Effects of Cl- channel blockers on endothelin-1-induced proliferation of rat vascular smooth muscle cells

The effects of Cl- channel blockers on endothelin-1 (ET-1)-induced proliferation of rat aortic vascular smooth muscle cells (VSMC) were examined. We found ET-1 concentration-dependently increased cell count and [3H]-thymidine incorporation into VSMC, with EC50 values of 24.8 and 11.4 nM, respectively. Both nifedipine and SK&F96365 inhibited 10 nM ET-1-induced [3H]-thymidine incorporation into VSMC with the maximal inhibitory concentrations of 1 and 10 microM, respectively. DIDS inhibited 10 nM ET-1-induced increase in cell count and [3H]-thymidine incorporation into VSMC in a concentration-dependent manner, whereas other Cl- channel blockers including IAA-94, NPPB, DPC, SITS and furosemide did not produce these effects. 3 microM DIDS reduced 10 nM ET-1-induced sustained increase in cytoplasmic Ca2+ concentration ([Ca2+]) by 52%. Pretreatment of VSMC with 1 microM nifedipine completely inhibited the DIDS effect on 10 nM ET-1-induced [3H]-thymidine incorporation into VSMC and sustained increase in [Ca2+]i, whereas pretreatment with 10 microM SK&F96365 did not completely block these effects of DIDS. DIDS did not affect ET-1-induced Ca2+ release and 30 mM KCl-induced increase in [Ca2+]i. Our data suggest that DIDS-sensitive Cl- channels mediate VSMC proliferation induced by ET-1 by mechanisms related to membrane depolarization and Ca2+ influx through voltage-dependent Ca2+ channels.     

Contribution of external and internal Ca2+ to changes in intracellular free Ca2+ produced by mitogens in Swiss 3T3 fibroblasts: the role of dihydropyridine sensitive Ca2+ channels

Changes in intracellular free Ca2+ concentration [( Ca2+]i) produced by growth factors and mitogens have been studied using aequorin-loaded Swiss 3T3 cells. Decreasing free Ca2+ in the external medium by using EGTA had no significant effect on the increase in [Ca2+]i produced by vasopressin, bradykinin, bombesin or prostaglandin E2, but reduced the increase in [Ca2+]i produced by platelet derived growth factor (PDGF) by 58%, by prostaglandin E1 44% and by prostaglandin F2 alpha 47%. The dihydropyridine Ca2+-channel antagonist nifedipine at 10 microM inhibited the [Ca2+]i response to PDGF by 41% in both the presence of and in the absence of external Ca2+. Methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl) pyridine-5-carboxylate (BAY K8644), a Ca2+-channel agonist, at 10 microM produced an increase in [Ca2+]i and decreased the [Ca2+]i response to PDGF by 39%. Nifedipine did not block 45Ca2+ uptake or release by inositol 1,4,5-trisphosphate in saponin-permeabilized Swiss 3T3 fibroblasts but BAY K8644 inhibited 45Ca2+ release by inositol 1,4,5-trisphosphate. The results suggest that the increase in [Ca2+]i caused by PDGF in Swiss 3T3 fibroblasts is due to the influx of external Ca2+ through dihydropyridine sensitive Ca2+ channels, as well as release of internal Ca2+.     

Novel effect of CP55,940, a CB1/CB2 cannabinoid receptor agonist,on intracellular free Ca2+ levels in bladder cancer cells

