate (InsP ₃) showed no changes in response to acidification with acetate (20 mmol/l) or alkalinization with TriMA (20 mmol/l). The InsP ₃ increase induced by CCH was unaltered at an acidic pH_i, but was augmented at an alkaline pH_i. Confocal measurements of cell volume showed no significant changes induced by TriMA or acetate. Slow-whole-cell patch-clamp experiments showed no additional effect of CCH on the membrane voltage (V _m) measured after TriMA or acetate application. We conclude that pH_i is a physiological modulator of hormonal effects in HT₂₉ cells, as the [Ca²⁺]_i responses to agonists were significantly changed at already slightly altered pH_i. The measurements of InsP ₃, cell volume and V _m show that pH_i must act distally to the InsP ₃ production, and not via changes of cell volume or V _m. Received: 21 March 1997 / Received after revision: 14 May 1997 / Accepted: 15 May 1997     

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     

Action of ANG II and ANP on colon epithelial cells

The interaction of angiotensin II (ANG II) and atrial natriuretic peptide (ANP) on intracellular pH (pH_i) and calcium ([Ca²⁺]_i) was investigated in T84 cells (a permanent cell line derived from human colon epithelium) using the fluorescent stains BCECF/AM and Fluo 4/AM, respectively. pH_i recovery rate mediated by the Na⁺/H⁺ exchanger (NHE) was examined following an NH₄Cl pulse. Under control conditions pH_i recovered at 0.114±0.005 pH units/min (n=35). ANG II (10^–12 or 10^–9 M) increased this value, whilst ANG II (10^–7 M) decreased it. These effects of ANG II were impaired by simultaneous addition of 1 M or 25 M HOE-694, indicating that the stimulatory and inhibitory effects of ANG II on pH_i recovery are mediated in part via the NHE1 and NHE2 isoforms. ANG II increased [Ca²⁺]_i concentration-dependently. ANP (10^–6 M) or dimethyl-BAPTA/AM (50 M) blocked the effects of ANG II on [Ca²⁺]_i and on the rate of pH_i recovery. Thapsigargin (10^–5 M) enhanced the effect of ANG II on [Ca²⁺]_i and reversed its stimulatory effect on the rate of pH_i recovery to an inhibitory one. External Ca²⁺-free solution did not affect the effects of ANG II on these parameters. These data suggest that the [Ca²⁺]_i increase induced by ANG II is dependent on intracellular calcium stores. They are compatible with the demonstration of two sites on the C-terminal of the Na⁺/H⁺ exchanger, one stimulating Na⁺/H⁺ activity by increases of [Ca²⁺]_i in the lower range (at 10^–12 or 10^–9 M ANG II) and the other inhibiting this activity at high [Ca²⁺]_i levels (at 10^–7 M ANG II). ANP or dimethyl-BAPTA/AM, by impairing the pathway mediating the increase in [Ca²⁺]_i, block both the stimulatory and inhibitory effects of ANG II.     

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.     

Electrogenic Na+/K+-transport in human endothelial cells

Na⁺/K⁺ pump currents were measured in endothelial cells from human umbilical cord vein using the whole-cell or nystatin-perforated-patch-clamp technique combined with intracellular calcium concentration ([Ca²⁺]_i) measurements with Fura-2/AM. Loading endothelial cells through the patch pipette with 40 mmol/l [Na⁺] did not induce significant changes of [Ca²⁺]_i. Superfusing the cells with K⁺-free solutions also did not significantly affect [Ca²⁺]_i. Reapplication of K⁺ after superfusion of the cells with K⁺-free solution induced an outward current at a holding potential of 0 mV. This current was nearly completely blocked by 100 mol/l dihydroouabain (DHO) and was therefore identified as a Na⁺/K⁺ pump current. During block and reactivation of the Na⁺/K⁺ pump no changes in [Ca²⁺]_i could be observed. Pump currents were blocked concentration dependently by DHO. The concentration for half-maximal inhibition was 21 mol/l. This value is larger than that reported for other tissues and the block was practically irreversible. Insulin (10–1000 U/l) did not affect the pump currents. An increase of the intracellular Na⁺ concentration ([Na⁺]_i) enhanced the amplitude of the pump current. Half-maximal activation of the pump current by [Na⁺]_i occurred at about 60 mmol/l. The concentration for half-maximal activation by extracellular K⁺ was 2.4±1.2 mmol/l, and 0.4±0.1 and 8.7±0.7 mmol/l for Tl⁺ and NH₄ ⁺ respectively. The voltage dependence of the DHO-sensitive current was obtained by applying linear voltage ramps. Its reversal potential was more negative than –150 mV. Pump currents measured with the conventional whole-cell technique were about four times smaller than pump currents recorded with the nystatin-perforated-patch method. If however 100 mol/l guanosine 5-O-(3-thiotriphosphate) (GTPS) were added to the pipette solution, the currents measured in the ruptured-whole-cell-mode were not significantly different from the currents measured with the perforated-patch technique. We suppose that the use of the perforated-patch technique prevents wash out of a guanine nucleotide-binding protein (G-protein)-connected intracellular regulator that is necessary for pump activation.     

