Regulation of histamine- and UTP-induced increases in Ins(1,4,5)P3, Ins (1,3,4,5)P4 and Ca2+ by cyclic AMP in DDT1 MF-2 cells.

1. Stimulation of P2U-purinoceptors with UTP or histamine H1-receptors with histamine gave rise to the formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) in DDT1 MF-2 smooth muscle cells. 2. Stimulation of P2U-purinoceptors or histamine H1-receptors caused an increase in cytoplasmic Ca2+, consisting of an initial peak, representing the release of Ca2+ from internal stores and a sustained phase representing Ca2+ influx. 3. The P2U-purinoceptor-mediated Ca(2+)-entry mechanism was more sensitive to UTP than Ca(2+)-mobilization (EC50: 3.3 microM +/- 0.4 microM vs 55.1 microM +/- 9.2 microM), in contrast to these processes activated by histamine H1-receptors (EC50: 5.8 microM +/- 0.6 microM vs 3.1 microM +/- 0.5 microM). 4. Pre-stimulation of cells with several adenosine 3':5'-cyclic monophosphate (cyclic AMP) elevating agents, reduced the histamine H1-receptor-mediated formation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Forskolin completely inhibited Ins(1,4,5)P3 formation (IC50: 158 +/- 24 nM) whereas Ins(1,3,4,5)P4 formation was inhibited by only 45% (IC50: 173 +/- 16 nM). The P2U-purinoceptor-mediated production of these inositol phosphates was not affected by cyclic AMP. 5. Forskolin and isoprenaline reduced the histamine-induced increase in cytoplasmic Ca2+, as measured in Ca2+ containing medium and in nominally Ca(2+)-free medium but did not change the UTP-induced increase in cytoplasmic Ca2+. 6. These results clearly demonstrate that cyclic AMP differentially regulates components of the histamine induced phospholipase C signal transduction pathway. Furthermore, cyclic AMP does not affect the phospholipase C pathway activated by stimulation of P2U-purinoceptors in DDT1 MF-2 cells.     

Nordihydroguaiaretic acid elevates osteoblastic intracellular Ca2+

Nordihydroguaiaretic acid (NDGA) is widely used as a pharmacological tool to inhibit lipoxygenases; however, recent evidence suggests that it increases renal intracellular [Ca2+]i via novel mechanisms. Here the effect of NDGA on Ca2+ signaling in MG63 osteoblastic cells was explored using fura-2 as a Ca2+ indicator. NDGA (2-50 microM) increased [Ca2+]i in a concentration-dependent manner. The signal comprised an initial rise and an elevated phase over a time period of 4 min. Removing extracellular Ca2+ reduced 2-50 microM NDGA-induced signals by 62+/-2%. After incubation with 50 microM NDGA in Ca2+-free medium for several minutes, addition of 3 mM CaCl2 induced an increase in [Ca2+]i. NDGA (50 microM)-induced [Ca2+]i increases were not changed by pretreatment with 10 microM of verapamil, diltiazem, nifedipine, nimodipine and nicardipine. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (1 microM) inhibited 50 microM NDGA-induced [Ca2+]i increases by 69+/-3%. Inhibition of phospholipase C with 2 microM U73122 had little effect on 50 microM NDGA-induced Ca2+ release. Several other lipoxygenase inhibitors had no effect on basal [Ca2+]i. At a concentration that did not increase basal [Ca2+]i, NDGA (1 microM) did not alter 10 microM ATP- or 1 microM thapsigargin-induced [Ca2+]i increases. Alteration of protein kinase C activity with 1 nM phorbol 12-myristate 13-acetate or 2 microM GF 109203X did not affect 50 microM NDGA-induced [Ca2+]i increases. Together, the results show that NDGA increased [Ca2+]i in osteoblasts in a lipoxygenase-independent manner, by releasing stored Ca2+ in a fashion independent of phospholipase C activity, and by causing Ca2+ influx.     

