Na+-dependent regulation of the free Mg2+ concentration in neuropile glial cells and P neurones of the leech Hirudo medicinalis

 To investigate the Mg²⁺ regulation in neuropile glial (NG) cells and pressure (P) neurones of the leech Hirudo medicinalis the intracellular free Mg²⁺ ([Mg²⁺]_i) and Na⁺ ([Na⁺]_i) concentrations, as well as the membrane potential (E _m), were measured using Mg²⁺- and Na⁺-selective microelectrodes. The mean steady-state values of [Mg²⁺]_i were found to be 0.91 mM (mean E _m=–63.6 mV) in NG cells and 0.20 mM (mean E _m=–40.6 mV) in P neurones with a [Na⁺]_i of 6.92 mM (mean E _m=–61.6 mV) and 7.76 mM (mean E _m=–38.5 mV), respectively. When the extracellular Mg²⁺ concentration ([Mg²⁺]_o) was elevated, [Mg²⁺]_i in P neurones increased within 5–20 min whereas in NG cells a [Mg²⁺]_i increase occurred only after long-term exposure (6 h). After [Mg²⁺]_o was reduced back to 1 mM, a reduction of the extracellular Na⁺ concentration ([Na⁺]_o) decreased the inwardly directed Na⁺ gradient and reduced the rate of Mg²⁺ extrusion considerably in both NG cells and P neurones. In P neurones Mg²⁺ extrusion was reduced to 15.4% in Na⁺-free solutions and to 6.0% in the presence of 2 mM amiloride. Mg²⁺ extrusion from NG cells was reduced to 6.2% in Na⁺-free solutions. The results suggest that the major [Mg²⁺]_i-regulating mechanism in both cell types is Na⁺/ Mg²⁺ antiport. In P neurones a second, Na⁺-independent Mg²⁺ extrusion system may exist. Received: 11 August 1998 / Received after revision: 14 October 1998 / Accepted: 15 October 1998     

Dynamics of Ca2+ i and pHi in Ehrlich ascites tumor cells after Ca2+-mobilizing agonists or exposure to hypertonic solution

 Intracellular free calcium concentration ([Ca²⁺]_i) and intracellular pH (pH_i) were monitored in Ehrlich ascites tumor cells using Fura-2 or 2′,7′,-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), or both probes in combination. An increase in [Ca²⁺]_i induced by thrombin or bradykinin, agonists known to elicit transient cell shrinkage in these cells, evoked a transient intracellular acidification, followed by an alkalinization. The latter was due to activation of a Na⁺/H⁺ exchanger and was inhibited under conditions preventing agonist-induced cell shrinkage without preventing the increase in [Ca²⁺]_i. In contrast, a smaller, slower increase in [Ca²⁺]_i elicited by thapsigargin did not cause cell shrinkage, and did not activate the Na⁺/H⁺ exchanger. Exposure to hypertonic solution was not associated with an increase in [Ca²⁺]_i, but elicited an intracellular alkalinization similar to that induced by thrombin or bradykinin, via activation of the Na⁺/H⁺ exchanger. Thus, activation of the exchanger by the Ca²⁺-mobilizing agonists is suggested to be secondary to the cell shrinkage induced by these compounds. NH₄Cl-induced intracellular alkalinization resulted in an increase in [Ca²⁺]_i, apparently via stimulation of Ca²⁺ influx, whereas shrinkage-induced intracellular alkalinization did not stimulate Ca²⁺ influx. Thus, cell shrinkage appears to inhibit the Ca²⁺ influx otherwise resulting from alkalosis. In agreement with that notion, thapsigargin-induced Ca²⁺ influx was inhibited by cell shrinkage. Received: 6 January 1998 / Received after revision: 10 March 1998 / Accepted: 11 March 1998     

Intracellular free Mg2+ concentration in skeletal muscle fibres of frog and crayfish

