The hepatic response to Ca2+ is inhibited by Mg2+ and enhanced by phenylephrine or ouabain

Net hepatic Ca2+ efflux, K+ uptake and glycogen breakdown in response to the alpha 1-adrenergic agonist phenylephrine were studied. Rat livers were perfused with CO2/bicarbonate-buffered solutions containing 10 microM Ca2+ and different amounts of Mg2+. K+-free medium and/or ouabain were used to block (Na+ + K+)-ATPase-dependent K+ uptake. In some experiments a sharp increase in extracellular Ca2+ concentrations was produced by infusing CaCl2 into the medium entering the liver. Perfusion with K+-free medium and ouabain enhanced the phenylephrine-induced Ca2+ efflux and diminished the glycogenolytic response, indicating a dissociation of Ca2+ release and glycogenolysis. Exogenous Ca2+ had practically no effect if livers were perfused with regular medium containing 1.2 mM Mg2+. In the presence of phenylephrine and if extracellular Mg2+ concentrations were lowered by omitting Mg2+ from the medium or by preperfusion with EGTA, exogenous Ca2+ was glycogenolytically effective and also produced a transient K+ uptake. Increased extracellular concentrations of Mg2+ inhibited the effects of exogenous Ca2+. In the presence of phenylephrine, higher concentrations of Mg2+ were needed than in the absence of alpha 1-adrenergic agonist to achieve a similar degree of inhibition. In one respect ouabain effects were comparable to those of phenylephrine: the glycoside also increased the metabolic response to exogenous Ca2+ and diminished the sensitivity towards Mg2+. Phenylephrine and ouabain may both enhance the permeability of plasma membranes for Ca2+.     

Effect of hemolysate on calcium inhibition of the (Na+ + K+)-ATPase of human red blood cells

The sensitivity of the (Na+ + K+)-ATPase in human red cell membranes to inhibition by Ca2+ is markedly increased by the addition of diluted cytoplasm from hemolyzed human red blood cells. The concentration of Ca2+ causing 50% inhibition of the (Na+ + K+)-ATPase is shifted from greater than 50 microM free Ca2+ in the absence of hemolysate to less than 10 microM free Ca2+ when hemolysate diluted 1:60 compared to in vivo concentrations is added to the assay mixture. Boiling the hemolysate destroys its ability to increase the sensitivity of the (Na+ + K+)-ATPase to Ca2+. Proteins extracted from the membrane in the presence of EDTA and concentrated on an Amicon PM 30 membrane increased the sensitivity of the (Na+ + K+)-ATPase to Ca2+ in a dose-dependent fashion, causing over 80% inhibition of the (Na+ + K+)-ATPase at 10 microM free Ca2+ at the highest concentration of the extract tested. The active factor in this membrane extract is Ca2+-dependent, because it had no effect on the (Na+ + K+)-ATPase in the absence of Ca2+. Trypsin digestion prior to the assay destroyed the ability of this protein extract to increase the sensitivity of the (Na+ + K+)-ATPase to Ca2+.     

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.     

Metabolic, cationic and secretory response to D-glucose in depolarized and Ca(2+)-deprived rat islets exposed to diazoxide

D-glucose stimulates insulin release from islets exposed to both diazoxide, to activate ATP-responsive K+ channels, and a high concentration of K+, to cause depolarization of the B-cell plasma membrane. Under these conditions, the insulinotropic action of D-glucose is claimed to occur despite unaltered cytosolic Ca2+ concentration, but no information is so far available on the changes in Ca2+ fluxes possibly caused by the hexose. In the present experiments, we investigated the effect of D-glucose upon 45Ca efflux from islets exposed to both diazoxide and high K+ concentrations. In the presence of diazoxide and at normal extracellular Ca2+ concentration, D-glucose (16.7 mmol/l) inhibited insulin release at 5 mmol/l K+, but stimulated insulin release of 90 mmol/l K+. In both cases, the hexose inhibited 45Ca outflow. In the presence of diazoxide, but absence of Ca2+, D-glucose (8.3 to 25.0 mmol/l) first caused a rapid decrease in insulin output followed by a progressive increase in secretory rate. This phenomenon was observed both at 5 mmol/l or higher concentrations (30, 60 and 90 mmol/l) of extracellular K+. It coincided with a monophasic decrease in 45Ca efflux and either a transient (at 5 mmol/l K+) or sustained (at 90 mmol/l K+) decrease in overall cytosolic Ca2+ concentration. The decrease in 45Ca efflux could be due to inhibition of Na(+)-Ca2+ countertransport with resulting localized Ca2+ accumulation in the cell web of insulin-producing cells. A comparable process may be involved in the secretory response to D-glucose in islets exposed to diazoxide and a high concentration of K+ in the presence of extracellular Ca2+.     

