Trans to cis proton concentration gradients accelerate ionophore A23187-mediated net fluxes of Ca2+ across the human red cell membrane

Ionophore A23187-mediated net influx of Ca2+ in ATP-depleted human red cells was studied as a function of the pH and the proton concentration gradient across the membranes. Utilizing the Ca2+-induced increase in K+ conductance of the cell membranes, various CCCP-mediated proton gradients were raised across the membranes of cells suspended in unbuffered salt solutions with different K+ concentrations. In ionophore-mediated equilibrium the concentration ratios of ionized Ca between ATP-depleted, DIDS-treated cells and their suspension medium were equal to the concentration ratios of protons raised to the second power. With no proton concentration gradient across the membranes the net influxes of Ca2+ as a function of pH resembled a titration curve of a weak acid, with half maximal net influx at pH 7.3, at 100 microM extracellular Ca2+. With cellular pH fixed at various values, the net influx of Ca2+ was determined as a function of the proton concentration gradient. A linear relationship between the logarithm of net influx and the difference between extracellular and cellular pH was found at all cellular pH values tested, but the proton concentration gradient acceleration was a function of the cellular pH. Accelerations between 10- and 40- times per unit delta pH were found and net effluxes were correspondingly decreased. The results are discussed in relation to present models of the mechanism of ionophore A23187-mediated Ca2+ transport. The importance of the proton concentration gradient dependency is discussed in relation to the induced oscillations in K+-conductance of human red cell membranes previously reported (Vestergaard-Bogind and Bennekou (1982) Biochim. Biophys. Acta 688, 37-44).     

Calcium-induced oscillations in K+ conductance and membrane potential of human erythrocytes mediated by the ionophore A23187

The time-dependence of ionophore A23187-induced changes in the conductance of the Ca2+-sensitive K+ channels of the human red cell has been monitored with ion-specific electrodes. The membrane potential was reflected in CCCP-mediated pH changes in a buffer-free extracellular medium, and changes in extracellular K+ activity and electrode potential of an extracellular Ca2+-electrode were recorded. Within a narrow range of ionophore-mediated Ca2+ influx, the above-mentioned parameters were found to oscillate when ionophore was added to a suspension of glucose-fed cells. The period of oscillation was about 2 min/cycle depending on ionophore concentration, and the amplitude of hyperpolarization was about 60 mV, corresponding to a maximal gK+ of the same magnitude as gCl-. Without CCCP present no oscillation in K+ conductance was observed. The Ca2+ affinity for the opening process was in the micromolar range. The closing of the K+ channels was a spontaneous process in that the depolarization was well under way before the Ca2+-ATPase-mediated Ca2+ net efflux started. Below the Ca2+ influx range for oscillations, no response was observed for up to 20 min after the addition of ionophore. Above the upper limit, a permanent hyperpolarization resulted with an extracellular K+ activity increasing monotonically as a function of time. In experiments with ATP-depleted cells, responses of the latter type ensued at all ionophore concentrations above the lower limit. Addition of surplus EGTA to suspensions of hyperpolarized cells restores the normal membrane potential in the case of glucose-fed cells, whereas the K+-channels in ATP-depleted cells remained open.     

Trans to Cis proton concentration gradients accelerate ionophore A23187-mediated net fluxes of Ca2+ across the human red cell membrane