The study was undertaken to explore the effect of CP55,940 ((-)-cis-3-[2-Hydroxy4-(1,1-dimethylheptyl) phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol), a drug commonly used as a CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca2+ levels ([Ca2+]i) in several cell types [Ca2+]i was measured in suspended cells by using the fluorescent dye fura-2 as an indicator. At concentrations between 1-50 microM, CP55,940 increased [Ca2+]i in a concentration-dependent manner with an EC50 of 8 microM. The [Ca2+]i signal comprised an initial rise, a slow decay, and a sustained phase. CP55940 (10 microM)-induced (Ca2+]i signal was not altered by 5 microM of two cannabinoid receptor antagonists (AM-251, N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; AM-281, 1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-m3thyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide). Extracellular Ca2+ removal decreased the maximum value of the Ca2+ signals by 50%. CPS5,940 (10 microM)-induced [Ca2+]i increase in Ca2+-free medium was inhibited by 80% by pretreatment with 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 10 microM CP55,940 in Ca2+-free medium for 6 min abolished thapsigargin-induced [Ca2+]i increase. Nifedipine (10 microM) and verapamil (10 microM) did not alter CP55,940 (10 microM)-induced [Ca2+]i increase. CP55, 940 (10 microM)-induced Ca2+ release was not affected when phospholipase C was inhibited by 2 microM U73122 (1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione). CP55,940 (5 microM) also increased [Ca22+] in Madin-Darby canine kidney cells, MG63 human osteosarcoma cells, and IMR-32 neuroblastoma cells. Collectively, CP,55940 induced significant [Ca2+]i increases in several cell types by releasing store Ca2+ from thapsigargin-sensitive pools and by causing Ca2+ entry. The CP55,940's action appears to be dissociated from stimulation of cannabinoid receptors     

Intracellular calcium release is required for caspase-3 and -9 activation

Increase in intracellular Ca2+ [Ca2+]i regulates many biological functions including apoptosis, but the protein(s) linking [Ca2+]i and apoptosis are not completely understood. We have previously shown that IP3R-deficient cells are resistant to T-cell receptor (TCR)-induced apoptosis due to lack of Ca2+ release from endoplasmic reticulum (ER) and calcineurin activation. Here we show that caspase-9 and -3 are not activated in IP3R-deficient cells after TCR stimulation, consistent with the resistance of these cells to apoptosis. However, we also demonstrate that Bcl-2 expression in IP3R-deficient cells is comparable to control cells. Taken together, these results strongly suggest that IP3R-mediated Ca2+ release plays a critical role in regulating the activity of caspases-3 and -9 independent of Bcl-2.     

Diospyrin derivative, an anticancer quinonoid, regulates apoptosis at endoplasmic reticulum as well as mitochondria by modulating cytosolic calcium in human breast carcinoma cells

Diospyrin diethylether (D7), a bisnaphthoquinonoid derivative, exhibited an oxidative stress-dependent apoptosis in several human cancer cells and tumor models. The present study was aimed at evaluation of the increase in cytosolic calcium [Ca(2+)](c) leading to the apoptotic cell death triggered by D7 in MCF7 human breast carcinoma cells. A phosphotidylcholine-specific phospholipase C (PC-PLC) inhibitor, viz. U73122, and an antioxidant, viz. N-acetylcysteine, could significantly prevent the D7-induced rise in [Ca(2+)](c) and PC-PLC activity. Using an endoplasmic reticulum (ER)-Ca(2+) mobilizer (thapsigargin) and an ER-IP3R antagonist (heparin), results revealed ER as a major source of [Ca(2+)](c) which led to the activation of calpain and caspase12, and cleavage of fodrin. These effects including apoptosis were significantly inhibited by the pretreatment of Bapta-AM (a cell permeable Ca(2+)-specific chelator), or calpeptin (a calpain inhibitor). Furthermore, D7-induced [Ca(2+)](c) was found to alter mitochondrial membrane potential and induce cytochrome c release, which was inhibited by either Bapta-AM or ruthenium red (an inhibitor of mitochondrial Ca(2+) uniporter). Thus, these results provided a deeper insight into the D7-induced redox signaling which eventually integrated the calcium-dependent calpain/caspase12 activation and mitochondrial alterations to accentuate the induction of apoptotic cell death.     

