Extracellular Ca²+ alleviates NaCl-induced stomatal opening through a pathway involving H₂O₂-blocked Na+ influx in Vicia guard cells

To gain further insights into the function of extracellular Ca2+ in alleviating salt stress, Vicia faba guard cell protoplasts (GCPs) were patch-clamped in a whole-cell configuration. The results showed that 100 mM NaCl clearly induced Na+ influx across the plasma membrane in GCPs and promoted stomatal opening. Extracellular Ca2+ at 10 mM efficiently blocked Na+ influx and inhibited stomatal opening, which was partially abolished by La3+ (an inhibitor of plasma membrane Ca2+ channel) or catalase (CAT, a H?O? scavenger), respectively. These results suggest that the plasma membrane Ca2+ channels and H?O? possibly mediate extracellular Ca2+-blocked Na+ influx in GCPs. Furthermore, extracellular Ca2+ activated the plasma membrane Ca2+ channels under NaCl stress, which was partially abolished by CAT. These results, taken together, indicate that hydrogen peroxide (H?O?) likely regulates Na+ uptake by activating plasma membrane Ca2+ channels in GCPs. In accordance with this hypothesis, H?O? could mimic extracellular Ca2+ to activate Ca2+ channels and block Na+ influx in guard cells. A single-cell analysis of cytosolic free Ca2+ ([Ca2+](cyt)) using Fluo 3-AM revealed that extracellular Ca2+ induced the accumulation of cytosolic Ca2+ under NaCl stress, but had few effects on the accumulation of cytosolic Ca2+ under non-NaCl conditions. All of these results, together with our previous studies showing that extracellular Ca2+ induced the generation of H?O? in GCPs during NaCl stress, indicate that extracellular Ca2+ alleviates salt stress, likely by activating the H?O?-dependent plasma membrane Ca2+ channels, and the increase in cytosolic Ca2+ appears to block Na+ influx across the plasma membrane in Vicia guard cells, leading to stomatal closure and reduction of water loss.     

Cytoprotective effect of reduced glutathione in hydrogen peroxide-induced endothelial cell injury

The kinetic effects of hydrogen peroxide (H2O2) on cultured endothelial cells isolated from bovine carotid artery were studied. The cytoprotective effects of glutathione (GSH) on H2O2-induced cell injury were also investigated. H2O2-induced a dose- and time-dependent cell injury in cultured endothelial cells. H2O2-induced cell injury was blocked by simultaneous treatment by catalase, but not by superoxide dismutase. H2O2 also induced endogenous PGI2 biosynthesis, and the maximum PGI2 production was reached after 1 h treatment. Stimulation of PGI2 production was parallel with arachidonate release from H2O2-treated cells. However the prostaglandin biosynthesis enzyme activity in cells was inhibited by H2O2 treatment. When the cells were treated with GSH, the intracellular GSH reached a plateau after 3 h treatment. Both H2O2-induced cell injury and PGI2 production were significantly inhibited by the 3 h pretreatment with GSH. The cytoprotective effect of GSH was completely inhibited by buthionine sulfoximine which is a specific inhibitor of gamma-glutamylcysteine synthetase. The results indicate that the cytoprotective effect of GSH on H2O2-induced cell injury in cultured bovine carotid artery endothelial cells depends on the increase in intracellular GSH content.     

H2O2-induced changes in mitochondrial activity in isolated mouse pancreatic acinar cells

This study employed confocal laser scanning microscopy to monitor the effect of H2O2 on cytosolic as well as mitochondrial calcium (Ca2+) concentrations, mitochondrial inner membrane potential (psi m) and flavine adenine dinucleotide (FAD) oxidation state in isolated mouse pancreatic acinar cells. The results show that incubation of pancreatic acinar cells with H2O2, in the absence of extracellular Ca2+ ([Ca2+],) led to an increase either in cytosolic and in mitochondrial Ca2+ concentration. Additionally, H2O2 induced a depolarization of mitochondria and increased oxidized FAD level. Pretreatment of cells with the mitochondrial inhibitors rotenone or cyanide inhibited the response induced by H2O2 on mitochondrial inner membrane potential but failed to block oxidation of FAD in the presence of H2O2. However, the H2O2-evoked effect on FAD state was blocked by pretreatment of cells with the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP). On the other hand, perfusion of cells with thapsigargin (Tps), an inhibitor of the SERCA pump, led to an increase in mitochondrial Ca2+ concentration and in oxidized FAD level, and depolarized mitochondria. Pretreatment of cells with thapsigargin inhibited H2O2-evoked changes in mitochondrial Ca2+ concentration but not those in membrane potential and FAD state. The present results have indicated that H2O2 can evoke marked changes in mitochondrial activity that might be due to the oxidant nature of H2O2. This in turn could represent the mechanism of action of ROS to induce cellular damage leading to cell dysfunction and generation of pathologies in the pancreas.     