High extracellular Ca2+ and Mg2+ stimulate accumulation of inositol phosphates in bovine parathyroid cells

We examined the effects of the divalent cations Ca2+ and Mg2+ on inositol phosphate accumulation in bovine parathyroid cells prelabelled with [3H]inositol to determine whether the high extracellular Ca2+ and Mg2+-evoked transients in cytosolic Ca2+ in these cells might result from increases in cellular IP3 levels. In the presence of Li+, both Ca2+ and Mg2+ produced rapid, 2-6-fold increases in IP3 and IP2 and a linear increase in IP of 6-8-fold at 30 min. Smaller (1.5-2-fold) increases in IP2 and IP3 were evident within 7.5-15 s upon exposure to high (3 mM) Ca2+ in the absence of Li+. The relative potencies of Ca2+ and Mg2+ (Ca2+ 3-fold more potent than Mg2+) in elevating inositol phosphates were similar to those for their effects in inhibiting PTH release. Fluoride (5 and 10 mM) also produced similar increases in inositol phosphate accumulation, presumably through activation of phospholipase C by a guanine nucleotide (G) protein-dependent process. Thus, high extracellular Ca2+ and Mg2+-induced spikes in cytosolic Ca2+ in bovine parathyroid cells may be mediated by increases in IP3, perhaps through a receptor-mediated process linked to phospholipase C by a G-protein.     

Nicorandil is a potent dilator of endothelin-induced vascular contraction

Nicorandil, an antianginal drug, is known to open K+ channel and to increase cGMP production. The effects of nicorandil on vascular contraction induced by endothelin (ET), a potent newly discovered vasoconstrictor peptide, were investigated using helical strips from rat thoracic aorta. ET at a concentration of 5 x 10(-9) M induced strong and persistent contraction in the presence of extracellular Ca2+ and similar persistent but smaller contraction in the absence of extracellular Ca2+. Nicorandil at concentrations greater than 10(-7) M, strongly and dose-dependently inhibited ET-induced contraction in the presence of extracellular Ca2+. Nicorandil also suppressed ET-induced contraction in the presence of 10(-4) M methylene blue, an inhibitor of cGMP production, in the presence of extracellular Ca2+ but not in the absence of extracellular Ca2+. ET-induced contraction was also inhibited to lesser extents by the Ca2+ channel blockers nicardipine and verapamil. Nicorandil also strongly suppressed ET-induced increase in cytosolic free Ca2+ concentration in cultured vascular smooth muscle cells. These results suggest that nicorandil is a potent dilator of ET-induced vasoconstriction.     