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

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

The rise of [Na+]i during ischemia and reperfusion in the rat heart—underlying mechanisms

Intracellular Na⁺ concentration ([Na⁺]_i) rises in the heart during ischemia, and on reperfusion, there is a transient rise followed by a return toward control. These changes in [Na⁺]_i contribute to ischemic and reperfusion damage through their effects on Ca²⁺ overload. Part of the rise of [Na⁺]_i during ischemia may be caused by increased activity of the cardiac Na⁺/H⁺ exchanger (NHE1), activated by the ischemic rise in [H⁺]_i. In support of this view, NHE1 inhibitors reduce the [Na⁺]_i rise during ischemia. Another possibility is that the rise of [Na⁺]_i during ischemia is caused by Na⁺ influx through channels. We have reexamined these issues by use of two different NHE1 inhibitors, amiloride, and zoniporide, in addition to tetrodotoxin (TTX), which blocks voltage-sensitive Na⁺ channels. All three drugs produced cardioprotection after ischemia, but amiloride (100 μM) and TTX (300 nM) prevented the rise in [Na⁺]_i during ischemia, whereas zoniporide (100 nM) did not. Both amiloride and zoniporide prevented the rise of [Na⁺]_i on reperfusion, whereas TTX was without effect. In an attempt to explain these differences, we measured the ability of the three drugs to block Na⁺ currents. At the concentrations used, TTX reduced the transient Na⁺ current (I _Na) by 11 ± 2% while amiloride and zoniporide were without effect. In contrast, TTX largely eliminated the persistent Na⁺ current (I _Na,P) and amiloride was equally effective, whereas zoniporide had a substantially smaller effect reducing I _Na,P to 41 ± 8%. These results suggest that part of the effect of NHE1 inhibitors on the [Na⁺]_i during ischemia is by blockade of I _Na,P. The fact that a low concentration of TTX eliminated the rise of [Na⁺]_i during ischemia suggests that I _Na,P is a major source of Na⁺ influx in this model of ischemia.     

Ca2+ regulated K+ and non-selective cation channels in the basolateral membrane of rat colonic crypt base cells