Relationship between cellular calcium and prostaglandin synthesis in cultured vascular smooth muscle cells

We have investigated the effects of extracellular and intracellular Ca deficits and of pharmacologic agents thought to inhibit Ca influx or intracellular Ca mobilization on vasopressin-evoked changes of cytosolic Ca2+ levels and PG synthesis in cultured rat mesenteric arterial vascular smooth muscle cells. Vasopressin rapidly increased cytosolic Ca2+ as well as PG synthesis. The increase of cytosolic Ca2+ and the rate of PG synthesis were both maximal within the first minute of incubation. An extracellular Ca deficit of short duration partially inhibited both vasopressin-evoked PG synthesis and the increase of cytosolic Ca2+ by 40 to 60%. Two procedures which deplete cells of some of their intracellular Ca, namely a 30 min incubation in EGTA-supplemented, Ca-lacking media, or a 1 min incubation with ionophore A23187 in Ca-deficient media, decreased PG synthesis by 65% to 100%. The addition of extracellular Ca to Ca-depleted cells restored the ability of vasopressin to stimulate PG synthesis. Two Ca channel antagonists, nifedipine or cinnarizine, had no effect on either vasopressin-evoked PG synthesis or increased cytosolic Ca2+, whereas TMB-8 (10 microM), a putative inhibitor of intracellular Ca mobilization, decreased PG synthesis by 75% by inhibiting acylhydrolase as well as cyclo-oxygenase activities, but had no effect on basal or vasopressin-evoked increase of cytosolic Ca2+, documenting that its inhibitory effect was not a consequence of decreased cytosolic Ca2+. These results demonstrate that decreased cellular Ca levels are associated with decreased cytosolic Ca2+ levels and PG synthesis, and support the hypothesis of a link between, on the one hand, cellular Ca and/or cytosolic Ca2+ and on the other hand, PG synthesis.     

Generation of ROS in response to CCK-8 stimulation in mouse pancreatic acinar cells

In the present study we have studied the changes in the intracellular reduction-oxidation state in mouse pancreatic acinar cells following stimulation with cholecystokinin octapeptide (CCK-8) and its dependence on Ca2+ mobilization. In our investigations cytosolic Ca2+ concentration and reactive oxygen species (ROS) production were determined by loading of cells with fura-2 and CM-H2DCF-DA, respectively. Changes in these parameters were determined by following changes in fluorescence in the cuvette of a spectrofluorimeter. The results show that stimulation of cells with CCK-8 and/or the sarco-endoplasmic reticulum Ca2+ pump inhibitor, thapsigargin (Tps), both induced changes in cytosolic free Ca2+ concentration and led to an increase in fluorescence of CM-H2DCF-DA, reflecting an increase in oxidation. In the presence of Tps, addition of CCK-8 did not significantly increase fluorescence compared to that evoked by the SERCA inhibitor. Similar results were obtained in the absence of extracellular Ca2+ and in the presence of EGTA. When the cells were challenged in the presence of the intracellular Ca2+ chelator BAPTA and in the absence of extracellular Ca2+ the responses to both CCK-8 and Tps were reduced although not completely inhibited. The mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone and the inhibitor of the electron transport chain, antimycin, evoked a marked increase in CM-H2DCF-DA fluorescence and completely inhibited CCK-8 and Tps-evoked responses, indicating that ROS are generated in the mitochondria. In summary, stimulation of mouse pancreatic acinar cells with CCK-8 leads to generation of ROS, and this effect may be derived from Ca2+ mobilization from intracellular stores and involves mitochondrial metabolism.     