Calcium inhibition of the ATPase and phosphatase activities of (Na+ + K+)-ATPase

In experiments performed at 37 degrees C, Ca2+ reversibly inhibits the Na+-and (Na+ + K+)-ATPase activities and the K+-dependent phosphatase activity of (Na+ + K+)-ATPase. With 3 mM ATP, the Na+-ATPase was less sensitive to CaCl2 than the (Na+ + K+)-ATPase activity. With 0.02 mM ATP, the Na+-ATPase and the (Na+ + K+)-ATPase activities were similarly inhibited by CaCl2. The K0.5 for Ca2+ as (Na+ + K+)-ATPase inhibitor depended on the total MgCl2 and ATP concentrations. This Ca2+ inhibition could be a consequence of Ca2+-Mg2+ competition, Ca . ATP-Mg . ATP competition or a combination of both mechanisms. In the presence of Na+ and Mg2+, Ca2+ inhibited the K+-dependent dephosphorylation of the phosphoenzyme formed from ATP, had no effect on the dephosphorylation in the absence of K+ and inhibited the rephosphorylation of the enzyme. In addition, the steady-state levels of phosphoenzyme were reduced in the presence both of NaCl and of NaCl plus KCl. With 3 mM ATP, Ca2+ alone sustained no more than 2% of the (Na+ + K+)-ATPase activity and about 23% of the Na+-ATPase activity observed with Mg2+ and no Ca2+. With 0.003 mM ATP, Ca2+ was able to maintain about 40% of the (Na+ + K+)-ATPase activity and 27% of the Na+-ATPase activity seen in the presence of Mg2+ alone. However, the E2(K)-E1K conformational change did not seem to be affected. Ca2+ inhibition of the K+-dependent rho-nitrophenylphosphatase activity of the (Na+ + K+)-ATPase followed competition kinetics between Ca2+ and Mg2+. In the presence of 10 mM NaCl and 0.75 mM KCl, the fractional inhibition of the K+-dependent rho-nitrophenylphosphatase activity as a function of Ca2+ concentration was the same with and without ATP, suggesting that Ca2+ indeed plays the important role in this process. In the absence of Mg2+, Ca2+ was unable to sustain any detectable ouabain-sensitive phosphatase activity, either with rho-nitrophenylphosphate or with acetyl phosphate as substrate.     

Involvement of Ca2+ entry and inositol trisphosphate-induced internal Ca2+ mobilization in muscarinic receptor-mediated catecholamine release in dog adrenal chromaffin cells.

Catecholamine (CA) release from adrenal medulla evoked by muscarinic receptor stimulation has been studied using isolated perfused adrenal gland and cultured chromaffin cells from dogs. Muscarine and oxotremorine (1-100 microM), and bethanechol (0.1-1 mM) dose-dependently stimulated CA release. Muscarine-evoked CA release was antagonized with M1-antagonist, pirenzepine and, to a lesser extent, with atropine; and was reduced either by removal of extracellular Ca2+ or treatment with Ca2+ channel blockers. Muscarine caused an increase of 45Ca uptake and 22Na uptake. Tetrodotoxin (TTX) did not affect muscarine-evoked increase of 22Na uptake and CA release. Under the absence of extracellular Ca2+, muscarine stimulated a 45Ca efflux. Muscarine-induced CA release was attenuated by treating the cells with 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate-HCl (TMB-8) which blocks Ca2+ release from the intracellular store. A phospholipase C inhibitor, neomycin, markedly reduced muscarine-induced CA release but not nicotine- and high K(+)-evoked release. Cinnarizine, a Ca2+ channel blocker, attenuated muscarine-evoked but not caffeine-induced CA release and 45Ca efflux in the absence of extracellular Ca2+. Muscarine caused an increase in intracellular free Ca2+ concentration ([Ca2+]i) in the presence of extracellular Ca2+. It caused a similar increase, but to a lesser extent, in the absence of extracellular Ca2+. The increase of [Ca2+]i induced by muscarine without extracellular Ca2+ was reduced by neomycin and cinnarizine. Polymixin B and retinal, which reduced 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced CA release, had little effect on muscarine-induced CA release. Muscarine increased cellular Ins(1,4,5)P3 production, and atropine inhibited this increase.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of platelet-activating factor on [14C]aminopyrine uptake by isolated guinea pig parietal cells

C16- and C18- platelet-activating factor (PAF) at 10(-6) M enhanced the uptake of [14C]aminopyrine (AP) by isolated guinea pig parietal cells. This increase was inhibited by a PAF antagonist CV-6209. In a medium with a low calcium (Ca2+) concentration (2 uM), this increase was not observed, which indicates that the increase of AP uptake by PAF is dependent on extracellular Ca2+. PAF and lyso-PAF showed no effect on AP uptake in the presence of histamine or carbachol.     