Nonylphenol-induced Ca2+ elevation and Ca2+-independent cell death in human osteosarcoma cells

The effect of the environmental toxicant nonylphenol on cytosolic free Ca2+ concentration ([Ca2+]i) and proliferation has not been explored in human osteoblast-like cells. This study examined whether nonylphenol alters Ca2+ levels and causes cell death in MG63 human osteosarcoma cells. [Ca2+]i and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. Nonylphenol at concentrations above 3 microM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 90% by removing extracellular Ca2+. The nonylphenol-induced Ca2+ influx was insensitive to blockade of L-type Ca2+ channel blockers. After pretreatment with 10 microM nonylphenol, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) failed to induce [Ca2+]i rises. Inhibition of phospholipase C with 2 microM U73122 did not change nonylphenol-induced [Ca2+]i rises. The nonylphenol-induced [Ca2+]i rises were enhanced or inhibited by phorbol myristate acetate or GF 109203X, respectively. At concentrations of 10 and 20 microM nonylphenol killed 55% and 100% cells, respectively. The cytotoxic effect of 10 microM nonylphenol was unaltered by pre-chelating cytosolic Ca2+ with BAPTA. Collectively, in MG63 cells, nonylphenol induced [Ca2+]i rises by causing Ca2+ release from intracellular stores and Ca2+ influx from extracellular space. Furthermore, nonylphenol can cause Ca2+-unrelated cytotoxicity in a concentration-dependent manner.     

Effect of the neuroprotective agent riluzole on intracellular Ca2+ levels in IMR32 neuroblastoma cells

Riluzole is an effective neuroprotective drug. Its effect on intracellular free Ca2+ levels ([Ca2+]i) has not been explored. This study examined the effect of riluzole on [Ca2+]i in IMR32 neuroblastoma cells using fura-2 as a Ca2+ probe. Riluzole 0.1-1 mM increased [Ca2+]i in a concentration-dependent manner. Removal of extracellular Ca2+ inhibited the response by 52 +/- 5%. The [Ca2+]i increase induced by 0.2 mM riluzole was unaltered by 0.1 mM La3+ or 10 microM verapamil, but was inhibited by 51 +/- 4% by 10 microM nifedipine. In Ca2+-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) reduced the 0.2 mM riluzole-induced Ca2+ release by 44 +/- 3%; this reduction was augmented to 66 +/- 5% by additionally depleting the Ca2+ stores in the Golgi complex with 50 microM brefeldin A. Inhibition of inositol 1,4,5-trisphosphate formation by 2 microM U73122, a phospholipase C inhibitor, did not affect Ca2+ release induced by 0.2 microM riluzole. It was concluded that the neuroprotective agent riluzole increased [Ca2+]i in IMR32 neuroblastoma cells concentration-dependently by releasing Ca2+ from multiple stores in an inositol 1,4,5-trisphosphate-independent manner and also by inducing nifedipine-sensitive Ca2+ influx.     

Effect of the antianginal drug bepridil on intracellular Ca2+ release and extracellular Ca2+ influx in human neutrophils

To understand more fully the effects of bepridil, an antiarrhythmic and antianginal drug, on myocardial ischemia-reperfusion injury and systemic immune responses, its effect on intracellular Ca2+ levels ([Ca2+]i) in human neutrophils was investigated by using fura-2 as a fluorescent probe. Bepridil (10-200 microM) increased [Ca2+]i in a concentration-dependent fashion. This signal was partly inhibited by removal of extracellular Ca2+. In a Ca(2+)-free medium, pretreatment with bepridil (100 microM) abolished the Ca2+ release induced by thapsigargin (1 microM), an endoplasmic reticulum Ca2+ pump inhibitor, and by carbonylcyanide m-chlorophenylhydrazone (2 microM), a mitochondrial uncoupler. Pretreatment with carbonylcyanide m-chlorophenylhydrazone and thapsigargin, respectively, partly inhibited bepridil-induced Ca2+ release. Addition of Ca2+ (3 mM) increased [Ca2+]i after pretreatment with bepridil (100 microM) in a Ca(2+)-free medium. Bepridil (100 microM)-induced Ca2+ release was not altered when phospholipase C was inhibited by U73122 (2 microM). Both Ca2+ release and Ca2+ entry induced by bepridil (100 microM) were augmented by activating protein kinase C with phorbol 12-myristate 13-acetate (10 nM), and were suppressed by inhibiting protein kinase C with GF 109203X (2 microM). Treatment with bepridil (10-20 microM) for 30 min increased the production of reactive oxygen intermediates (ROI) by more than 50%. Collectively, it was found that bepridil increased [Ca2+]i concentration-dependently in human neutrophils by releasing Ca2+ from the endoplasmic reticulum, mitochondria and, possibly, other compartments in a phospholipase C-independent manner. Bepridil also activated Ca2+ influx. The activity of protein kinase C may regulate bepridil-induced Ca2+ release and Ca2+ entry.     