The free intracellular Mg²⁺ concentration ([Mg²⁺]_i) was investigated in frog sartorius and crayfish phasic and tonic skeletal muscle fibres, using a new Mg²⁺-sensitive microelectrode based on the ionophore ETH 5214 [Hu et al. (1989) Anal Chem 61:574–576]. In Ringer solution containing 0.5 mmol/l MgCl₂, the mean [Mg²⁺]_i of the frog muscle fibres was 1.3 mmol/l. In phasic crayfish muscle fibres, [Mg²⁺]_i was about twice as high (mean 3.5 mmol/l) as in tonic fibres (mean 1.5 mmol/l), measured in van Harreveld solution containing 1.2 mmol/l MgCl₂. Long-lasting (3–12 h) incubation of frog skeletal muscle fibres in Na⁺-free solution produced a reversible increase of [Mg²⁺]_i by a factor of about 1.7. A tenfold rise of extracellular Mg²⁺ led to an increase in [Mg²⁺]_i in the presence as well as in the absence of Na⁺. In these experiments, mean [Mg²⁺]_i values of 3.2 mmol/l were never exceeded. Thus, [Mg²⁺]_i remained at least 60 times lower than predicted from a passive distribution across the cell membrane. The results suggest the existence of a Na⁺-dependent and a Na⁺-independent Mg²⁺ extrusion mechanism, which is regulated by actual Mg²⁺ concentrations.     

The effect of intracellular pH on cytosolic Ca2+ in HT29 cells

 The influence of intracellular pH (pH_i) on intracellular Ca²⁺ activity ([Ca²⁺]_i) in HT₂₉ cells was examined microspectrofluorometrically. pH_i was changed by replacing phosphate buffer by the diffusible buffers CO₂/HCO₃ ^–or NH₃/NH₄ ⁺ (pH 7.4). CO₂/HCO₃ ^–buffers at 2,5 or 10% acidified pH_i by 0.1, 0.32 and 0.38 pH units, respectively, and increased [Ca²⁺]_i by 8–15 nmol/l. This effect was independent of the extracellular Ca²⁺ activity and the filling state of thapsigargin-sensitive Ca²⁺ stores. Removing the CO₂/HCO₃ ^–buffer alkalinized pH_i by 0.14 (2%), 0.27 (5%), and 0.38 (10%) units and enhanced [Ca²⁺]_i to a peak value of 20, 65, and 143 nmol/l, respectively. Experiments carried out with Ca²⁺-free solution and with thapsigargin showed that the [Ca²⁺]_i transient was due to release from intracellular pools and stimulated Ca²⁺ entry. NH₃/NH₄ ⁺ (20 mmol/l) induced a transient intracellular alkalinization by 0.6 pHunits and increased [Ca²⁺]_i to a peak (Δ [Ca²⁺]_i = 164 nmol/l). The peak [Ca²⁺]_i increase was not influenced by removal of external Ca²⁺, but the decline to basal [Ca²⁺]_i was faster. Neither the phospholipase C inhibitor U73122 nor the inositol 1,4,5-trisphosphate (InsP ₃) antagonist theophylline had any influence on the NH₃/NH₄ ⁺-stimulated [Ca²⁺]_i increase, whereas carbachol-induced [Ca²⁺]_i transients were reduced by more than 80% and 30%, respectively. InsP ₃ measurements showed no change of InsP ₃ during exposure to NH₃/NH₄ ⁺, whereas carbachol enhanced the InsP ₃ concentration, and this effect was abolished by U73122. The pH_i influence on ”capacitative” Ca²⁺ influx was also examined. An acid pH_i attenuated, and an alkaline pH_i enhanced, carbachol- and thapsigargin-induced [Ca²⁺]_i influx. We conclude that: (1) an alkaline pH_i releases Ca²⁺ from InsP ₃-dependent intracellular stores; (2) the store release is InsP ₃ independent and occurs via an as yet unknown mechanism; (3) the store release stimulates capacitative Ca²⁺ influx; (4) the capacitative Ca²⁺ influx activated by InsP ₃ agonists is decreased by acidic and enhanced by alkaline pH_i. The effects of pH_i on [Ca²⁺]_i should be of relevance under many physiological conditions. Received: 17 June 1996 / Received after revision and accepted: 30 August 1996     

Sodium-dependent recovery of ionised magnesium concentration following magnesium load in rat heart myocytes