Decreasing extracellular Na+ concentration triggers inositol polyphosphate production and Ca2+ mobilization

Removing extracellular Na+ (Na+o) evoked a large increase in cytosolic free Ca2+ concentration ([Ca2+]i in human skin fibroblasts. Decreasing [Na+]o from 120 to 14 mM caused the half-maximal peak increase in [Ca2+]i. Removing Na+o strongly stimulated 45Ca2+ efflux and decreased total cell Ca2+ by about 40%. Bradykinin caused changes in [Ca2+]i, total Ca2+, and 45Ca2+ fluxes similar to those evoked by removing Na+o. Prior stimulation of the cells with bradykinin prevented Na+o removal from increasing [Ca2+]i and vice versa. Na+o removal rapidly increased [3H]inositol polyphosphate production. Loading the cells with Na+ had no effect on the increase in 45Ca2+ efflux produced by Na+o removal. Therefore, decreasing [Na+]o probably stimulates a "receptor(s)" which is sensitive to extracellular, not intracellular, Na+. Removing Na+o also mobilized intracellular Ca2+ in smooth muscle and endothelial cells cultured from human umbilical and dog coronary arteries, respectively.     

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.     

1,25-Dihydroxyvitamin D3 increases the toxicity of hydrogen peroxide in the human monocytic line U937: the role of calcium and heat shock

1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) increases synthesis of heat shock proteins in monocytes and U937 cells and protects these cells from thermal injury. We examined whether 1,25-(OH)2D3 would also modulate the susceptibility of U937 cells to H2O2-induced oxidative stress. Cell viability was assessed by trypan blue exclusion and [3H]thymidine incorporation into DNA. Prior incubation for 24 h with 1,25-(OH)2D3 (25 pM or higher) unexpectedly increased H2O2 toxicity. Since cellular Ca2+ may be a mediator of cell injury we investigated effects of altering extracellular Ca2+ ([Ca2+]e) on 1,25-(OH)2D3-enhanced H2O2 toxicity as well as effects of 1,25-(OH)2D3 and H2O2 on cytosolic free Ca2+ concentration ([Ca2+]f). Basal [Ca2+]f in medium containing 1.5 mM Ca as determined by fura-2 fluorescence was higher in 1,25-(OH)2D3-pretreated cells than control cells (137 versus 112 nM, P less than 0.005). H2O2 induced a rapid increase in [Ca2+]f (to greater than 300 nM) in both 1,25-(OH)2D3-treated and control cells, which was prevented by a reduction in [Ca2+]e to less than basal [Ca2+]f. The 1,25(OH)2D3-induced increase in H2O2 toxicity was also prevented by preincubation with 1,25-(OH)2D3 in Ca2+-free medium or by exposing the cells to H2O2 in the presence of EGTA. Preexposure of cells to 45 degrees C for 20 min, 4 h earlier, partially prevented the toxic effects of H2O2 particularly in 1,25-(OH)2D3-treated cells, even in the presence of physiological levels of [Ca2+]e. Thus 1,25-(OH)2D3 potentiates H2O2-induced injury probably by increasing cellular Ca2+ stores. The 1,25-(OH)2D3-induced amplification of the heat shock response likely represents a mechanism for counteracting the Ca2+-associated enhanced susceptibility to oxidative injury due to 1,25-(OH)2D3.     

Requirement of hydrogen peroxide generation in TGF-beta 1 signal transduction in human lung fibroblast cells: involvement of hydrogen peroxide and Ca2+ in TGF-beta 1-induced IL-6 expression

Stimulation of human lung fibroblast cells with TGF-beta1 resulted in a transient burst of reactive oxygen species with maximal increase at 5 min after treatment. This reactive oxygen species increase was inhibited by the antioxidant, N-acetyl-l -cysteine (NAC). TGF-beta1 treatment stimulated IL-6 gene expression and protein synthesis in human lung fibroblast cells. Antioxidants including NAC, glutathione, and catalase reduced TGF-beta1-induced IL-6 gene expression, and direct H2O2 treatment induced IL-6 expression in a dose-dependent manner. NAC also reduced TGF-beta1-induced AP-1 binding activity, which is involved in IL-6 gene expression. It has been reported that Ca2+ influx is stimulated by TGF-beta1 treatment. EGTA suppressed TGF-beta1- or H2O2-induced IL-6 expression, and ionomycin increased IL-6 expression, with simultaneously modulating AP-1 activity in the same pattern. PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase/extracellular signal-related kinase kinase 1, suppressed TGF-beta1- or H2O2-induced IL-6 and AP-1 activation. In addition, TGF-beta1 or H2O2 increased MAPK activity which was reduced by EGTA and NAC, suggesting that MAPK is involved in TGF-beta1-induced IL-6 expression. Taken together, these results indicate that TGF-beta1 induces a transient increase of intracellular H2O2 production, which regulates downstream events such as Ca2+ influx, MAPK, and AP-1 activation and IL-6 gene expression.     