Ionophore A23187-mediated net influx of Ca²⁺ in ATP-depleted human red cells was studied as a function of the pH and the proton concentration gradient across the membranes. Utilizing the Ca²⁺-induced increase in K⁺ conductance of the cell membranes, various CCCP-mediated proton gradients were raised across the membranes of cells suspended in unbuffered salt solutions with different K⁺ concentrations. In ionophore-mediated equilibrium the concentration ratios of ionized Ca between ATP-depleted, DIDS-treated cells and their suspension medium were equal to the concentration ratios of protons raised to the second power. With no proton concentration gradient across the membranes the net influxes of Ca²⁺ as a function of pH resembled a titration curve of a weak acid, with half maximal net influx at pH 7.3, at 100 μM extracellular Ca²⁺. With cellular pH fixed at various values, the net influx of Ca²⁺ was determined as a function of the proton concentration gradient. A linear relationship between the logarithm of net influx and the difference between extracellular and cellular pH was found at all cellular pH values tested, but the proton concentration gradient acceleration was a function of the cellular pH. Accelerations between 10- and 40- times per unit ΔpH were found and net effluxes were correspondingly decreased. The results are discussed in relation to present models of the mechanism of ionophore A23187-mediated Ca²⁺ transport. The importance of the proton concentration gradient dependency is discussed in relation to the induced oscillations in K⁺-conductance of human red cell membranes previously reported (Vestergaard-Bogind and Bennekou (1982) Biochim. Biophys. Acta 688, 37ndash;44).     

Delayed activation of calcium pump during transient increases in cellular Ca2+ concentration and K+ conductance in hyperpolarizing human red cells

The net Ca2+ influx was increased in human red cells in suspension by adding moderate concentrations of the Ca2+ ionophore A23187, and due to the increased cellular Ca2+ concentration [( Ca]i) the K+ channels opened (the 'Gardos effect'). At low K+ concentration and with the protonophore CCCP in the buffer-free medium the cells hyperpolarized and the extracellular pH (pH0) increased, enhancing the A23187-mediated net Ca2+ influx. This elicited a prolonged response, viz. a primary transient increase of pH0 and [Ca]i followed by one or more spontaneous pH0 and [Ca]i transients. We explored the pump-mediated Ca2+ efflux by blocking the A23187-mediated Ca2+ flux with CoCl2 at appropriate times during the prolonged response. The Ca2+ pumping was higher during the descendent than during the ascendent phase of the primary transient at equal values of [Ca]i. The data were analyzed using a mathematical model that accounts for the prolonged oscillatory response, including pH0 and [Ca]i. In conclusion, the activation of the Ca2+ pump is delayed due to slow binding of cellular calmodulin, which is a hysteretic response to a rapid increase of the cellular Ca2+ concentration. This mechanism may be important for generation and execution of transient signals in other types of cell.     

Ca2+-activated K+ conductance of human red cell membranes exhibits two different types of voltage dependence

The voltage dependence for outward-going current of the Ca-activated K+ conductance (gK(Ca] of the human red cell membrane has been examined over a wide range of membrane potentials (Vm at constant values of [K+]ex, [K+]c and pHc, the intact cells being preloaded to different concentrations of ionized calcium. Outward-current conductances were calculated from initial net effluxes of K+ and the corresponding (Vm - EK) values. The basic conductance, defined as the outward-current conductance at (Vm - EK) greater than or equal to 20 mV and [K+]ex greater than or equal to 3 mM (B. Vestergaard-Bogind, P. Stampe and P. Christophersen, J. Membrane Biol. 95:121-130, 1987) was found to be a function of cellular ionized Ca. At all degrees of Ca activation gK(Ca) was an apparently linear function of voltage (Vm range -40 to +70 mV), the absolute level as well as the slope decreasing with decreasing activation. In a simple two-state model the constant voltage dependence can, at the different degrees of Ca activation, be accounted for by a Boltzmann-type equilibrium function with an equivalent valence of approximately 0.4, assuming chemical equilibrium at Vm = 0 mV. Alternatively, the phenomenon might be explained by a voltage-dependent block of the outward current by an intracellular ion. Superimposed upon the basic conductance is the apparently independent inward-rectifying steep voltage function with an equivalent valence of approximately 5 and chemical equilibrium at the given EK value.     