 We have previously shown that a new type of K⁺ channel, present in the basolateral membrane of the colonic crypt base (blm), is necessary for cAMP-activated Cl^- secretion. Under basal conditions, and when stimulated by carbachol (CCH) alone, this channel is absent. In the present patch clamp-study we examined the ion channels present in the blm under cell-attached and in cell-excised conditions. In cell-attached recordings with NaCl-type solution in the pipette we measured activity of a K⁺ channel of 16 ± 0.3 pS (n = 168). The activity of this channel was sharply increased by CCH (0.1 mmol/l, n = 26). Reduction of extracellular Ca²⁺ to 0.1 mmol/l (n = 34) led to a reversible reduction of activity of this small channel (SK_Ca). It was also inactivated by forskolin (5 μmol/l, n = 38), whilst the K⁺ channel noise caused by the very small K⁺ channel increased. Activity of non-selective cation channels (NS_cat) was rarely observed immediately prior to the loss of attached basolateral patches and routinely in excised patches. The NS_cat, with a mean conductance of 49 ± 1.0 pS (n = 96), was Ca²⁺ activated and required >10 μmol/l Ca²⁺ (cytosolic side = cs). It was reversibly inhibited by ATP (<1 mmol/l, n = 13) and by 3′,5-dichloro-diphenylamine-2-carboxylate (10–100 μmol/l, n = 5). SK_Ca was also Ca²⁺ dependent in excised inside-out basolateral patches. Its activity stayed almost unaltered down to 1 μmol/l (cs) and then fell sharply to almost zero at 0.1 μmol/l Ca²⁺ (cs, n = 12). SK_Ca was inhibited by Ba²⁺ (n = 31) and was charybdotoxin sensitive (1 nmol/l) in outside-out basolateral patches (n = 3). Measurements of the Ca²⁺ activity ([Ca²⁺]_i) in these cells using fura-2 indicated that forskolin and depolarization, induced by an increase in bath K⁺ concentration to 30 mmol/l, reduced [Ca²⁺]_i markedly (n = 8–10). Hyperpolarization had the opposite effect. The present data indicate that the blm of these cells contains a small-conductance Ca²⁺-sensitive K⁺ channel. This channel is activated promptly by very small increments in [Ca²⁺]_i and is inactivated by a fall in [Ca²⁺]_i induced by forskolin. Received: 15 April 1996 / Received after revision and accepted: 17 June 1996     

HCO<Subscript>3</Subscript><Superscript>−</Superscript>-dependent transient acidification induced by ionomycin in rat submandibular acinar cells

Ionomycin (IM, 5 μM), which exchanges 1 Ca²⁺ for 1 H⁺, changed intracellular pH (pH_i) with Ca²⁺ entry into rat submandibular acinar cells. IM-induced changes in pH_i consisted of two components: the first is an HCO₃ ⁻-dependent transient pH_i decrease, and the second is an HCO₃ ⁻-independent gradual pH_i increase. IM (1 μM), which activates store-operated Ca²⁺ channels, induced an HCO₃ ⁻-dependent and transient pH_i decrease without any HCO₃ ⁻-independent pH_i increase. Thus, a gradual pH_i increase was induced by the Ca²⁺/H⁺ exchange. The HCO₃ ⁻-dependent and transient pH_i decrease induced by IM was abolished by acetazolamide, but not by methyl isobutyl amiloride (MIA) or diisothiocyanatostilbene disulfonate (DIDS), suggesting that the Na⁺/H⁺ exchange, the Cl⁻/HCO₃ ⁻ exchange, or the Na⁺-HCO₃ ⁻ cotransport induces no transient pH_i decrease. Thapsigargin induced no transient pH_i decrease. Thus, IM, not Ca²⁺ entry, reduced pH_i transiently. IM reacts with Ca²⁺ to produce H⁺ in the presence of \textCO ₂ /\textHCO ₃ ^- :  [ \textH - \textIM ] ^- + \text Ca ²⁺  + \textCO ₂ \rightleftarrows [ \textH-\textCa - \textIM ] ⁺ ·\textHCO ₃ ^- + \textH ⁺ {\text{CO}}_{ 2} /{\text{HCO}}_{ 3}{^{ - }} : \, \left[ {{\text{H}} - {\text{IM}}} \right]^{ - } + {\text{ Ca}}^{ 2+ } \,+ {\text{CO}}_{ 2} \rightleftarrows \left[ {{\text{H}}-{\text{Ca}} - {\text{IM}}} \right]^{ + } \cdot {\text{HCO}}_{ 3}{^{ - } }+ {\text{H}}^{ + } . In this reaction, a monoprotonated IM reacts with Ca²⁺ and CO₂ to produce an electroneutral IM complex and H⁺, and then H⁺ is removed from the cells via CO₂ production. Thus, IM transiently decreased pH_i. In conclusion, in rat submandibular acinar cells IM (5 μM) transiently reduces pH_i because of its chemical characteristics, with HCO₃ ⁻ dependence, and increases pH_i by exchanging Ca²⁺ for H⁺, which is independent of HCO₃ ⁻.     