Our objectives were to investigate regulation of intracellular ionised Mg²⁺ concentration ([fMg²⁺]_i) in cardiac muscle and cardiac Na⁺/Mg²⁺ antiport stoichiometry. [fMg²⁺]_i was measured at 37°C in isolated rat ventricular myocytes with mag-fura-2. Superfusion of myocytes with Na⁺ and Ca²⁺ free solutions containing 30 mM Mg²⁺ for 15 min more than doubled [fMg²⁺]_i from its basal level (0.75 mM). Re-addition of Na⁺ caused [fMg²⁺]_i to fall exponentially with time to basal level, the rate increasing linearly with [Na⁺]. Log(recovery rate) increased linearly with log([Na⁺]), the slope of 1.06 (95% confidence limits, 0.94–1.17) suggesting one Na⁺ ion is exchanged for each Mg²⁺. [fMg²⁺]_i recovery was complete even if the membrane potential was depolarised to 0 mV or if superfusate [Mg²⁺] was increased to 3 mM. Recovery was rapid in normal Tyrode (0.3 min^–1) with a Q₁₀ of 2.2. It was completely inhibited by 200 M imipramine but was unaffected by 20 M KB-R7943 or 1 M SEA0400, suggesting the Na⁺ /Ca²⁺ antiporter is not involved. Membrane depolarisation by increasing superfusate [K⁺] to 70 mM, or voltage clamp to 0 mV, increased recovery rate in Na⁺ containing solutions more than threefold. We conclude [fMg²⁺]_i recovery is by Mg²⁺ efflux on a 1 Na⁺:1 Mg²⁺ antiport.     

Intracellular Na+ modulates large conductance Ca2+-activated K+ currents in human umbilical vein endothelial cells

We studied the effects of Na⁺ influx on large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in cultured human umbilical vein endothelial cells (HUVECs) by means of patch clamp and SBFI microfluorescence measurements. In current-clamped HUVECs, extracellular Na⁺ replacement by NMDG⁺ or mannitol hyperpolarized cells. In voltage-clamped HUVECs, changing membrane potential from 0 mV to negative potentials increased intracellular Na⁺ concentration ([Na⁺]_i) and vice versa. In addition, extracellular Na⁺ depletion decreased [Na⁺]_i. In voltage-clamped cells, BK_Ca currents were markedly increased by extracellular Na⁺ depletion. In inside-out patches, increasing [Na⁺]_i from 0 to 20 or 40 mM reduced single channel conductance but not open probability (NPo) of BK_Ca channels and decreasing intracellular K⁺ concentration ([K⁺]_i) gradually from 140 to 70 mM reduced both single channel conductance and NPo. Furthermore, increasing [Na⁺]_i gradually from 0 to 70 mM, by replacing K⁺, markedly reduced single channel conductance and NPo. The Na⁺–Ca²⁺ exchange blocker Ni²⁺ or KB-R7943 decreased [Na⁺]_i and increased BK_Ca currents simultaneously, and the Na⁺ ionophore monensin completely inhibited BK_Ca currents. BK_Ca currents were significantly augmented by increasing extracellular K⁺ concentration ([K⁺]_o) from 6 to 12 mM and significantly reduced by decreasing [K⁺]_o from 12 or 6 to 0 mM or applying the Na⁺–K⁺ pump inhibitor ouabain. These results suggest that intracellular Na⁺ inhibit single channel conductance of BK_Ca channels and that intracellular K⁺ increases single channel conductance and NPo. GH Liang and MY Kim contributed equally to this publication and therefore share the first authorship.     

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

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

Role of physiological HCO3 –buffer on intracellular pH and histamine release in rat peritoneal mast cells

 The purpose of this study was to examine how intracellular pH (pH_i) regulation and histamine release are affected by HCO₃ ^–in rat peritoneal mast cells. The pH_i was measured using the pH-sensitive dye 2′, 7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). We observed a pH_i of 6.88±0.012 (n=24) in resting mast cells exposed to a HEPES buffer (pH 7.4), but a sustained drop of 0.21 pH units to 6.67±0.015 (n=23) when we exposed the mast cells to a HEPES/HCO₃ ^–buffer equilibrated at all time with 5% CO₂ (pH 7.4). This fall in pH_i is inhibited by the carbonic anhydrase inhibitor dichlorphenamide and is Na⁺-independent, indicating the involvement of Na⁺-independent Cl^–/HCO₃ ^–exchange activity. Furthermore removal of external Cl^–in the presence but not in the absence of HCO₃ ^–reversed the Cl^–/HCO₃ ^–exchange and induced an alkaline load. The recovery from this alkaline load was dependent on external Cl^–but independent of Na⁺. Both the alkalinization and the recovery were inhibited by the anion transport inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS). In addition, ³⁶Cl^–uptake measurements confirm the presence of a Cl^–/HCO₃ ^–exchanger. Histamine release stimulated by antigen and compound 48/80 was substantially reduced in the presence of HEPES/ HCO₃ ^–buffer (pH_o 7.4, pH_i 6.66). Histamine release was increased, however, when pH_i was clamped to 6.66 in HCO₃ ^–-free media (pH_o 6.9). We conclude that: (1) Na⁺-independent Cl^–/HCO₃ ^–exchange determines steady-state pH_i in rat peritoneal mast cells; and (2) the reduction in histamine release observed in the presence of HCO₃ ^–is not due to its effect on pH_i per se, but rather on other changes in ion transport. Received: 29 January 1998 / Received after revision and accepted: 3 April 1998     

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 ⁻.     