Calcium-dependent K+ efflux from rat submandibular gland. The effects of trifluoperazine and quinidine

The Ca2+-dependent K+ efflux from rat submandibular gland was studied using a K+-sensitive electrode. A K+ efflux was induced by either adrenalin or by using the divalent cation ionophore A23187 plus added Ca2+ to bypass the receptor mechanism. Trifluoperazine, which was used to investigate the role of calmodulin, was found to block the adrenalin-induced K+ efflux but not the A23187/Ca2+-induced K+ efflux. The adrenalin-induced K+ efflux was abolished by quinidine and the A23187/Ca2+-induced K+ efflux was significantly reduced by quinidine. In other experiments, the presence of indomethacin did not inhibit the adrenalin-induced K+ efflux, and exogenously added arachidonic acid did not induce a K+ efflux. It is concluded that neither prostaglandin synthesis, nor a cytosolic Ca2+-calmodulin complex is involved in the agonist-induced K+ efflux from rat submandibular gland. A similarity between the Ca2+-dependent K+ efflux mechanism of erythrocyte ghosts and submandibular tissue is indicated by their common response to quinidine.     

Mitochondrial glutathione status during Ca2+ ionophore-induced injury to isolated hepatocytes

In this study the Ca2+ ionophore, A23187, was used to determine the effects of disrupted Ca2+ homeostasis on cellular thiols. Isolated rat hepatocytes were incubated with varying concentrations of extracellular Ca2+ and A23187 to induce accumulation or loss of cellular Ca2+. These treatments resulted in loss of mitochondrial and cytosolic glutathione (GSH), loss of protein-thiols, and cell injury. This injury was dependent on the concentrations of ionophore and extracellular Ca2+. A correlation was found between cell injury and the loss of mitochondrial GSH, while the loss of cytosolic glutathione preceded both these events. The time course of protein-thiol loss appeared secondary to the loss of non-protein thiols. In the absence of extracellular Ca2+, the antioxidants alpha-tocopherol and diphenyl-p-phenylenediamine both totally prevented A23187-induced cell injury and loss of mitochondrial GSH, and thus protected the cells from the effects of mobilization of intracellular Ca2+. In the presence of extracellular Ca2+, cell injury as well as the loss of mitochondrial GSH were only partially prevented by antioxidant treatment. The mitochondrial Ca2+ channel blocker, ruthenium red, protected hepatocytes from A23187-induced injury in the absence of extracellular Ca2+. Leupeptin, an inhibitor of Ca2+-activated proteases, and dibucaine, a phospholipase inhibitor, did not affect cytotoxicity. Our results indicate that the level of mitochondrial GSH may be important for cell survival during ionophore-induced perturbation of cellular Ca2+ homeostasis.     

The effects of Ca2+ and Sr2+ on Ca2+-sensitive biochemical changes in human erythrocytes and their membranes.

1. The Ca2+-dependency of K+ efflux, microvesiculation and breakdown of polyphosphoinositides and of ankyrin have been measured in intact human erythrocytes exposed to ionophore A23187 and HEDTA [N'-(2-hydroxyethyl)ethylenediamine NNN'-triacetate]-Ca2+ buffers. Half-maximal responses were observed at pCa values of 6.4, 4.1, 5.0 and 4.8 respectively. 2. The Ca2+ dependencies of K+ efflux and breakdown of polyphosphoinositides and ankyrin measured in erythrocyte ghosts without addition of ionophore showed almost identical values with those seen in whole cells treated with ionophore. 3. We conclude that ionophore A23187 is able to cause rapid equilibration of extracellular and intracellular [Ca2+] in intact cells and that in the presence of a suitable Ca2+ buffer, ionophore A23187 can be used to precisely fix the intracellular concentration of Ca2+ in erythrocytes. 4. The relatively high concentration of Ca2+ required to produce microvesiculation in intact cells may indicate that microvesiculation could be at least partly dependent on a direct interaction of Ca2+ with phospholipid. 5. Results obtained with Sr2+ paralleled those with Ca2+, although higher Sr2+ concentrations were required to achieve the same effects as Ca2+. Mg2+ produced none of the changes seen with Ca2+ or Sr2+.     