Interrelationships of platelet-derived growth factor isoform-induced changes in c-fos expression, intracellular free calcium, and mitogenesis

Both increases in c-fos proto-oncogene expression and intracellular free calcium ([Ca2+]i) have been implicated as necessary components of the signal transduction pathway by which platelet-derived growth factor (PDGF) stimulates DNA synthesis in cultured BALB/c3T3 fibroblasts. To determine the interrelationship between PDGF-induced increases in c-fos proto-oncogene expression and [Ca2+]i, purified, recombinant BB and AA homodimeric isoforms of PDGF were used to evaluate the dose-response relationships and mechanisms of growth factor-induced changes in these two parameters as well as DNA synthesis. Concentration-dependent increases in [Ca2+]i, c-fos expression, and [3H]thymidine incorporation were observed with both BB and AA PDGF isoforms. BB PDGF was consistently more potent and efficacious than the AA isoform in eliciting a given response. The [Ca2+]i dependency of PDGF-induced increases in c-fos expression and DNA synthesis was determined by pretreatment of cells with agents that inhibit increases in [Ca2+]i: BAPTA, Quin-2, and TMB-8. Under these conditions, PDGF-induced DNA synthesis was blocked, whereas c-fos expression was enhanced. Conversely, in cells made deficient in protein kinase C (PKC) activity by prolonged treatment with phorbol ester, BB and AA PDGF-induced c-fos expression was inhibited by 75-80%, while PDGF-induced increases in [Ca2+]i and DNA synthesis were unaffected or enhanced. Additionally, the PKC-independent component of PDGF-stimulated c-fos expression was found to be independent of increases in [Ca2+]i. These data suggest that 1) both BB and AA PDGF isoforms elicit alterations in [Ca2+]i and c-fos proto-oncogene expression through the same or similar mechanisms in BALB/c3T3 fibroblasts, 2) PDGF-stimulated increases in [Ca2+]i are not required for c-fos expression, and 3) distinct pathways regulate PDGF-induced c-fos expression and mitogenesis, with c-fos expression being substantially PKC-dependent yet [Ca2+]i-independent, while mitogenesis is [Ca2+]i-dependent yet PKC-independent.     

Calcium antagonists cause dry mouth by inhibiting resting saliva secretion

Ca2+ antagonists cause dry mouth by inhibiting saliva secretion. The present study was undertaken to elucidate the mechanism by which Ca2+ antagonists cause dry mouth. Since the intracellular Ca2+ concentration ([Ca2+]i) is closely related to saliva secretion, [Ca2+]i was measured with a video-imaging analysis system by using human submandibular gland (HSG) cells as the material. The Ca2+ antagonist, nifedipine, inhibited the elevation in [Ca2+]i induced by 1-10 microM carbachol (CCh), but had no inhibitory effect on that induced by 30 and 100 microM CCh. The other kinds of Ca2+ antagonists, verapamil (10 microM), diltiazem (10 microM), and the inorganic Ca2+ channel blocker, CdCl2 (50 microM), also inhibited the [Ca2+]i elevation induced by 10 microM CCh. The Ca2+ channel activator, Bay K 8644 (5 microM), significantly enhanced the CCh (10 microM)-induced [Ca2+]i elevation. Endothelin-1 and norepinephrine also increased the CCh (10 microM)-induced [Ca2+]i elevation. SKF-96365 reversed the enhancement of the CCh (10 microM)-induced [Ca2+]i elevation caused by AlF4- and phenylephrine. The phospholipase Cbeta (PLCbeta) inhibitor, U-73122 (5 microM), significantly inhibited the [Ca2+]i elevation induced by 100 microM CCh compared with that induced by 10 microM CCh, while the PLCbeta activator, m-3M3FBS (20 microM), significantly increased the [Ca2+]i elevation induced by 100 microM CCh compared with that induced by 10 microM CCh. We therefore conclude that non-selective cation and voltage-dependent Ca2+ channels are involved in resting salivation and that Ca2+ antagonists depress H2O secretion by blocking the Ca2+ channels and thereby cause dry mouth.     