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.     

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     

Sodium-sensitive, calcium-independent potassium conductance in the leech giant glial cell

 We have measured membrane current, membrane potential and intracellular Na⁺ and Ca²⁺ concentrations, [Na⁺]_i and [Ca²⁺]_i, of the giant glial cell in the nervous system of the leech Hirudo medicinalis using conventional microelectrodes and the fluorescent dyes sodium-binding benzofuran isophthalate (SBFI) and fura-2. When the Na⁺ was removed from the saline, the membrane conductance increased twofold from 1.29±0.1 μS to 2.57±0.18 μS (mean ± SEM; n=27). The rise in membrane conductance was accompanied by a current, which reversed around –74 mV, and the amplitude of K⁺-induced depolarizations or currents increased during Na⁺ removal, suggesting an increase in the K⁺ conductance of the glial membrane. We also monitored [Ca²⁺]_i when removing external Na⁺ in the presence and absence of external Ca²⁺, and during injection of the Ca²⁺-chelator BAPTA into the cells. Our results indicate that Na⁺ modulates a K⁺ conductance of these glial cells, independent of intra- and extracellular Ca²⁺. Received: 1 April 1998 / Received after revision and accepted: 22 May 1998     

Subcellular localization of glutamate-stimulated intracellular magnesium concentration changes in cultured rat forebrain neurons using confocal microscopy

Glutamate can stimulate increases in intracellular magnesium concentration ([Mg²⁺]_i) and induce neurotoxicity, both independent of Ca²⁺ changes. Although Mg²⁺ is essential within the cell, very little is known about how it is regulated, especially in neurons. Therefore we used the fluorescent indicator, magindo-1 and confocal microscopy to examine possible intracellular pools of Mg²⁺ in cultured neurons that can be dynamically regulated by glutamate. The magindo-1 fluorescence signal was present throughout the cell body and extends into the neuronal processes. The magindo-1 405 nm/490 nm ratio signal was similar in the cytoplasm and nucleus, suggesting that resting [Mg²⁺]_i is uniform across the neuron. The addition of 100 μM glutamate/10 μM glycine in an extracellular Ca²⁺- and Na⁺-free buffer stimulated an increase in [Mg²⁺]_i in both the nuclear and cytoplasmic regions of similar magnitude and duration. This glutamate exposure also stimulated a [Mg²⁺]_i increase in neuronal processes which was inhibited by the N-methyl-d-aspartate receptor antagonist, MK-801 (10 μM). The glutamate-stimulated [Mg²⁺]_i increase in both the cell body and neuronal processes was dependent on the extracellular Mg²⁺ concentration.These findings suggest glutamate-stimulated [Mg²⁺]_i changes may not only impact cytoplasmic processes, but also directly trigger nuclear events involved, for example, in neuronal injury.     

Ionic mechanism of ouabain-induced swelling of leech Retzius neurons

By using electrophysiological and microfluorimetric methods, we found that leech Retzius neurons swell after inhibition of the Na⁺–K⁺ pump by the cardiac glycoside ouabain. To explore the mechanism of this swelling, we measured the effect of ouabain on [Na⁺]_i, [K⁺]_i, and [Cl⁻]_i, as well as on the membrane potential, by applying triple-barrelled ion-sensitive microelectrodes. As shown previously, ouabain induced a marked [Na⁺]_i increase, a [K⁺]_i decrease, and a membrane depolarization, and it also evoked an increase in [Cl⁻]_i. The analysis of the data revealed a net uptake of NaCl, which quantitatively explained the ouabain-induced cell swelling. In the absence of extracellular Na⁺ or Cl⁻, NaCl uptake was excluded, and the cell volume remained unaffected. Likewise, NaCl uptake and, hence, cell swelling did not occur when the Na⁺–K⁺ pump was inhibited by omitting bath K⁺. Also, in K⁺-free solution, [Na⁺]_i increased and [K⁺]_i dropped, but [Cl⁻]_i slightly decreased, and after an initial, small membrane depolarization, the cells hyperpolarized for a prolonged period. It is concluded that the ouabain-induced NaCl uptake is caused by the depolarization of the plasma membrane, which augments the inwardly directed electrochemical Cl⁻ gradient.     