Mechanisms of calcium transport across the basolateral membrane of the rabbit cortical thick ascending limb of Henle's loop

Although net Ca²⁺ absorption takes place in the thick ascending limb of Henle's loop, detailed mechanisms are unknown. Because it has been reported that the Ca²⁺ entry step across the luminal membrane is mediated by Ca²⁺ channels inserted by stimulation with parathyroid hormone, we studied the mechanism of Ca²⁺ transport across the basolateral membrane of rabbit cortical thick ascending limb (CTAL) perfused in vitro by using microscopic fluorometry of cytosolic Ca²⁺ ([Ca²⁺]_i) with fura-2. The resting [Ca²⁺]_i in this segment was 49.8±4.5 nmol/l. Neither Na⁺ removal from the bathing solution nor addition of ouabain (0.1 mmol/l) to the bath increased [Ca²⁺]_i, indicating that a Na⁺/Ca²⁺ exchanger in the basolateral membrane may not contribute in any major way to [Ca²⁺]_i of CTAL. To confirm our technical accuracy, similar protocols were conducted in the connecting tubule, where the existence of a Na⁺/Ca²⁺ exchanger has been reported. In this segment, Na⁺ removal from the bath increased cell Ca²⁺ from 148.6 ±6.4 nmol/l to 647.6±132.0 nmol/l, confirming the documented fact. [Ca²⁺]_i in the CTAL was markedly increased when 1 mmol/l NaCN was added to the bath in the absence of glucose. Calmodulin inhibitors (trifluoperazine or W-7) increased [Ca²⁺]_i. When the bath pH was made alkaline, [Ca²⁺]_i was also increased. This response was abolished when Ca²⁺ was eliminated from the bath, indicating that the Ca²⁺ entry across the basolateral membrane is dependent on bath pH. Increase in [Ca²⁺]_i induced by an alkaline bath was inhibited by increased the bath K⁺ from 5 nmol/l to 50 mmol/l, suggesting that the Ca²⁺ entry system is voltage-dependent. However, the pH-dependent [Ca²⁺]_i increase was unaffected by 0.1–10 mol/l nicardipine in the bath. We conclude that Ca²⁺ transport across the basolateral membrane of CTAL is mediated by a pump-and-leak system of Ca²⁺ rather than a Na⁺/Ca²⁺ exchanger secondarily linked to a Na⁺, K⁺ pump.     

Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells

The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pH_i) and intracellular Ca²⁺ activity ([Ca²⁺]_i) of HT₂₉ cells was investigated microspectrofluorimetrically using pH- and Ca²⁺- sensitive fluorescent indicators, [i.e. 2′,7′-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2 respectively]. Membrane voltage (V _m) was studied by the patch-clamp technique. Secondary and tertiary amines led to a rapid and stable concentration-dependent alkalinization which was independent of their pK _a value. Trimethylamine (20 mmol/l) increased pH_i by 0.78 ± 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pH_i and a slow and incomplete recovery: pH_i stayed 0.26 ± 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were without influence on pH_i. All tested secondary and tertiary amines (dimethyl-, diethyl-, trimethyl-, and triethyl-amine) induced a [Ca²⁺]_i transient which reached a peak value within 10–25 s and then slowly declined to a [Ca²⁺]_i plateau. The initial Δ[Ca²⁺]_i induced by trimethylamine (20 mmol/l) was 160 ± 15 nmol/l (n = 17). The [Ca²⁺]_i peak was independent of the Ca²⁺ activity in the bath solution, but the [Ca²⁺]_i plateau was significantly lower under Ca²⁺-free conditions and could be immediately interrupted by application of CO₂ (10%; n = 6), a manoeuvre to acidify pH_i in HT₂₉ cells. Emptying of the carbachol- or neurotensin-sensitive intracellular Ca²⁺ stores completely abolished this [Ca²⁺]_i transient. Tetramethylamine led to higher [Ca²⁺]_i changes than the other amines tested and only this transient could be completely blocked by atropine (10⁻⁶ mol/l). Trimethylamine (20 mmol/l) hyperpolarized V _m by 22.5 ± 3.7 mV (n = 16) and increased the whole-cell conductance by 2.3 ± 0.5 nS (n = 16). We conclude that secondary and tertiary amines induce stable alkaline pH_i changes, release Ca²⁺ from intracellular, inositol-1,4,5-trisphosphate-sensitive Ca²⁺ stores and increase Ca²⁺ influx into HT₂₉ cells. The latter may be related to both the store depletion and the hyperpolarization. Received: 11 September 1995/Received after revision and accepted: 18 December 1995     