Calcium-Mediated Mitochondrial Permeability Transition Involved in Hydrogen Peroxide-Induced Apoptosis in Tobacco Protoplasts

In the present study, we focused on whether Intracellular free Ca^2＋ （[Ca^2＋],） regulates the formation of mltochondrlal permeability transition pore （MPTP） In H2O2-induced apoptosis In tobacco protoplasts. It was shown that the decrease In mltochondrlal membrane potential （△ψm） preceded the appearance of H2O2-Induced apoptosls; pretreatment with the specific MPTP Inhibitor cyclosporine A, which also Inhibits Ca^2＋ cycling by the mitochondria, effectively retarded apoptosls and the decrease In △ψm. Apoptosls and decreased △ψm were exacerbated by CaCl2, whereas the plasma membrane voltage-dependent Ca^2＋ channel blocker lanthanum chloride （LaCl3） attentuated these responses. Chelation of extracellular Ca^2＋ with EGTA almost totally Inhibited apoptosls and the decrease In △ψmInduced by H2O2. The time-course of changes In [Ca^2＋]l In apoptosls was detected using the Ca^2＋ probe Fiuo-3 AM. These studies showed that [Ca^2＋]1 was Increased at the very early stage of H2O2-Induced apoptosls. The EGTA evidently Inhibited the Increase In [Ca^2＋]1 Induced by H=O=, whereas It was only partially Inhibited by LaCl3. The results suggest that H2O2 may elevate cytoplasmic free Ca^2＋ concentrations In tobacco protoplasts, which mainly results from the entry of extracellular Ca^2＋, to regulate mltochondrlal permeability transition. The signaling pathway of [Ca^2＋]1-medlated mltochondrlal permeability transition was associated with H2O2-Induced apoptosis In tobacco protoplaete.     

Lipoxin A4 stimulates a cytosolic Ca2+ increase in human bronchial epithelium

Lipoxins are biologically active eicosanoids possessing anti-inflammatory properties. Using a calcium imaging system we investigated the effect of lipoxin A(4) (LXA(4)) on intracellular [Ca(2+)] ([Ca(2+)](i)) of human bronchial epithelial cell. Exposure of the cells to LXA(4) produced a dose-dependent increase in [Ca(2+)](i) followed by a recovery to basal values in primary culture and in 16HBE14o(-) cells. The LXA(4)-induced [Ca(2+)](i) increase was completely abolished after pre-treatment of the 16HBE14o(-) cells with pertussis toxin (G-protein inhibitor). The [Ca(2+)](i) response was not affected by the removal of external [Ca(2+)] but completely inhibited by thapsigargin (Ca(2+)-ATPase inhibitor) treatment. Pre-treatment of the bronchial epithelial cells with either MDL hydrochloride (adenylate cyclase inhibitor) or (R(p))-cAMP (cAMP-dependent protein kinase inhibitor) inhibited the Ca(2+) response to LXA(4). However, the response was not affected by chelerytrine chloride (protein kinase C inhibitor) or montelukast (cysteinyl leukotriene receptor antagonist). The LXA(4) receptor mRNA was detected, by RT-PCR, in primary culture of human bronchial epithelium and in immortalized 16HBE14o(-) cells. The functional consequences of the effect of LXA(4) on intracellular [Ca(2+)](i) have been investigated on Cl(-) secretion, measured using the short-circuit techniques on 16HBE14o(-) monolayers grown on permeable filters. LXA(4) produced a sustained stimulation of the Cl(-) secretion by 16HBE14o(-) monolayers, which was inhibited by BAPTA-AM, a chelator of intracellular calcium. Taken together our results provided evidence for the stimulation of a [Ca(2+)](i) increase by LXA(4) through a mechanism involving its specific receptor and protein kinase A activation and resulting in a subsequent Ca(2+)-dependent Cl(-) secretion by human airway epithelial cells.     