Mechanism underlying histamine-induced intracellular Ca2+ movement in PC3 human prostate cancer cells.

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in PC3 human prostate cancer cells and the underlying mechanism were evaluated using fura-2 as a Ca2+ dye. Histamine at concentrations between 0.1 and 50 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 1 microM. The [Ca2+]i response comprised an initial rise and a slow decay, which returned to baseline within 3 min. Extracellular Ca2+ removal inhibited 50% of the [Ca2+]i signal. In the absence of extracellular Ca2+, after cells were treated with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 10 microM histamine did not increase [Ca2+]i. After pretreatment with 10 microM histamine in a Ca2+-free medium for several minutes, addition of 3 mM Ca2+ induced [Ca2+]i increases. Histamine (10 microM)-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 2 microM 1-(6-((17 beta-3- methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 10 microM pyrilamine but was not altered by 50 microM cimetidine. Collectively, the present study shows that histamine induced [Ca2+]i transients in PC3 human prostate cancer cells by stimulating H1 histamine receptors leading to Ca2+ release from the endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, and by inducing Ca2+ entry.     

Tamoxifen-induced Ca2+ mobilization in bladder female transitional carcinoma cells

This study examined the effect of tamoxifen, an anti-breast cancer drug, on Ca2+ handling in bladder female transitional cancer cells. Changes in cytosolic free Ca2+ levels were recorded by using the Ca2+-sensitive dye fura-2. In a dose-dependent manner, tamoxifen induced intracellular free Ca2+ concentrations ([Ca2+]i) increases between 5 and 20 microM with an EC50 of 10 microM. External Ca2+ removal reduced the response by 60+/-6%. Addition of 3 mM Ca2+ caused a [Ca2+]i increase after pretreatment with 10 microM tamoxifen in Ca2+-free medium. In Ca2+-free medium, pretreatment with 10 microM tamoxifen abolished the [Ca2+]i increase induced by 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 1 microM thapsigargin prevented tamoxifen from releasing more Ca2+. Inhibition of phospholipase C-dependent inositol 1,4,5-tris-phosphate formation with 2 microM U73122 did not alter 10 microM tamoxifen-induced Ca2+ release. The [Ca2+]i increase induced by 5 microM tamoxifen was not altered by 10 microM La3+, nifedipine, verapamil, and diltiazem. Collectively, it was found that tamoxifen increased [Ca2+]i in bladder cancer cells by releasing Ca2+ from the endoplasmic reticulum Ca2+ stores in a manner independent of phospholipase C activity, and by inducing Ca2+ entry from external medium.     

A major role for the rho-associated coiled coil forming protein kinase in G-protein-mediated Ca2+ sensitization through inhibition of myosin phosphatase in rabbit trachea

1 G protein-mediated Ca2+ sensitization of airway smooth muscle contraction was investigated with respect to the relative importance of Rho-associated coiled coil forming protein kinase (ROCK) and protein kinase C (PKC). We examined the effects of Y-27632, a ROCK inhibitor, and GF 109203X, a PKC inhibitor, on guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-induced contraction in alpha-toxin- or beta-escin-permeabilized rabbit trachea. 2 Although pre-treatment with Y-27632 dose-dependently inhibited GTPgammaS (10 microM)-induced Ca2+ sensitization of alpha-toxin-permeabilized trachea, a Y-27632-insensitive component (approximately 16% of the maximum contraction) was retained during the early phase of the GTPgammaS response in the presence of Y-27632 (100 microM). 3 GF 109203X (5 microM) abolished 1 microM 4beta-phorbol 12, 13-dibutyrate (PDBu)-induced, but only partially inhibited the GTPgammaS-induced Ca2+ sensitization. A combination of Y-27632 (100 microM) and GF 109203X (5 microM) totally abolished the GTPgammaS response. 4 GTPgammaS caused only a small contraction in the absence of Ca2+. Wortmannin (30 microM), a myosin light chain kinase (MLCK) inhibitor, completely inhibited Ca2+-induced contraction. ATP-triggered contraction of the strip which had been treated with calyculin A (1 microM), a phosphatase inhibitor, in rigor solutions was markedly slowed by worthmannin (30 microM), but not by Y-27632 (100 microM), in the presence of GTPgammaS and Ca2+. 5 GTPgammaS, but not PDBu, contracted the beta-escin-permeabilized trachea in the absence of Ca2+, but the presence of Ca2+-independent MLCK. 6 We conclude that ROCK plays a primary role in G-protein-mediated Ca2+ sensitization, which requires MLCK activity, with minor contribution of PKC to the early phase of contraction, and PDBu utilizes conventional PKC(s) in airway smooth muscle.     