Evidence for a Na+/Ca2+ exchange mechanism in frog skin epithelium

 In the present study we investigated the possible existence of a Na⁺/Ca²⁺ exchange mechanism in the basolateral membrane of the frog skin epithelium and whether such a mechanism plays a role in the regulation of transepithelial Na⁺ transport. Cytosolic calcium ([Ca²⁺]_i) was measured with the probe fura-2 in a set-up in which pieces of tissue were mounted on the stage of an epifluorescence microscope. Na⁺ transport was measured as the amiloride-sensitive short-circuit current (I _sc) using a conventional voltage clamp. Basal [Ca²⁺]_i was 65±6 nM (n=15). Removal of Na⁺ from the mucosal solution had no effect on [Ca²⁺]_i. When Na⁺ was removed from the serosal solution, [Ca²⁺]_i increased biphasically to a peak of 220±38 nM (n=8, P=0.006). Readdition of Na⁺ to the serosal solution returned [Ca²⁺]_i to control level. The serosal Na⁺ gradient and changes in [Ca²⁺]_i were closely correlated; stepwise changes in serosal Na⁺ were followed by stepwise changes in [Ca²⁺]_i. These observations indicate the existence of a Na⁺/Ca²⁺ exchange mechanism in the basolateral membrane of the frog skin epithelium. The transepithelial Na⁺ transport decreased from 13.2±1.8 to 9.2±1.5 μA cm^–2 (n=8, P=0.049) when Na⁺ was omitted from the serosal solution. When this protocol was repeated in the absence of serosal Ca²⁺, Na⁺ transport decreased similarly from 16.7±1.7 to 11.6 ±1.8 μA cm^–2 (n=6, P=0.004). We conclude that it is unlikely that the observed decrease in I _sc after removal of serosal Na⁺ is due to an increase in [Ca²⁺]_i per se. Received: 10 July 1998 / Received after revision: 23 September 1998 / Accepted: 25 September 1998     

Anin vitro study into the mechanisms of lidocaine and befol actions on sodium exchange in normal and hypoxia-exposed rat cardiomyocytes

Using benzofuran isophthalate, a fluorescent probe for sodium ions, intracellular (sarcoplasmic) Na⁺ concentrations ([Na⁺]_i) were estimated in cardiomyocytes isolated from the left ventricle of rats. Lidocaine (1–100 μM) had little effect on [Na⁺]_i in resting (unstimulated) cardiocytes, while befol lowered it by virtue of its inhibitory effect on Na/H exchange. In cardiomyocytes exposed to “chemical” hypoxia (produced by 5 mM KCN+30 mM 2-deoxyglucoses). [Na⁺] were three times higher than in resting cells, and the Na-blocking effects of both lidocaine and befol were much stronger. When these two drugs were used together, potentiation of these effects was observed, which may be accounted for by their action on different Na-transporting systems. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 5, pp. 45–47, July, 1996     

Intracellular Na+ measurements in smooth muscle using SBFI – changes in [Na+], Ca2+ and force in normal and Na+-loaded ureter

 Our understanding of the control and effects of intracellular [Na⁺] ([Na⁺]_i) in intact smooth muscle is limited by the lack of data concerning [Na⁺]_i. The initial aim of this work was therefore to investigate the suitability of using the Na⁺-sensitive fluorophore SBFI in intact smooth muscle. We find this to be a good method for measuring [Na⁺]_i in ureteric smooth muscle. Resting [Na⁺]_i was found to be around 10 mM and rose to 25 mM when the Na⁺-K⁺-ATPase was inhibited by ouabain. This relatively low [Na⁺]_i in the absence of Na⁺-K⁺-ATPase suggests that other cellular processes, such as Na⁺-Ca²⁺ exchange, play a role in maintaining [Na⁺]_i under these conditions. Simultaneous measurements of [Na⁺]_i or [Ca²⁺] _i and force showed that Na⁺-Ca²⁺ exchange can play a functional role in ureteric smooth muscle. We found that the greater the driving force for Na⁺ exit and hence Ca²⁺ entry, the larger the contraction. In addition the Na⁺-Ca²⁺ exchanger activity under these conditions was found to be pH sensitive: acidification reduced the contraction and concomitant changes in [Ca²⁺] and [Na⁺]_i. We conclude that SBFI is a useful method for monitoring [Na] in smooth muscle and that Na⁺-Ca²⁺ exchange may play a functional role in the ureter. Received: 26 August 1997 / Received after revision: 27 October 1997 / Accepted: 28 October 1997     

Does adrenaline modulate the Na+-Ca2+ exchanger in isolated toad pacemaker cells?