Effect of (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol (CP55,940) on intracellular Ca2+ levels in human osteosarcoma cells

The study was undertaken to explore the effect of CP55,940 ((-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol), a drug commonly used as a CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca2+ levels ([Ca2+]i) in MG63 human osteoblast-like epithelial cells. [Ca2+]i was measured in suspended cells by using the fluorescent dye fura-2 as an indicator. At concentrations between 2-20 microM, CP55,940 increased [Ca2+]i in a concentration-dependent manner with an EC50 of 8 microM. The [Ca2+] signal comprised an initial rise, a slow decay, and a sustained phase. CP55940 (10 microM)-induced [Ca2+]i signal was not altered by 5 microM of two cannabinoid receptor antagonists (AM-251, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole3-carboxamide; AM-281, 1-(2,4-Dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide). Extracellular Ca2+ removal decreased the maximum value of the Ca2+ signals by 50%. CP55,940 induced quench of fura-2 fluorescence by Mn2+ (50 microM), suggesting the presence of Ca2+ influx across the plasma membrane. CP55,940 (10 microM)-induced [Ca2+]i increase in Ca(2+)-free medium was inhibited by 84% by pretreatment with 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 10 microM CP55,940 in Ca(2+)-free medium abolished thapsigargin-induced [Ca2+]i increase. At 1 microM, nifedipine, verapamil, and diltiazem did not alter CP55, 940 (10 microM)-induced [Ca2+]i increase. CP55,940 (20 microM)-induced Ca2+ release was not affected when phospholipase C was inhibited by 2 microM U73122 (1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino) hexyl)-1H-pyrrole-2,5-dione). CP55,940 (20 microM) did not induce acute cell death after incubation for 30 min as assayed by trypan blue exclusion. Collectively, CP55,940 induced significant [Ca2+]i increases in osteoblasts by releasing store Ca2+ from thapsigargin-sensitive stores and by causing Ca2+ entry. The CP55,940's action appears to be independent of stimulation of CB1 cannabinoid receptors.     

Protease-activated receptor-1-induced calcium signaling in gingival fibroblasts is mediated by sarcoplasmic reticulum calcium release and extracellular calcium influx

Thrombin is a serine protease activated during injury and inflammation. Thrombin and other proteases generated by periodontal pathogens affect the behavior of periodontal cells via activation of protease-activated receptors (PARs). We noted that thrombin and PAR-1 agonist peptide stimulated intracellular calcium levels ([Ca2+]i) of gingival fibroblasts (GF). This increase of [Ca2+]i was inhibited by EGTA and verapamil. U73122 and neomycin inhibited thrombin- and PAR-1-induced [Ca2+]i. Furthermore, 2-APB (75-100 microM, inositol triphosphate [IP3] receptor antagonist), thapsigargin (1 microM), SKF-96365 (200 microM) and W7 (50 and 100 microM) also suppressed the PAR-1- and thrombin-induced [Ca2+]i. However, H7 (100, 200 microM) and ryanodine showed little effects. Blocking Ca2+ efflux from mitochondria by CGP37157 (50, 100 microM) inhibited both thrombin- and PAR-1-induced [Ca2+]i. Thrombin induced the IP3 production of GF within 30-seconds of exposure, which was inhibited by U73122. These results indicate that mitochondrial calcium efflux and calcium-calmodulin pathways are related to thrombin and PAR-1 induced [Ca2+]i in GF. Thrombin-induced [Ca2+]i of GF is mainly due to PAR-1 activation, extracellular calcium influx via L-type calcium channel, PLC activation, then IP3 binding to IP3 receptor in sarcoplasmic reticulum, which leads to intracellular calcium release and subsequently alters cell membrane capacitative calcium entry.     