Interleukin-8 primes oxidative burst in neutrophil-like HL-60 through changes in cytosolic calcium

In response to a variety of stimuli, neutrophils release large amount of reactive oxygen species (ROS) generated by NADPH oxidase. This process known as the respiratory burst is dependent on cytosolic free calcium concentration ([Ca(2+)](i)). Proinflammatory cytokines such as interleukin-8 (IL-8) may modulate ROS generation through a priming phenomenon. The aim of this study was to determine the effect of human IL-8 on ROS production in neutrophil-like dimethylsulfoxide-differentiated HL-60 cells (not equalHL-60 cells) and further to examine the role of Ca(2+) mobilization during the priming. IL-8 at 10 nM induced no ROS production but a [Ca(2+)](i) rise (254 +/- 36 nM). IL-8 induced a strongly enhanced (2 fold) ROS release during stimulation with 1 microM of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF). This potentiation of ROS production is dependent of extracellular Ca(2+) (17.0+/-4.5 arbitrary units (A.U.) in the absence of Ca(2+) versus 56.6 +/- 3.9 A.U. in the presence of 1.25 mM of Ca(2+)). Also, IL-8 enhanced fMLF-stimulated increase in [Ca(2+)](i) (375 +/- 35 versus 245 +/- 21 nM, 0.1 microM of fMLF). IL-8 had no effect on not equalHL-60 cells in response to 1 microM of thapsigargin (472 +/- 66 versus 470 +/- 60 nM). In conclusion, Ca(2+) influx is necessary for a full induction of neutrophil priming by IL-8.     

Release of O2- by human umbilical cord blood-derived eosinophils: role of intra- and extracellular calcium.

The aim of our study was to investigate the physiologic mechanisms involved in eosinophil activation as an essential prerequisite to disrupting the biochemical cascade that triggers inflammation, thereby attenuating the effect of this activation or, ideally, preventing it from occurring. We have, therefore, examined the nature of the fMLP- and PAF-induced [Ca2+]i rise and the relationship between the [Ca2+]i rise and O2- production in human umbilical cord blood-derived eosinophils cultured in the presence of IL-3 and IL-5. These cells responded to fMLP or PAF (1 microM each) with an increase in [Ca2+]i (217.3 +/- 22.1 and 197.8 +/- 22.1 nM respectively) which was associated with production of O2- (40.2 +/- 8.2 and 35.2 +/- 7.6 pmol/min/10(6) cells respectively). The role of Ca2+ in the induced respiratory burst was studied by changing the availability of Ca2+ in the intra- and extracellular compartments. Removal or chelation of extracellular Ca2+ induced a reduction of both the fMLP and PAF-induced [Ca2+]i rise and O2- production. Chelation of intracellular Ca2+ induced a concentration-dependent inhibition of fMLP- and PAF-induced [Ca2+]i rise and caused a decrease in O2- production. SK&F 96365 had a stimulatory effect on PAF-induced [Ca2+]i rise and on fMLP-induced O2- production, this phenomenon was not observed with extracellular Ca2+ removal or chelation. Furthermore, Ni2+ exhibited an inhibition of both fMLP and PAF-induced [Ca2+]i rise and O2- production. Finally, both fMLP and PAF induced an increase in divalent cation influx that was further augmented by thapsigargin. Our results indicate that fMLP and PAF dependent O2- production in human eosinophils require intra- and extracellular Ca2+ and that Ca2+ influx is necessary for optimal activation.     