Cyclic AMP and Ca2+-activated K+ transport in a human colonic epithelial cell line

Addition of either vasoactive intestinal peptide (VIP) or the Ca2+ ionophore, A23187, to confluent monolayers of the T84 epithelial cell line derived from a human colon carcinoma increased the rate of 86Rb+ or 42K+ efflux from preloaded cells. Stimulation of the rate of efflux by VIP and A23187 still occurred in the presence of ouabain and bumetanide, inhibitors of the Na+,K+-ATPase and Na+,K+,Cl- cotransport, respectively. The effect of A23187 required extracellular Ca2+, while that of VIP correlated with its known effect on cyclic AMP production. Other agents which increased cyclic AMP production or mimicked its effect also increased 86Rb+ efflux. VIP- or A23187-stimulated efflux was inhibited by 5 mM Ba2+ or 1 mM quinidine, but not by 20 mM tetraethylammonium, 4 mM 4-aminopyridine, or 1 microM apamin. Under appropriate conditions, VIP and A23187 also increased the rate of 86Rb+ or 42K+ uptake. Stimulation of the initial rate of uptake by either agent required high intracellular K+ and was not markedly affected by the imposition of transcellular pH gradients. The effect of A23187, but not VIP or dibutyryl cyclic AMP, was refractory to depletion of cellular energy stores. A23187-stimulated uptake was not significantly affected by anion substitution, however, stimulation of uptake by VIP required the presence of a permeant anion. This result may be due to the simultaneous activation of a cyclic AMP-dependent Cl- transport system. The kinetics of both VIP- and A23187-stimulated uptake and efflux were consistent with a channel-rather than a carrier-mediated K+ transport mechanism. The results also suggest that cyclic AMP and Ca2+ may activate two different kinds of K+ transport systems. Finally, both transport systems have been localized to the basolateral membrane of T84 monolayers, a result compatible with their possible regulatory role in hormone-activated electrogenic Cl- secretion.     

Calcium regulates cAMP-induced potassium ion efflux in Dictyostelium discoideum

The chemoattractant cAMP elicits a transient efflux of K+ in cell suspensions of Dictyostelium discoideum. This cellular response displayed half-maximal activity at about 1 microM cAMP and saturated at 100 microM cAMP, cAMP-stimulated K+ efflux, measured with a K+-sensitive electrode, depended on the extracellular free Ca2+ concentration ([Ca2+]0) and was maximal in the presence of EGTA. Usually more than 90% of the K+ release could be inhibited by the addition of Ca2+. Half-maximal reduction occurred at about 2 microM [Ca2+]0. Inhibition was also observed in the presence of caffeine or A23187, drugs known to elevate the intracellular free Ca2+ concentration ([Ca2+]i). Under conditions where [Ca2+]0 was maintained at a low level, half-maximal inhibition was 1 mM for caffeine and 3 microM for A23187. These results indicate that Cai2+ is involved in the regulation of K+ efflux. Simultaneous measurements of Ca2+ uptake and K+ efflux induced by cAMP as well as free running oscillations of both ions revealed that initiation and termination of Ca2+ uptake slightly preceded those of K+ efflux.     

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum.

ATP and the divalent cations Mg2+ and Ca2+ regulated K+ stimulation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K+-stimulated ATPase activity of the Ca2+ pump by two mechanisms. First, ATP chelated free Mg2+ and, at low ionized Mg2+ concentrations, K+ was shown to be a potent activator of ATP hydrolysis. In the absence of K+ ionized Mg2+ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K+ the concentration of ionized Mg2+ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K+-stimulation. With a saturating concentration of ionized Mg2+, stimulation by K+ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K+ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold. Activation of K+-stimulated ATPase activity by Ca2+ was maximal at an ionized Ca2+ concentration of approx. 1 microM. At very high concentrations of either Ca2+ or Mg2+, basal Ca2+-dependent ATPase activity persisted, but the enzymic response to K+ was completely inhibited. The results provide further evidence that the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme.     