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     

Roles of Na+/H+ exchange in regulation of p38 mitogen-activated protein kinase activity and cell death after chemical anoxia in NIH3T3 fibroblasts

Activation of Na⁺/H⁺ exchange (NHE) plays a major role in cell death following ischemia/hypoxia in many cell types, yet counteracts apoptotic cell death after other stimuli. To address the role of NHE activity in regulation of cell death/survival, we examined the causal relationship between NHE, p38 mitogen-activated protein kinase (MAPK), ERK1/2, p53, and Akt activity, and cell death, after chemical anoxia in NIH3T3 fibroblasts. The NHE1 inhibitor 5′-(N-ethyl-N-isopropyl) amiloride (EIPA) (5 μM), as well as removal of extracellular Na⁺ [replaced by N-methyl-d-glucamine (NMDG⁺)], prevented recovery of intracellular pH (pH_i) during chemical anoxia (10 mM NaN₃ ± 10 mM glucose), indicating that activation of NHE was the dominating mechanism of pH_i regulation under these conditions. NHE activation by chemical anoxia was unaffected by inhibitors of p38 MAPK (SB203580) and extracellular signal-regulated kinase (ERK) (PD98059). In contrast, chemical anoxia activated p38 MAPK in an NHE-dependent manner, while ERK1/2 activity was unaffected. Anoxia-induced cell death was caspase-3-independent, mildly attenuated by EIPA, potently exacerbated by SB203580, and unaffected by PD98059. Ser¹⁵ phosphorylation of p53 was increased by anoxia in an NHE- and p38 MAPK-independent manner, while Akt activity was unaffected. It is suggested that after chemical anoxia in NIH3T3 fibroblasts, NHE activity is required for activation of p38 MAPK, which in turn protects the cells against anoxia-induced death. In spite of this, NHE inhibition slightly attenuates anoxia-induced cell death, likely due to the involvement of NHE in other anoxia-induced death pathways.     

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]     

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.     

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.     

Na+ dependence of single-channel current and channel density generate saturation of Na+ uptake in A6 cells

 In high-resistance, salt-absorbing epithelia the apical amiloride-sensitive Na⁺ channel is the key site for regulation of salt and water balance. The saturation of macroscopic Na⁺ transport through these channels was investigated using A6 epithelial monolayers. The relation between transepithelial Na⁺ transport (I _Na) and apical Na⁺ concentration ([Na⁺]_ap) under short-circuit conditions was studied. Michaelis-Menten analysis of the saturable short-circuit current (I _sc) yielded an apparent Michaelis-Menten constant (K _m ^I ) of 5 mmol/l and a maximal current (I _max) of 8 μA/cm². The microscopic parameters underlying I _Na, namely the single-channel current (i) and the open channel density (N _o), were investigated by the analysis of current fluctuations induced by the electroneutral amiloride analogue CDPC (6-chloro-3,5-diaminopyrazine-2-carboxamide). A two-state model analysis yielded the absolute values of i (0.18 ± 0.01 pA) and N _o (65.38 ± 9.57 million channels/cm² of epithelium) at [Na⁺]_ap = 110 mmol/l containing 50 μmol/l CDPC. Our data indicate that in A6 cells both i and N _o depend on [Na⁺]_ap. Between 3 and ≈ 20 mmol/l the density of conducting pores, N _o, decreases sharply and behaves again as an almost [Na⁺]_ap-independent parameter at higher [Na⁺]_ap. The single-channel current clearly saturates with an apparent Michaelis-Menten constant, K _m ⁱ , of ≈ 17 mmol/l. Thus, the [Na⁺]_ap dependence of N _o as well as the limited transport capacity of the amiloride-sensitive Na⁺ channel are both responsible for the saturation of I _Na. Received: 2 June 1997 / Received after revision: 12 November 1997 / Accepted: 10 December 1997