Calcium-dependent,swelling-activated K+ conductance in human neuroblastoma cells

In most mammalian cells, regulatory volume decrease (RVD) is mediated by swelling-activated Cl(-) and K(+) channels. Previous studies in the human neuroblastoma cell line CHP-100 have demonstrated that exposure to hypoosmotic solutions activates Cl(-) channels which are sensitive to Ca(2+). Whether a Ca(2+)-dependent K(+) conductance is activated after cell swelling was investigated in the present studies. Reducing the extracellular osmolarity from 290 to 190 mOsm/kg H(2)O rapidly activated 86Rb effluxes. Hypoosmotic stress also increased cytosolic Ca(2+) in fura-2 loaded cells. Pretreatment with 2.5 mM EGTA and nominally Ca(2+) free extracellular solution significantly decreased the hypoosmotically induced rise in cytosolic Ca(2+) and the swelling-activated 86Rb efflux. In cell-attached patch-clamp studies, decreasing the extracellular osmolarity activated a K(+) conductance that was blocked by Ba(2+). In addition, the swelling-activated K(+) channels were significantly inhibited in the presence of nominally free extracellular Ca(2+) and 2.5mM EGTA. These results suggest that in response to hypoosmotic stress, a Ca(2+)-dependent K(+) conductance is activated in the human neuroblastoma cell line CHP-100.     

Oxidatively stressed lymphocytes remain in G0/G1a on mitogenic stimulation

Thiol modifiers and oxidants inhibit lymphocyte activation. To investigate which of the many cell functions sensitive to oxidation are critical in this inhibition, mouse splenic lymphocytes were treated with oxidants prior to exposure to mitogen, and progression into the cell cycle was assayed. Different treatments were used to chemically dissect different potential targets within the cell: copper phenanthroline (CuP), to oxidize surface sulfhydryls; N-ethyl maleimide (NEM), to alkylate extra- and intracellular thiols; and hydrogen peroxide, which generates the highly reactive hydroxyl radical within the cell. Progression into the cell cycle was assayed with acridine orange (AO) and assays of interleukin-2 (IL-2) production and IL-2 receptor (IL-2R) expression. The contribution of ADP-ribosylation to inhibition of mitogenesis was assessed using 3-aminobenzamide (3AB) to inhibit adenosine 5'-diphosphate (ADP)-ribose transferases. The results indicate that the CuP and NEM treatments both produce two independent inhibitory effects, that is, a failure in the production of and response to IL-2. Cells treated with these compounds were able to progress only through G1a upon mitogenic stimulation. H2O2 had more complex effects. Both ADP-ribosylation and modulations of cytosolic Ca2+ were involved in the inhibitory effects. With lower inhibitory doses of H2O2, lymphocytes were completely unresponsive to mitogen and failed to exit Go upon mitogenic stimulation. If intra- and extracellular Ca2+ were buffered before treatment with H2O2, higher concentrations were required, and under these conditions cells were able to enter G1a but could not progress into G1b. Under neither of these conditions could cells produce IL-2 or express IL-2R.     

Pharmacological inhibition of intracellular calcium release blocks acid-induced bone resorption

In vivo chronic metabolic acidosis induces net Ca2+ efflux from bone, and incubation of neonatal mouse calvariae in medium simulating physiological metabolic acidosis induces bone resorption. It appears that activation of the proton (H+) receptor OGR1 in the osteoblast leads to an increase in intracellular Ca2+, which is associated with an increase in cyclooxygenase 2 (COX2) and PGE2-induced receptor activator of NF-κB ligand (RANKL) and H+-induced osteoclastic bone resorption. To support this hypothesis, we tested whether intracellular Ca2+ signaling was integral to H+-induced bone resorption by determining whether 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) and 2-aminoethoxydiphenyl borate (2-APB), inhibitors of inositol trisphosphate-mediated Ca2+ signaling, would block H+-induced bone resorption in cultured neonatal calvariae and, if so, would do so by inhibiting H+-induced stimulation of COX2 and RANKL in osteoblastic cells. We found that H+-induced bone resorption is significantly inhibited by TMB-8 and 2-APB. Both compounds also inhibit H+-induced stimulation of COX2 protein in calvariae and COX2 mRNA and protein levels in primary osteoblasts. H+-induced stimulation of RANKL in calvarial cultures, as well as primary cells, is also completely inhibited by TMB-8 and 2-APB. These results support the hypothesis that H+ stimulation of net Ca2+ efflux from bone, mediated by COX2- and subsequent PGE2-induced RANKL production, is initiated in the osteoblast via activation of Ca2+ signaling.     