Artocarpol A stimulation of superoxide anion generation in neutrophils involved the activation of PLC, PKC and p38 mitogen-activated PK signaling pathways

1 Artocarpol A (ART), a natural phenolic compound isolated from Artocarpus rigida, stimulated a slow onset and long-lasting superoxide anion generation in rat neutrophils, whereas only slightly activated the NADPH oxidase in a cell-free system. 2 Pretreatment of neutrophils with pertussis toxin (1 microg ml(-1)), 50 microM 2'-amino-3'-methoxyflavone (PD 98059), or 1 microM 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126) had no effect on ART-stimulated superoxide anion generation. ART (30 microM) did not induce extracellular signal-regulated kinase (ERK) phosphorylation. 3 4-(4-Fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB 203580) markedly attenuated the ART-stimulated superoxide anion generation (IC50 value of 4.3+/-0.3 microM). Moreover, ART induced p38 mitogen-activated PK (MAPK) phosphorylation and activation. 4 The superoxide anion generation in response to ART was also substantially inhibited in a Ca2+-free medium, and by pretreatment with 1 microM 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U-73122) and 100 microM 2-aminoethyldiphenyl borate (2-APB). ART (30 microM) stimulated the [Ca2+]i elevation in the presence or absence of external Ca2+, and also increased the D-myo-inositol 1,4,5-trisphosphate formation. 5 2-[1-(3-Dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF 109203X) greatly inhibited the ART-stimulated superoxide anion generation (IC50 value of 7.8+/-1.0 nM). ART increased the recruitment of PKC-alpha, -betaI, and -betaII to the plasma membrane of neutrophils, and stimulated Ca2+-dependent PKC activation in the cytosol preparation. 6 ART induced the phosphorylation of p47phox, which was attenuated by GF 109203X. Moreover, ART evoked the membrane association of p47(phox), which was inhibited by GF 109203X and SB 203580. 7 These results indicate that the ART stimulation of superoxide anion generation involved the activation of p38 MAPK, PLC/Ca2+, and PKC signaling pathways in rat neutrophils.     

Effects of the antianginal drug fendiline on Ca2+ movement in hepatoma cells.

This study investigated the effect of the anti-anginal drug, fendiline, on intracellular free Ca2+ levels ([Ca2+]i) in HA/ 22 human hepatoma cells by using fura-2 as a fluorescent Ca2+ dye. Fendiline (1-100 microM) increased [Ca2+]i with an EC50 of 25 microM. Removal of extracellular Ca2+ reduced the [Ca2+]i signals by 51 +/- 5%. Fendiline (10 microM)-induced Ca2+ release was abolished by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Inhibition of phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122) did not alter 10 microM fendiline-induced Ca2+ release. Several other calmodulin antagonists, such as phenoxybenzamine (100-200 microM), trifluoperazine (5-50 microM), and fluphenazine-N-chloroethane (2-100 microM), had no effect on [Ca2+]i. Together, it was found that fendiline increased [Ca2+]i in human hepatoma cells by discharging Ca2+ from the endoplasmic reticulum in an inositol 1,4,5-trisphosphate-independent manner and by inducing Ca2+ entry. This effect of fendiline does not appear to be via antagonism of calmodulin.     

The ether lipid ET-18-OCH3 increases cytosolic Ca2+ concentrations in Madin Darby canine kidney cells.