β-Adrenergic stimulation of pacemaker cells from the sinus venosus of the cane toad (Bufo marinus) increases intracellular calcium ([Ca²⁺]_i) and firing rate. The increase in [Ca²⁺]_i could contribute to the increased firing rate by increasing the inward Na⁺-Ca²⁺ exchange current (I _Na-Ca) during diastole. In this study we measured [Ca²⁺]_i and membrane currents in single, isolated, voltage-clamped pacemaker cells. We show that I _Na-Ca increases during β-adrenergic stimulation. To test whether this increase in I _Na-Ca is caused by elevated [Ca²⁺]_i or by changes in the properties of the Na⁺-Ca²⁺ exchanger, we made rapid applications of caffeine and plotted the I _Na-Ca against [Ca²⁺]_i. This relationship was linear during the declining phase of the [Ca²⁺]_i signal caused by caffeine and was not significantly different in the presence or absence of β stimulation. These results show that I _Na-Ca is increased during β-adrenergic stimulation and will contribute to the increased firing rate. However the increase in I _Na-Ca appears to be a consequence of the increase in [Ca²⁺]_i and is not caused by changes in the intrinsic properties of the Na⁺-Ca²⁺ exchanger. Received: 18 January 1999 / Received after revision: 9 April 1999 / Accepted: 22 April 1999     

Inwardly rectifying K+ currents of alveolar type II cells isolated from fetal guinea-pig lung: regulation by G protein- and Mg2+-dependent pathways

 K⁺ currents in alveolar type II cells, isolated from fetal guinea-pig lung, were studied using the whole-cell patch-clamp technique. Inwardly rectifying (IR) K⁺ currents were observed when cells were bathed in symmetrical KCl-rich solutions. When extracellular K⁺ was replaced by Na⁺, inward currents were greatly decreased and the zero-current potential moved from 0 mV to –69 mV, indicating high K⁺ selectivity. In recordings with an intracellular KCl-rich solution, containing 1.12 mM Mg²⁺ and 10^–8 M free Ca²⁺, IR K⁺ currents slowly diminished with time. Addition of the irreversible G protein activator, guanosine 5’-O-(3-thiotriphosphate) (GTP [γ-S]), to the intracellular solution accelerated the rate of current run-down. In experiments where the intracellular solution was Mg²⁺ free, current run-down was abolished. The rate of current run-down was found to increase with increasing free intracellular [Mg²⁺]. Raising the intracellular free [Ca²⁺] to 10^–6 M under Mg²⁺-free conditions had no effect on the K⁺ currents. Extracellular Ba²⁺ blocked the IR K⁺ currents in a concentration- and voltage-dependent manner. Tolbutamide, a blocker of ATP-sensitive K⁺ (K_ATP) channels, had no effect on the currents. The single channel underlying the whole-cell IR K⁺ currents displayed inward rectification and had a conductance of 31 pS in symmetrical KCl-rich solutions. We demonstate that mRNA coding for IRK1 is expressed in this cell preparation. Possible functions for this channel are discussed. Received: 28 June 1996 / Received after revision and accepted: 9 September 1996     

The use of antioxidants to prevent glutamate-induced derangement of calcium ion metabolism in rat cerebral cortex synaptosomes

Glutamate is shown to induce increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i), increases in⁴⁵Ca²⁺ influx, decreases in the activity of Na⁺, K⁺,-ATPase activity, and activation of the Na⁺/Ca²⁺ exchanger in rat cerebral cortex synaptosomes. NMDA receptor antagonists virtually prevented these effects. Preincubation of synaptosomes with α-tocopherol, superoxide dismutase, and ganglioside GM₁ normalized [Ca²⁺]_i,⁴⁵Ca²⁺, influx, and Na⁺, K⁺-ATPase activity in rat cerebral cortex synaptosomes exposed to glutamate. Glutamate and GM₁ activated the Na⁺/K⁺ exchanger, and their effects were additive. Calcium ions entering cerebral cortex nerve cells via NMDA receptors during exposure to high glutamate concentrations appeared to be only the trigger for the processes activating free-radical reactions. Activation of these reactions led to increases in Ca²⁺ influx into cells, decreases in Na⁺, K⁺-ATPase activity, and significant increases in [Ca²⁺]_i, though this could be prevented by antioxidants and gangliosides. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 85, No. 4, pp. 488–496, April. 1999.