Extracellular ATP increases cytoplasmic free Ca2+ concentration in clonal insulin-producing RINm5F cells. A mechanism involving direct interaction with both release and refilling of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool.

Effects of extracellularly applied ATP (added as disodium salt) on stimulus-secretion coupling were investigated in clonal insulin-producing RINm5F cells. Cytoplasmic free Ca2+ concentration [( Ca2+]i), electrical activity, membrane potential, formation of InsP3 and insulin release were measured. Addition of ATP in a Ca2(+)-containing medium promoted a rapid rise in [Ca2+]i, which was followed by a slow decline towards the basal level. In a Ca2(+)-free medium, the ATP-induced increase in [Ca2+]i was smaller, but still enough to elicit insulin secretion. Upon normalization of the extracellular Ca2+ concentration, the response to ATP recovered instantaneously. The presence of glucose in the incubation medium was a prerequisite to obtain a pronounced effect of ATP in the absence of extracellular Ca2+. However, glucose did not enhance the response to ATP in a Ca2(+)-containing medium. The effect of ATP was dose-dependent, with a clearly detectable increase in [Ca2+]i at 1 microM and a maximal response being obtained at 200 microM-ATP. The response to ATP was unaffected by activating adenylate cyclase by forskolin, but was abolished by 10 nM of the phorbol ester phorbol 12-myristate 13-acetate. The effects of ATP on [Ca2+]i could not be accounted for by a generalized increase in plasma-membrane permeability, as evident from the failure of the nucleotide to increase the fluorescence of the nuclear stain ethidium bromide. After stimulation with ATP there was an increase in membrane potential, in both the absence and the presence of extracellular Ca2+. Blockage of the voltage-activated Ca2+ channals with D-600, in a Ca2(+)-containing medium, decreased the effect of ATP on [Ca2+]i slightly. Patch-clamp measurements using the cell-attached patch configuration revealed that the RINm5F cells produce spontaneous action potentials, the frequency of which increased markedly on addition of ATP. Whole-cell recordings demonstrated that the increase in spike frequency was not associated with the development of an inward current, but was rather accountable for by a decrease in the activity of the ATP-regulated K+ channels. Addition of 200 microM-ATP stimulated phospholipase C activity, as evident from the formation of InsP3, both in the absence and in the presence of extracellular Ca2+. Thus in the absence of extracellular Ca2+ the stimulatory effect of ATP on insulin release can be explained by InsP3-induced mobilization of intracellularly bound Ca2+. Hence, in the RINm5F cells extracellular ATP acts in a manner similar to other Ca2(+)-mobilizing agents.(ABSTRACT TRUNCATED AT 400 WORDS)     

IgE receptor-mediated phosphatidylinositol hydrolysis and exocytosis from rat basophilic leukemia cells are independent of extracellular Ca2+ in a hypotonic buffer containing a high concentration of K+

In isotonic buffer, IgE receptor-mediated exocytosis from rat basophilic leukemia cells is dependent on extracellular Ca2+, with half-maximal degranulation requiring 0.4 mM Ca2+. No significant exocytosis occurs in the absence of extracellular Ca2+. This absolute requirement for Ca2+ is eliminated by suspending the cells in a hypotonic buffer containing 60 to 80 mM K+; Na+ cannot substitute for K+. Optimal Ca2(+)-independent exocytosis occurs in a buffer containing 20 mM dipotassium Pipes, pH 7.1, 40 mM KCl, 5 mM glucose, 7 mM Mg acetate, 0.1% BSA, and 1 mM EGTA. The cells maintain this Ca2(+)-independent exocytosis even if they are preincubated with 1 mM EGTA for 40 min at 37 degrees C before triggering. Exocytosis is eliminated as isotonicity is approached by adding sucrose, NaCl, KCl, or potassium glutamate to the buffer. Quin 2 fluorescence measurements reveal only a very small rise in [Ca2+]i when the cells are triggered in hypotonic buffer in the absence of extracellular Ca2+ and the presence of 1 mM EGTA. In isotonic buffer, degranulation does not occur under conditions that lead to such a small rise in [Ca2+]i. Sustained IgE receptor-mediated phosphatidylinositol hydrolysis, which is also Ca2+ dependent in isotonic buffer, becomes independent of Ca2+ in the hypotonic buffer. In fact, the rate of phosphatidylinositol hydrolysis in hypotonic buffer in the absence of Ca2+ (and presence of 1 mM EGTA) is twice that observed in isotonic buffer in the presence of 1 mM Ca2+. These data show that in hypotonic buffer, the requirement of IgE receptor-mediated PI hydrolysis for extracellular Ca2+ is eliminated, and degranulation proceeds with a [Ca2+]i of 0.1 microM, the baseline level of [Ca2+]i found in resting cells. These results are consistent with the hypothesis that, in isotonic buffer, the Ca2+ requirement for mast cell degranulation is for the generation of second messengers via hydrolysis of membrane phosphatidylinositols.     