The effect of the ether lipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH3) on the intracellular free Ca2+ concentration ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was studied using fura-2 as the Ca2+ probe. In Ca2+ medium, ET-18-OCH3 induced a significant rise in [Ca2+]i at concentrations between 10-100 microM with a concentration-dependent delay of 45-175 s. The [Ca2+]i signal was composed of a gradual rise and a sustained plateau. In Ca2+-free medium, ET-18-OCH3 (10-100 microM) induced a Ca2+ release from internal Ca2+ stores with a concentration-dependent delay of 45-175 s. This discharge of internal Ca2+ triggered capacitative Ca2+ entry in a concentration-dependent manner. This capacitative Ca2+ entry was not inhibited by econazole (25 microM), 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF96365; 50 microM), nifedipine (10 microM), verapamil (10 microM), diltiazem (10 microM) and cadmium (0.5 microM). Methyl 2-(phenylthio)ethyl-1,4-dihydro-2,4,6-trimethylpyridine-3,5-dicarboxylat e (PCA-4248), a platelet-activating factor (PAF) receptor antagonist, inhibited 25 microM ET-18-OCH3-induced [Ca2+]i rise in a concentration-dependent manner between 1-20 microM, with 20 microM exerting a complete block. The [Ca2+]i rise induced by ET-18-OCH3 (25 microM) was not altered when the production of inositol 1,4,5-trisphosphate (IP3) was suppressed by the phospholipase C inhibitor U73122 (2 microM), but was partly inhibited by the phospholipase D inhibitor propranolol (0.1 mM) or the phospholipase A2 inhibitor aristolochic acid (20-40 microM). In Ca2+-free medium, pretreatment with 25 microM ET-18-OCH3 completely depleted the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin-sensitive Ca2+ store. In contrast, pretreatment with thapsigargin abolished 0.1 mM ATP-induced [Ca2+]i rise without altering the ET-18-OCH3-induced [Ca2+]i rise. This suggests that ET-18-OCH3 depleted thapsigargin-sensitive Ca2+ stores and also released Ca2+ from thapsigargin-insensitive stores. The thapsigargin-insensitive stores involve mitochondria because the mitochondria uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM) induced a release of mitochondrial Ca2+ which was abolished by pretreatment with 25 microM ET-18-OCH3. ET-18-OCH3 (25 microM) induced a significant Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength confirming that ET-18-OCH3 induced capacitative Ca2+ entry. La3+ (0.1 mM) or Gd3+ (50 microM) abolished the ET-18-OCH3-induced Mn2+ quench and [Ca2+]i rise. Our data imply that ET-18-OCH3 induced a [Ca2+]i rise in MDCK cells by activating PAF receptors leading to an internal Ca2+ release followed by capacitative Ca2+ entry. Phospholipase D and phospholipase A2, but not phospholipase C, might be involved in mediating the capacitative Ca2+ entry. La3+ abolished the ET-18-OCH3-induced [Ca2+]i rise presumably by inhibiting PAF receptors.     

Protein kinase C potentiates capacitative Ca2+ entry that links to steroidogenesis in bovine adrenocortical cells

I investigated the role of protein kinase C (PKC) in regulation of the capacitative Ca2+ entry and steroidogenesis in bovine adrenocortical (BA) cells. Thapsigargin (TG)-treatment depleted intracellular Ca2+ stores followed by induction of Ca2+ influx from the extracellular pool and also increasing of Mn2+ influx as an indicator of divalent cation influx in BA cells. Calphostin C, a PKC inhibitor, inhibited the TG-induced [Ca2+]i elevation dose-dependently (0.1-1 microM) and attenuated Mn2+ entry. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, potentiated the elevation of [Ca2+]i and enhanced Mn2+ entry by TG treatment. These results suggest that PKC may modulate capacitative Ca2+ entry in BA cells. In the presence of extracellular Ca2+, TG enhanced cortisol production in BA cells. Calphostin C attenuated the TG-induced steroidogenesis dose-dependently (0.25-1 microM). PMA enhanced the steroidogenesis dose-dependently (1-100 nM). These results suggested that PKC may have a modulatory effect on the capacitative Ca2+ entry that links to steroidogenesis in BA cells.     

Quantitative comparisons of muscarinic and bradykinin receptor-mediated Ins (1,4,5)P3 accumulation and Ca2+ signalling in human neuroblastoma cells.