CP55,940 increases intracellular Ca2+ levels in Madin-Darby canine kidney cells

The effect of CP55,940, a presumed CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca2+ levels ([Ca2+]i) in Madin-Darby canine kidney cells was examined by using the fluorescent dye fura-2 as a Ca2+ indicator. CP55,940 (2-50 microM) increased [Ca2+]i concentration-dependently with an EC50 of 8 microM. The [Ca2+]i signal comprised an initial rise and a sustained phase. Extracellular Ca2+ removal decreased the maximum [Ca2+]i signals by 32+/-12%. CP55,940 (20 microM)-induced [Ca2+]i signal was not altered by 5 microM of two cannabinoid receptor antagonists, AM-251 and AM-281. CP55,940 (20 microM)-induced [Ca2+]i increase in Ca2+-free medium was inhibited by 86+/-3% by pretreatment with 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 20 microM CP55,940 in Ca2+-free medium for 6 min abolished thapsigargin-induced [Ca2+]i increases. CP55,940 (20 microM)-induced intracellular Ca2+ release was not inhibited when inositol 1,4,5-trisphosphate formation was abolished by suppressing phospholipase C with 2 microM U73122. Collectively, this study shows that CP,55940 induced significant [Ca2+]i increases in canine renal tubular cells by releasing stored Ca2+ from the thapsigargin-sensitive pools in an inositol 1,4,5-trisphosphate-independent manner, and also by causing extracellular Ca2+ entry. The CP55,940's action appears to be dissociated from stimulation of cannabinoid receptors.     

Inhibition of prostaglandin E1-induced elevation of cytoplasmic free calcium ion by protein kinase C-activating phorbol esters and diacylglycerol in Swiss 3T3 fibroblasts

In quiescent cultures of Swiss 3T3 cells, prostaglandin E1 (PGE1) known to elevate cAMP increased rapidly cytoplasmic free Ca2+ concentration ([Ca2+]i) as measured with the fluorescent Ca2+ indicator quin2. The primary source of the PGE1-induced elevation of [Ca2+]i was extracellular. Pretreatment of the cells with various doses of 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein kinase C-activating phorbol ester, inhibited the PGE1-induced elevation of [Ca2+]i in a dose-dependent manner. Inversely, TPA enhanced slightly the PGE1-induced increase of cAMP. TPA alone did not affect the basal level of [Ca2+]i or cAMP in the absence of PGE1. The inhibitory action of TPA on the PGE1-induced elevation of [Ca2+]i was mimicked by other protein kinase C-activating agents such as phorbol 12,13-dibutyrate and 1-oleoyl-2-acetylglycerol. 4 alpha-Phorbol 12,13-didecanoate known to be inactive for protein kinase C was ineffective in this capacity. Prolonged treatment of the cells with phorbol 12,13-dibutyrate resulted in the down-regulation and disappearance of protein kinase C. In these protein kinase C-deficient cells, PGE1 still elevated [Ca2+]i to the same extent as that in the control cells, but TPA did not inhibit the PGE1-induced elevation of [Ca2+]i. These results strongly suggest that protein kinase C serves as an inhibitor for PGE1-induced Ca2+ influx in Swiss 3T3 cells.     

Inhibition of endothelium-dependent vasorelaxation by extracellular K(+): a novel controlling signal for vascular contractility