Gastrin induces intracellular Ca2+ release and acid secretion regulation by the microtubular-microfilamentous system

In the absence of extracellular Ca2+ gastrin induced accumulation of amino [14C]pyrine as an index of acid secretion and evoked a cytosolic Ca2+ rise (measured by quin2/AM), suggesting that an intracellular Ca2+ release after the activation of the gastrinergic pathway may trigger acid secretion. In isolated parietal cells pretreatment with colchicine or cytochalasin B abolished the amino [14C]pyrine accumulation and the rise in cytosolic Ca2+ concentration evoked by gastrin. The results suggest that the microtubular-microfilamentous system regulates gastrin-induced intracellular Ca2+ release and acid secretion.     

L-type amino acids stimulate gastric acid secretion by activation of the calcium-sensing receptor in parietal cells

Parietal cells are the primary acid secretory cells of the stomach. We have previously shown that activation of the calcium-sensing receptor (CaSR) by divalent (Ca(2+)) or trivalent (Gd(3+)) ions stimulates acid production in the absence of secretagogues by increasing H(+),K(+)-ATPase activity. When overexpressed in HEK-293 cells, the CaSR can be allosterically activated by L-amino acids in the presence of physiological concentrations of extracellular Ca(2+) (Ca(o)(2+); 1.5-2.5 mM). To determine whether the endogenously expressed parietal cell CaSR is allosterically activated by L-amino acids, we examined the effect of the amino acids L-phenylalanine (L-Phe), L-tryptophan, and L-leucine on acid secretion. In ex vivo whole stomach preparations, exposure to L-Phe resulted in gastric luminal pH significantly lower than controls. Studies using D-Phe (inactive isomer) failed to elicit a response on gastric pH. H(+)-K(+)-ATPase activity was monitored by measuring the intracellular pH (pH(i)) of individual parietal cells in isolated rat gastric glands and calculating the rate of H(+) extrusion. We demonstrated that increasing Ca(o)(2+) in the absence of secretagogues caused a dose-dependent increase in H(+) extrusion. These effects were amplified by the addition of amino acids at various Ca(o)(2+) concentrations. Blocking the histamine-2 receptor with cimetidine or inhibiting system L-amino acid transport with 2-amino-2-norbornane-carboxylic acid did not affect the rate of H(+) extrusion in the presence of L-Phe. These data support the conclusion that amino acids, in conjunction with a physiological Ca(o)(2+) concentration, can induce acid secretion independent of hormonal stimulation via allosteric activation of the stomach CaSR.     