Increase in cytosolic Ca2+ levels through the activation of non-selective cation channels induced by oxidative stress causes mitochondrial depolarization leading to apoptosis-like death in Leishmania donovani promastigotes

Reactive oxygen species are important regulators of protozoal infection. Promastigotes of Leishmania donovani, the causative agent of Kala-azar, undergo an apoptosis-like death upon exposure to H2O2. The present study shows that upon activation of death response by H2O2, a dose- and time-dependent loss of mitochondrial membrane potential occurs. This loss is accompanied by a depletion of cellular glutathione, but cardiolipin content or thiol oxidation status remains unchanged. ATP levels are reduced within the first 60 min of exposure as a result of mitochondrial membrane potential loss. A tight link exists between changes in cytosolic Ca2+ homeostasis and collapse of the mitochondrial membrane potential, but the dissipation of the potential is independent of elevation of cytosolic Na+ and mitochondrial Ca2+. Partial inhibition of cytosolic Ca2+ increase achieved by chelating extracellular or intracellular Ca2+ by the use of appropriate agents resulted in significant rescue of the fall of the mitochondrial membrane potential and apoptosis-like death. It is further demonstrated that the increase in cytosolic Ca2+ is an additive result of release of Ca2+ from intracellular stores as well as by influx of extracellular Ca2+ through flufenamic acid-sensitive non-selective cation channels; contribution of the latter was larger. Mitochondrial changes do not involve opening of the mitochondrial transition pore as cyclosporin A is unable to prevent mitochondrial membrane potential loss. An antioxidant like N-acetylcysteine is able to inhibit the fall of the mitochondrial membrane potential and prevent apoptosis-like death. Together, these findings show the importance of non-selective cation channels in regulating the response of L. donovani promastigotes to oxidative stress that triggers downstream signaling cascades leading to apoptosis-like death.     

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.     

Relationship between cellular calcium and prostaglandin synthesis in cultured vascular smooth muscle cells

We have investigated the effects of extracellular and intracellular Ca deficits and of pharmacologic agents thought to inhibit Ca influx or intracellular Ca mobilization on vasopressin-evoked changes of cytosolic Ca²⁺ levels and PG synthesis in cultured rat mesenteric arterial vascular smooth muscle cells. Vasopressin rapidly increased cytosolic Ca²⁺ as well as PG synthesis. The increase of cytosolic Ca²⁺ and the rate of PG synthesis were both maximal within the first minute of incubation. An extracellular Ca deficit of short duration partially inhibited both vasopressin-evoked PG synthesis and the increase of cytosolic Ca²⁺ by 40 to 60%. Two procedures which deplete cells of some of their intracellular Ca, namely a 30 min incubation in EGIA-supplemented, Ca-lacking media, or a 1 min incubation with ionophore A23187 in Ca-deficient media, decreased PG synthesis by 65% to 100%. The addition of extracellular Ca to Ca-depleted cells restored the ability of vasopressin to stimulate PG synthesis. Two Ca channel antagonists, nifedipine or cinnarizine, had no effect on either vasopressin-evoked PG synthesis or increased cytosolic Ca²⁺, whereas TMB-8 (10 μM), a putative inhibitor of intracellular Ca mobilization, decreased PG synthesis by 75% by inhibiting acylhydrolase as well as cyclo-oxygenase activities, but had no effect on basal or vasopressin-evoked increase of cytosolic Ca²⁺, documenting that its inhibitory effect was not a consequence of decreased cytosolic Ca²⁺.These results demonstrate that decreased cellular Ca levels are associated with decreased cytosolic Ca²⁺ levels and PG synthesis, and support the hypothesis of a link between, on the one hand, cellular Ca and/or cytosolic Ca²⁺ and on the other hand, PG synthesis.     