Lymphocyte membrane potential and Ca2+-sensitive potassium channels described by oxonol dye fluorescence measurements

A method is described for quantitative measurement of lymphocyte transmembrane electrical potential difference (psi) by flow cytometric recording of the oxonol dye fluorescence of single cells. Both the simultaneous collection and analysis of multiple optical parameters and the use of a negatively charged oxonol probe allowed more accurate measurement of psi than may be obtained by bulk cell suspension techniques employing cationic voltage indicators. Mouse spleen and human blood lymphocyte psi was calculated to be -70 mV. T and B lymphocytes maintain a constant psi as extracellular K+ is varied from 2 to 10 mM and the deviation from K+ equilibrium potentials (EK) is shown to result from Na+ permeability. At [K+]o values greater than 10 mM, lymphocytes behave as K+ electrodes. Examination of lymphocyte subsets showed that hyperpolarization induced by the Ca2+ ionophore A23187 occurs only in T cells. This response was identified as activation of a Ca2+-sensitive K+ channel by pharmacologic manipulations. Hence, T cells depolarized by 4-aminopyridine (4-AP, 10 mM) were observed to return to resting psi by A23187-induced elevation of [Ca2+]i. Cells depolarized by quinine (100 microM) were unaffected by A23187. The Ca2+-activated channel does not contribute to resting psi in T cells since it may be selectively blocked by quinine (20 microM) or modulated by calmodulin antagonists (5 microM trifluperazine) without affecting resting psi.     

Spontaneous inactivation of the Ca2+-sensitive K+ channels of human red cells at high intracellular Ca2+ activity

Ionophore A23187-mediated Ca²⁺-induced oscillations in the conductance of the Ca²⁺-sensitive K⁺ channels of human red cells were monitored with ion specific electrodes. The membrane potential was continuously reflected in CCCP-mediated pH changes in the buffer-free medium, changes in extracellular K⁺ activity were followed with a K⁺-selective electrode, and changes in the intracellular concentration of ionized calcium were calculated on the basis of cellular ⁴⁵Ca content. An increased cellular ⁴⁵Ca content at the successive minima of the oscillations where the K⁺ channels are closed indicates that the activation of the channels might be a $\text{(}\text{dCa}{}^{\mathrm{2+}}\text{/}\text{d}\text{t)-}\text{sensitive}$ process and that accommodation to enhanced levels of intracellular free calcium may occur. An incipient inactivation of the K⁺ channels at intracellular ionized calcium levels of about 10 μM and a concurrent membrane potential of about ?65 mV was observed. At a membrane potential of about ?70 mV and an intracellular concentration of about 2·10^?4M no inactivation of K⁺ channels took place. Inactivation of the K⁺ channels is suggested to be a compound function of the intracellular level of free calcium and the membrane potential. The observed sharp peak values in cellular ⁴⁵Ca content support the notion that a necessary component of the oscillatory system is a Ca²⁺ pump operating with a significant delay in the activation/inactivation process in response to changes in cellular concentration of ionized calcium.     

Participation of the microtubular-microfilamentous system on intracellular Ca2+ transport and acid secretion in dispersed parietal cells

Biphasic responses of amino[14C]pyrine accumulation and oxygen consumption were registered by gastrin stimulation in dispersed parietal cells from guinea pig gastric mucosa, and this was mimicked with the calcium ionophore A23187. The characteristics of these phases (first phase and second phase) were distinguished by the differences in the requirements of extracellular Ca2+. The first phase evoked by gastrin or ionophore A23187 was independent of extracellular Ca2+, whereas the second phase was not. In the first phase, fluorescence of a cytosolic Ca2+ indicator (quin2-AM) increased with the stimulation of ionophore A23187 and carbamylcholine chloride in the presence of extracellular Ca2+. In addition, an increase in cytosolic Ca2+ induced by ionophore A23187, but not by carbamylcholine chloride was also observed in the absence of extracellular Ca2+, suggesting that Ca2+ pool(s) in parietal cells might be present in the intracellular organelle. Cytochalasin B and colchicine, but not oligomycin, could eliminate this cytosolic Ca2+ increase induced by A23187 in a Ca2+-free medium. On the other hand, in a Ca2+-free medium, addition of ATP after pretreatment with digitonin could diminish the cytosolic Ca2+ increase brought about by A23187. This was also observed with oligomycin-treated cells, but not with cytochalasin B-treated cells. Similarly, subcellular fractionation of a parietal cell which had been pretreated with cytochalasin B or colchicine in an intact cell system reduced the rate of ATP-dependent Ca2+ uptake. These observations indicate that intracellular Ca2+ transport in dispersed parietal cells may be regulated by the microtubular-microfilamentous system. In conclusion, this study demonstrates the possibility of the existence of intracellular Ca2+ transport mediated by gastrin or ionophore A23187 and regulated by the microtubular-microfilamentous system in parietal cells.     