Regulation of cytosolic Ca2+ in clonal human muscle cell cultures

Human muscle cells were grown in culture and clonally selected for fusion potential. The concentration of cytoplasmic ionized calcium, [Ca2+]i, was measured in monolayers of fused myotubes using the Ca2+ indicator indo-1. The contributions of independent routes of Ca2+ influx and efflux to/from the cytoplasm on [Ca2+]i were investigated. The resting [Ca2+]i was 170-190 nM in different cell clones. Acetylcholine increased [Ca2+]i by about 2-fold in the presence of absence of extracellular Ca2+. Cell depolarization by K+ elevated [Ca2+]i about 3-fold, and this increase was largely dependent on extracellular Ca2+. Replacing Na+ by N-methylglucammonium+ raised [Ca2+]i greater than 5-fold, and 50% of this increase was dependent on extracellular Ca2+. All these increases in [Ca2+]i were transient, returning to basal [Ca2+]i within 2 min. It is concluded that cells in culture [Ca2+]i can be elevated transiently by acetylcholine through Ca2+ release from intracellular stores, and by K through Ca2+ influx. The return to basal [Ca2+]i is due to Na+/Ca2+ exchange and Ca2+-ATPase activity.     

Tetracaine stimulates insulin secretion through the mobilization of Ca2+ from thapsigargin- and IP3-insensitive Ca2+ reservoir in pancreatic beta-cells

The effect of tetracaine on 45Ca efflux, cytoplasmic Ca2+ concentration [Ca2+]i, and insulin secretion in isolated pancreatic islets and beta-cells was studied. In the absence of external Ca2+, tetracaine (0.1-2.0 mM) increased the 45Ca efflux from isolated islets in a dose-dependentOFF efflux caused by 50 mM K+ or by the association of carbachol (0.2 mM) and 50 mM K+. Tetracaine permanently increased the [Ca2+]i in isolated beta-cells in Ca2+-free medium enriched with 2.8 mM glucose and 25 microM D-600 (methoxiverapamil). This effect was also observed in the presence of 10 mM caffeine or 1 microM thapsigargin. In the presence of 16.7 mM glucose, tetracaine transiently increased the insulin secretion from islets perfused in the absence and presence of external Ca2+. These data indicate that tetracaine mobilises Ca2+ from a thapsigargin-insensitive store and stimulates insulin secretion in the absence of extracellular Ca2+. The increase in 45Ca efflux caused by high concentrations of K+ and by carbachol indicates that tetracaine did not interfere with a cation or inositol triphosphate sensitive Ca2+ pool in beta-cells.     

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.     

Cadmium as a tool for studying calcium-dependent cation permeability of the human red blood cell membrane.

Three Ca(2+)-dependent procedures known to increase cation permeability of red blood cell membranes were tested with Cd2+ ions which equal Ca2+ ions both in their charge and the crystal radius, 1. Increase of non-selective permeability for monovalent cations by incubating the red cells in a Ca(2+)-free sucrose medium. Addition of Cd2+ to the suspension of leaky cells failed to restore the initial impermeability of the red cell membrane while a repairing effect of Ca2+ was evident both in the presence and absence of Cd2+. Thus, in low electrolyte medium, Cd2+ could neither mimic Ca2+, nor prevent the latter from interacting with membrane structures which control cation permeability. 2. Increase of the K(+)-selective permeability by propranolol plus Ca2+. Cd2+ added to a Ca(2+)-free Ringer type medium containing propranolol enhanced K+ permeability similar to that obtained with Ca2+. No changes of membrane permeability could be detected in the presence of 0.5 mmol/l Cd2+ in absence of propranolol. The Cd(2+)-stimulated K+ channels were different from those induced by Ca2+. They proved to be insensitive to quinine, exhibited a low K+/Na+ selectivity, and showed no tendency to self-inactivation. 3. Stimulation of K+ permeability by electron donors plus Ca2+. Substitution of Ca2+ by Cd2+ yielded results similar to those obtained with propranolol. The ability of Cd2+ to overtake the role of Ca2+ appears to depend on the system studied. It supplies information allowing to distinguish between the diverse Ca(2+)-dependent systems in cell membranes.     