1. Muscarinic and bradykinin receptor-mediated Ins(1,4,5)P3 accumulation, Ca2+ mobilization and Ca2+ entry have been examined in human SH-SY5Y neuroblastoma cells. This has allowed both direct comparison of signalling events by two receptor types potentially linked to the same transduction pathway and an investigation of the interactions between the components of this pathway. 2. Stimulation of muscarinic receptors with carbachol produced biphasic accumulations of Ins(1,4,5)P3 consisting of a rapid peak followed by a lower sustained phase. Both phases were dose-dependent but the potency of elevation at peak was significantly less than that of the sustained phase. Bradykinin also dose-dependently stimulated Ins(1,4,5)P3 accumulation but responses were smaller and not sustained. 3. Lowering of [Ca2+]e reduced basal Ins(1,4,5)P3 levels. Peak Ins(1,4,5)P3 elevation in response to carbachol and bradykinin were lowered by an amount approximating this reduction over the entire dose-response curves. Sustained Ins(1,4,5)P3 elevation in response to carbachol showed a more marked absolute reduction. Agonist potencies were unaffected by lowering [Ca2+]e. Thus, a consistent but small amount of PLC activity during rapid activation appears to be sensitive to lowered [Ca2+]e, whilst activity during sustained stimulation is greatly facilitated by external Ca2+, probably through Ca2+ entry. 4. The temporal- and dose-dependency of carbachol-mediated Ins(1,4,5)P3 accumulations were unaffected by loading cells with fura-2, thus allowing direct comparison of Ins(1,4,5)P3 and [Ca2+]i changes monitored by fura-2. 5. Changes in [Ca2+]i by both agonists revealed temporal patterns that were similar to Ins(1,4,5)P3 accumulations. Only carbachol stimulated a marked sustained [Ca2+]i signal and this was fully dependent on external Ca2+. 6. All agonist-mediated [Ca2+]i elevations occurred with significantly greater potency than that of the respective Ins(1,4,5)P3 accumulations. Further examination of peak elevations in response to carbachol indicated that this was independent of Ca2+ entry. Thus, a major site for amplification of the potency of rapid agonist-mediated responses lies at the level of the Ins(1,4,5)P3 receptor. 7. The transient nature of Ins(1,4,5)P3 and [Ca2+]i peaks followed by either lower but sustained levels with carbachol or a return to basal levels with bradykinin suggests rapid but partial desensitization of the muscarinic receptor and complete desensitization of the bradykinin receptor. This indicates receptor-specific desensitization. Further analysis of this was provided by detecting accumulations of [3H]-inositol phosphates ([3H]-InsPs) in Li(+)-blocked, myo-[3H]-inositol labelled cells. Carbachol produced a rapid accumulation over the first minute, followed by a slower linear accumulation for at least 29 min.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of triethyltin on Ca2+ movement in Madin-Darby canine kidney cells

The effects of the environmental toxicant, triethyltin, on Ca2+ mobilization in Madin-Darby canine kidney (MDCK) cells have been examined. Triethyltin induced an increase in cytosolic free Ca2+ levels ([Ca2+]i) at concentrations larger than 2 microM in a concentration-dependent manner. Within 5 min, the [Ca2+]i signal was composed of a gradual rise and a sustained phase. The [Ca2+]i signal was partly reduced by removing extracellular Ca2+. In Ca(2+)-free medium, pretreatment with thapsigargin (1 microM), an endoplasmic reticulum Ca2+ pump inhibitor, reduced 50 microM triethyltin-induced [Ca2+]i increase by 80%. Conversely, pretreatment with triethyltin abolished thapsigargin-induced Ca2+ release. Pretreatment with U73122 (2 microM) to inhibit phospholipase C-coupled inositol 1,4,5-trisphosphate formations failed to alter 50 microM triethyltin-induced Ca2+ release. Incubation with triethyltin at a concentration (1 microM) that did not increase basal [Ca2+]i for 3 min did not alter ATP (10 microM)- and bradykinin (1 microM)-induced [Ca2+]i increases. Collectively, this study shows that triethyltin altered Ca2+ movement in renal tubular cells by releasing Ca2+ from multiple stores in an inositol 1,4,5-trisphosphate-independent manner, and by inducing Ca2+ influx.     