A role for Na+-dependent Ca2+ extrusion in protection against neuronal excitotoxicity

The hypothesis that Na+-dependent calcium extrusion is important in protecting against neuronal excitotoxicity was tested. In cocultures of embryonic rat hippocampal neurons and mouse neuroblastoma hybrid (NCB-20) cells, calcium ionophore A23187 (1 microM) or high levels of extracellular K+ killed hippocampal neurons selectively, leaving NCB-20 cells unscathed. Hippocampal neurons showed large, sustained rises in intracellular calcium in response to A23187 or K+, whereas NCB-20 cells showed only transient calcium responses. The ability of NCB-20 cells to reduce the calcium load and to survive exposure to A23187 or K+ were dependent on extracellular Na+, suggesting that an active Na+/Ca2+ exchange mechanism was important in protecting against cell death. Finally, removal of extracellular Na+ reduced the threshold for glutamate neurotoxicity in hippocampal neurons, demonstrating the importance of Na+/Ca2+ exchange in protecting against excitotoxicity. Taken together, these findings suggest that differences in cell calcium-regulating systems may determine whether a neuron lives or degenerates in the face of an excitatory challenge.     

Calcium ion-flux across phosphatidylcholine membranes mediated by ionophore A23187.

The antibiotic A23187 carries Ca2+ across Müller-Rudin membranes made from 1,2-dierucoyl-sn-glycero-3-phosphocholine and n-decane. The conductance of the membranes is not increased by the Ca2+-transport. The flux depends linearly on Ca2+ concentration and ionophore concentration (above pH 6). It increases with increasing pH, approximately by a factor of 4-5 between pH 6 and pH 8. Maximal Ca2+-fluxes of about 10(-10) mol-cm-2-s-1 were found. A counter transport of H+ could not be detected. The complex formation between A23187 and Ca2+ in egg phosphotidylcholine vesicles was studied spectroscopically. The results are consistent with the formation of a 2:1 complex. Optical absorption measurements on single phophatidylcholine membranes were used to calculate the concentration of membrane-bound ionophore A23187.     

Ca2+ uptake in bovine adrenocortical microsomes: formation of phosphorylated intermediate of Ca2+ dependent ATPase

Bovine adrenocortical microsomes were prepared and partially purified by discontinuous sucrose density gradient. Light fractions of the microsomes at the interface between 15 and 30% sucrose solution, exhibited ATP dependent Ca2+ uptake. The Ca2+ uptake was dependent on temperature and stimulated by free Ca2+ (the concentration for half maximal activation = 1.0 microM) and Mg2+. The Ca2+ uptake was inhibited by ADP but not affected by 10 mM NaN3 or 0.5 mM ouabain. Calcium release from the microsomes was accelerated by a Ca2+ ionophore, A23187, but not by a Ca2+ antagonist, diltiazem. A microsomal protein with a molecular weight of 100-110 kDa was phosphorylated by [gamma-32P]ATP in the presence of Ca2+, and the Ca2+ dependency was over the same range as the Ca2+ uptake (the concentration for half maximal activation = 3.0 microM). The phosphorylated protein (EP) was stable at acidic pH but labile at alkaline pH and sensitive to hydroxylamine. The rate of EP formation at 0 degrees C in the presence of 1 microM ATP and 10 microM Ca2+ (half time = 0.2 s) was less than that in the sarcoplasmic reticulum (SR) of rabbit skeletal muscle (half time = 0.1 s). The rate of EP decomposition at 0 degrees C after adding EGTA was about 6.7 times slower (rate constant: kd = 4.3 X 10(-3) s-1) than that of SR. It was suggested that adrenocortical microsomes contain a Ca2+ dependent ATPase which function as a Ca2+ pump with similar properties to that of SR.     