Role of calcium in the preparation and secretory activity of cells isolated from the gastric mucosa of rabbits

The role of calcium in the preparation and the acid secretory activity of parietal cells was studied using cells isolated from rabbit gastric mucosa. The preparation of isolated cells was performed by enzymatic dissociation (collagenase) in the presence of EDTA; without EDTA, only isolated gastric glands were obtained. The acid secretory activity of parietal cells was determined by the 14C-aminopyrine accumulation method; the stimulation induced by histamine or isobutylmethylxanthine (IBMX) was not significantly affected by a reduction of extracellular Ca2+ level (20% diminution in a Ca2+-free medium). The carbachol induced stimulation was highly dependent upon the concentration of extracellular Ca2+: incubation of parietal cells in a Ca2+-free medium reduced the response to 100 microM carbachol by about 60%.     

Effects of Ca2+ on the sodium pump observed in cardiac myocytes isolated from guinea pigs

It is presently unknown whether Ca2+ plays a role in the physiological control of Na+/K+-ATPase or sodium pump activity. Because the enzyme is exposed to markedly different intra- and extracellular Ca2+ concentrations, tissue homogenates or purified enzyme preparations may not provide pertinent information regarding this question. Therefore, the effects of Ca2+ on the sodium pump were examined with studies of [3H]ouabain binding and 86Rb+ uptake using viable myocytes isolated from guinea-pig heart and apparently maintaining ion gradients. In the presence of K+, a reduction of the extracellular Ca2+ increased specific [3H]ouabain binding observed at apparent binding equilibria: a half-maximal stimulation was observed when extracellular Ca2+ was lowered to about 50 microM. The change in [3H]ouabain binding was caused by a change in the number of binding sites accessible by ouabain instead of a change in their affinity for the glycoside. Ouabain-sensitive 86Rb+ uptake was increased by a reduction of extracellular Ca2+ concentration. Benzocaine in concentrations reported to reduce the rate of Na+ influx failed to influence the inhibitory effect of Ca2+ on glycoside binding. When [3H]ouabain binding was at equilibrium, the addition of Ca2+ decreased and that of EGTA increased the glycoside binding. Mn2+, which does not penetrate the cell membrane, had effects similar to Ca2+. In the absence of K+, cells lose their tolerance to Ca2+. Reducing Ca2+ concentration prevented the loss of rod-shaped cells but failed to affect specific [3H]ouabain binding observed in the absence of K+. These results indicate that a large change in extracellular Ca2+ directly affects the sodium pump in cardiac myocytes isolated from guinea pigs.     

Novel role for K+-dependent Na+/Ca2+ exchangers in regulation of cytoplasmic free Ca2+ and contractility in arterial smooth muscle

Cytoplasmic free Ca2+ ([Ca2+]cyt) is essential for the contraction and relaxation of blood vessels. The role of plasma membrane Na+/Ca2+ exchange (NCX) activity in the regulation of vascular Ca2+ homeostasis was previously ascribed to the NCX1 protein. However, recent studies suggest that a relatively newly discovered K+-dependent Na+/Ca2+ exchanger, NCKX (gene family SLC24), is also present in vascular smooth muscle. The purpose of the present study was to identify the expression and function of NCKX in arteries. mRNA encoding NCKX3 and NCKX4 was demonstrated by RT-PCR and Northern blot in both rat mesenteric and aortic smooth muscle. NCXK3 and NCKX4 proteins were also demonstrated by immunoblot and immunofluorescence. After voltage-gated Ca2+ channels, store-operated Ca2+ channels, and Na+ pump were pharmacologically blocked, when the extracellular Na+ was replaced with Li+ (0 Na+) to induce reverse mode (Ca2+ entry) activity of Na+/Ca2+ exchangers, a large increase in [Ca2+]cyt signal was observed in primary cultured aortic smooth muscle cells. About one-half of this [Ca2+]cyt signal depended on the extracellular K+. In addition, after the activity of NCX was inhibited by KB-R7943, Na+ replacement-induced Ca2+ entry was absolutely dependent on extracellular K+. In arterial rings denuded of endothelium, a significant fraction of the phenylephrine-induced and nifedipine-resistant aortic or mesenteric contraction could be prevented by removal of extracellular K+. Taken together, these data provide strong evidence for the expression of NCKX proteins in the vascular smooth muscle and their novel role in mediating agonist-stimulated [Ca2+]cyt and thereby vascular tone.