IL-1beta signaling in cat lower esophageal sphincter circular muscle

In a cat model of acute experimental esophagitis, resting in vivo lower esophageal sphincter (LES) pressure and in vitro tone are lower than in normal LES, and the LES circular smooth muscle layer contains elevated levels of IL-1beta that decrease the LES tone of normal cats. We now examined the mechanisms of IL-1beta-induced reduction in LES tone. IL-1beta significantly reduced acetylcholine-induced Ca(2+) release in Ca(2+)-free medium, and this effect was partially reversed by catalase, demonstrating a role of H(2)O(2) in these changes. IL-1beta significantly increased the production of H(2)O(2), and the increase was blocked by the p38 MAPK inhibitor SB-203580, by the cytosolic phospholipase A(2) (cPLA(2)) inhibitor AACOCF3, and by the NADPH oxidase inhibitor apocynin, but not by the MEK1 inhibitor PD-98059. IL-1beta significantly increased the phosphorylation of p38 MAPK and cPLA(2). IL-1beta-induced cPLA(2) phosphorylation was blocked by SB-203580 but not by AACOCF3, suggesting sequential activation of p38 MAPK-phosphorylating cPLA(2). The IL-1beta-induced reduction in LES tone was partially reversed by AACOCF3 and by the Ca(2+)-insensitive PLA(2) inhibitor bromoenol lactone (BEL). IL-1beta significantly increased cyclooxygenase (COX)-2 and PGE(2) levels. The increase in PGE(2) was blocked by SB-203580, AACOCF3, BEL, and the COX-2 inhibitor NS-398 but not by PD-98059 or the COX-1 inhibitor valeryl salicylate. The data suggested that IL-1beta reduces LES tone by producing H(2)O(2), which may affect Ca(2+)-release mechanisms and increase the synthesis of COX-2 and PGE(2). Both H(2)O(2) and PGE(2) production depend on sequential activation of p38 MAPK and cPLA(2). cPLA(2) activates NADPH oxidases, producing H(2)O(2), and may produce arachidonic acid, converted to PGE(2) via COX-2.     

Glucose increases cytosolic calcium concentration and inositol lipid metabolism in HIT-T15 cells

We have investigated the effects of glucose on cytosolic free calcium concentration in the insulin-secreting cell line HIT-T15. Addition of glucose (10 mM) caused a 20-75% increase in cytosolic [Ca2+] within 5 minutes compared to controls in the absence of glucose. A maximal increase in cytosolic [Ca2+] was obtained with 5 mM glucose. The magnitude of the response was markedly dependent upon the concentration of extracellular Ca2+, and the rise in cytosolic [Ca2+] was inhibited by verapamil. Cytosolic [Ca2+] was greatly increased by depolarization of the cells with KCl (50 mM), whereas carbamylcholine had no apparent effect. Glucose and KCl were also effective in stimulating insulin release from HIT cells, although carbamylcholine was again ineffective. The secretory response to glucose was also found to be directly related to the concentration of extracellular [Ca2+]. Glucose and KCl, but not carbamylcholine, were found to slightly enhance the production of [3H]-inositol trisphosphate in HIT cells pre-labelled with myo-[3H]-inositol, indicating a modest stimulation of inositol lipid hydrolysis.     

The role of Ca2+ in dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells

Cultured Friend cells can be induced by dimethyl sulfoxide (Me2SO) and several other agents to mature along the erythroid pathway. Evidence has been presented that an increase in Ca2+ influx is an early and necessary prelude to the commitment to maturation by these cells (Levenson, R., Housman, D., and Cantley, L. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5948-5952). The simplest hypothesis supporting all the available data is that Me2SO and other inducers elevate the cytosolic Ca2+ concentration. We have now measured cytosolic Ca2+ using the fluorescent indicator quin-2, and find, contrary to expectation, a small decrease upon treatment of cells with Me2SO. Cytosolic Ca2+ was increased by raising the Ca2+ in the medium, but was not dramatically altered by addition of ouabain or monensin or by incubation in Na+-free medium. Measurement of total cell Ca2+ by a triple-labeling technique using 3H2O and 125I-albumin to determine cell water and extracellular space, respectively, revealed no significant change upon treatment with Me2SO for up to 40 h. A decrease in the initial rate of 45Ca2+ influx was observed in Me2SO-treated cells, when measured at 4 degrees C. These data do not support the hypothesis that an increase in cell Ca2+ is necessary for the induction of Friend cell differentiation or that Na+/Ca2+ exchange is a significant regulator of cytosolic Ca2+ in Friend cells.