Effects of A23187 and Ca2+ on volume- and thiol-stimulated, ouabain-resistant K+C1- fluxes in low K+ fluxes in low K+ sheep erythrocytes

Ouabain-resistant (OR), C1- -dependent K+ (K+C1-) transport measured by Rb+ influx in isosmotic and anisosmotic media was stimulated by the Ca2+ ionophore A23187 and EGTA (ethylene-glycol-tetracetic acid) in low K+ (LK) but not in high K+ (HK) sheep red cells. Increasing external Ca2+ concentrations, [Ca2+]o, from about 10(-7) to 10(-3)M in presence of A23187 and in absence of EGTA inhibited OR Rb+ influx, in LK red cells osmotically shrunken or swollen as well as treated with the thiol reagent N-ethylmaleimide (NEM). Hence the volume- and the NEM-stimulated K+C1- transport system in LK cells can be experimentally modulated by cellular Ca2+ or other Me2+, which may interact with sites on the K+C1- transporter under the control of membrane sulfhydryl (SH) groups.     

Inhibition of translational initiation in eukaryotic cells by calcium ionophore

Ca2+ has been recently reported to be required for high rates of translational initiation in GH3 pituitary cells (Chin, K.-V., Cade, C., Brostrom, C.O., Galuska, E.M., and Brostrom, M.A. (1987) J. Biol. Chem. 262, 16509-16514). In the present investigation low concentrations of the Ca2+ ionophores, A23187 and ionomycin, were found to rapidly suppress the Ca2+-dependent component of protein synthesis in GH3 cells. More ionophore was required to inhibit amino acid incorporation into protein as extracellular Ca2+ was increased. Pre-existing inhibitions of protein synthesis produced by low concentrations of ionophore at low extracellular Ca2+ concentrations were reversed by adjustment to high extracellular Ca2+. Treatment with ionophore reduced the cellular contents of polysomes and 43 S preinitiation complex to values equivalent to those found for Ca2+-depleted cells. Average ribosomal transit times were unaffected by ionophore, and treated cells retained the ability to accumulate polysomes when incubated with cycloheximide. Cell types, such as HeLa and Chinese hamster ovary, that normally display only a modest Ca2+-dependent component of protein synthesis, manifested a strong underlying Ca2+ dependence in amino acid incorporation and polysome formation following treatment with low concentrations of ionophore. Protein synthesis in GH3 or HeLa cells during recovery from heat shock and arsenite treatment was not affected by cellular Ca2+ depletion or ionophore treatment. On the basis of these results, Ca2+ ionophore is proposed to inhibit Ca2+-dependent translational initiation through facilitating the mobilization of sequestered intracellular Ca2+.     

Effect of hypoglycemic sulfonylureas on Ca2+ fluxes across lipid bilayers

Black lipid membranes and liposomes loaded with Ca2+ or 5,6-carboxyfluorescein were used for exploring the mechanism of action of insulin-releasing sulfonylureas. Unlike the Ca2+/H+ exchanging ionophore A-23187, tolbutamide did not stimulate the net efflux of Ca2+ from the liposomes. Glibenclamide caused a sustained release of Ca2+, but this effect could be attributed to labilization of the liposomal membrane as indicated by a quantitatively similar loss of the stability marker 5,6-carboxyfluorescein. Unlike the neutral ionophore nonactin or the channel forming quasi-ionophore gramicidin A, the sulfonylureas did not alter the conductance of black lipid membranes in medium containing Na+, K+, Ca2+, Mg2+, and Cl-. It is concluded that the sulfonylureas tested lack ionophore properties but that glibenclamide can labilize membranes.