The effects of extracellular K+ on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) were examined in mouse aorta, mouse aorta endothelial cells (MAEC), and human umbilical vein endothelial cells (HUVEC). In mouse aortic rings precontracted with prostaglandin F2alpha or norepinephrine, an increase in extracellular K+ concentration ([K+]o) from 6 to 12 mM inhibited EDR concentration dependently. In endothelial cells, an increase in [K+]o inhibited the agonist-induced [Ca2+]i increase concentration dependently. Similar to K+, Cs+ also inhibited EDR and the increase in [Ca2+]i concentration dependently. In current-clamped HUVEC, increasing [K+]o from 6 to 12 mM depolarized membrane potential from -32.8 +/- 2.7 to -8.6 +/- 4.9 mV (n = 8). In voltage-clamped HUVEC, depolarizing the holding potential from -50 to -25 mV decreased [Ca2+]i significantly from 0.95 +/- 0.03 to 0.88 +/- 0.03 microM (n = 11, P < 0.01) and further decreased [Ca2+]i to 0.47 +/- 0.04 microM by depolarizing the holding potential from -25 to 0 mV (n = 11, P < 0.001). Tetraethylammonium (1 mM) inhibited EDR and the ATP-induced [Ca2+]i increase in voltage-clamped MAEC. The intermediate-conductance Ca2+-activated K+ channel openers 1-ethyl-2-benzimidazolinone, chlorozoxazone, and zoxazolamine reversed the K+-induced inhibition of EDR and increase in [Ca2+]i. The K+-induced inhibition of EDR and increase in [Ca2+]i was abolished by the Na+-K+ pump inhibitor ouabain (10 microM). These results indicate that an increase of [K+]o in the physiological range (6-12 mM) inhibits [Ca2+]i increase in endothelial cells and diminishes EDR by depolarizing the membrane potential, decreasing K+ efflux, and activating the Na+-K+ pump, thereby modulating the release of endothelium-derived vasoactive factors from endothelial cells and vasomotor tone.     

Effect of m-3m3FBS on Ca2+ handling and viability in OC2 human oral cancer cells

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca2+ concentrations ([Ca2+]i) in OC2 human oral cancer cells is unclear. This study explored whether m-3M3FBS changed basal [Ca2+]i levels in suspended OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. M-3M3FBS at concentrations between 10-60 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. M-3M3FBS-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, and by the phospholipase A2 inhibitor aristolochic acid. In Ca2+-free medium, 30 μM m-3M3FBS pretreatment inhibited the [Ca2+]i rise induced by the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin and 2,5-di-tert-butylhydroquinone (BHQ). Conversely, pretreatment with thapsigargin, BHQ or cyclopiazonic acid partly reduced m-3M3FBS-induced [Ca2+]i rise. Inhibition of inositol 1,4,5-trisphosphate formation with U73122 did not alter m-3M3FBS-induced [Ca2+]i rise. At concentrations between 5 and 100 μM m-3M3FBS killed cells in a concentration-dependent manner. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Propidium iodide staining data suggest that m-3M3FBS (20 or 50 μM) induced apoptosis in a Ca2+-independent manner. Collectively, in OC2 cells, m-3M3FBS induced [Ca2+]i rise by causing inositol 1,4,5-trisphosphate-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-sensitive store-operated Ca2+ channels. M-3M3FBS also induced Ca2+-independent cell death and apoptosis.     

A mechanism of Ca2+ release from Ca2+ stores coupling to the Na+/Ca2+ exchanger in cultured smooth muscle cells.

We previously observed Ca2+ release from intracellular Ca2+ stores caused by reduction in extracellular Na+ concentration ([Na+]o). The purpose of this study was to determine whether lowering [Na+]o can elicit Ca2+ release from Ca2+ stores via the Na+/Ca2+ exchanger and to elucidate the mechanisms related to the Ca2+ release pathway in cultured longitudinal smooth muscle cells obtained from guinea pig ileum. Low [Na+]o-induced Ca2+ release was inhibited by antisense oligodeoxynucleotides for Na+/Ca2+ exchanger type 1 (anti-NCX). Application of anti-NCX to cells attenuated both the number of Ca2+ responding cells and the expression of the exchanger. Moreover, microinjection of heparin, a blocker of inositol 1,4,5-trisphosphate (IP3) receptors, into the cells inhibited low [Na+]o-induced Ca2+ release. These findings suggest that low [Na+]o-induced Ca2+ release occurs through an IP3-induced Ca2+ release mechanism due to changes in the Ca2+ flux regulated by the Na+/Ca2+ exchanger.