Econazole induces increases in free intracellular Ca2+ concentrations in human osteosarcoma cells

Econazole is an antifungal drug with different in vitro effects. However, econazole's effect on osteoblast-like cells is unknown. In human MG63 osteosarcoma cells, the effect of econazole on intracellular Ca2+ concentrations ([Ca2+]i) was explored by using fura-2. At a concentration of 0.1 microM, econazole started to cause a rise in [Ca2+]i in a concentration-dependent manner. Econazole-induced [Ca2+]i rise was reduced by 74% by removal of extracellular Ca2+. The econazole-induced Ca2+ influx was mediated via a nimodipine-sensitive pathway. In Ca2+ -free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca+ -ATPase, caused a [Ca2+]i rise, after which the increasing effect of econazole on [Ca2+]i was abolished. Pretreatment of cells with econazole to deplete Ca2+ stores totally prevented thapsigargin from releasing Ca2+. U73122, an inhibitor of phospholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca2+ mobilizer)-induced, but not econazole-induced, [Ca2+]i rise. Econazole inhibited 76% of thapsigargin-induced store-operated Ca2+ entry. These findings suggest that in MG63 osteosarcoma cells, econazole increases [Ca2+]i by stimulating Ca2+ influx and Ca2+ release from the endoplasmic reticulum via a phospholipase C-independent manner. In contrast, econazole acts as a potent blocker of store-operated Ca2+ entry.     

Effect of fluoxetine on intracellular Ca2+ levels in bladder female transitional carcinoma (BFTC) cells.

The effect of the antidepressant fluoxetine on Ca2+ signaling in cultured cells was largely unknown. The effect of various concentrations of fluoxetine on [Ca 2+] i in populations of bladder female transitional cancer (BFTC) cells was evaluated by using fura-2 as a Ca2+ probe. Fluoxetine increased [Ca 2+] i concentration dependently (20-100 microM) with an EC50 value of 30 microM. The response was inhibited by 50-60% on extracellular Ca2+ removal. In Ca2+ -free medium, pretreatment with 1 microM thapsigargin (an inhibitor of the endoplasmic reticulum Ca2+ pump) abolished 50 microM fluoxetine-induced Ca2+ release; whereas pretreatment with fluoxetine did not alter the thapsigargin-induced Ca2+ response. Addition of 3 mM Ca2+ increased [Ca 2+] i after pretreatment with 50 microM fluoxetine in Ca2+ -free medium, suggestive of fluoxetine-induced capacitative Ca2+ entry. Suppression of inositol 1,4,5-trisphosphate formation by 2 microM U73122 (a phospholipase C inhibitor) did not affect 50 microM fluoxetine-induced Ca2+ release. Collectively, this study shows that fluoxetine increased [Ca 2+] i in bladder cancer cells in a concentration-dependent fashion, by releasing Ca2+ from thapsigargin-sensitive Ca2+ stores in an IP3-independent manner, and by inducing Ca2+ influx from extracellular medium.     

Inositol 1,4,5-triphosphate-stimulated calcium release from permeabilized cerebellar granule cells.

1. Muscarinic cholinoceptor stimulation of phosphoinositide hydrolysis in rat cultured cerebellar granule cells results in a rapid, transient accumulation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which has been implicated in the release of non-mitochondrial intracellular Ca2+ stores. In the present study, the release of Ca2+ from intracellular stores and the Ins(1,4,5)P3 receptor responsible for this process have been investigated. 2. Monolayers of saponin-permeabilized granule cells accumulate 45Ca2+ in an ATP-dependent manner and the sequestered 45Ca2+ can be concentration-dependently released by Ins(1,4,5)P3 by a stereospecific and heparin-sensitive mechanism. The EC50 for Ins(1,4,5)P3-stimulated 45Ca2+ release was 80 +/- 3 nM. 3. Radioligand binding studies performed on a crude granule cell membrane fraction indicated the presence of an apparently homogeneous population of stereo-specific Ins(1,4,5)P3 receptors (KD 54.7 +/- 2.0 nM; Bmax 1.37 +/- 0.29 pmol mg-1 protein). 4. This study provides evidence for Ins(1,4,5)P3-sensitive intracellular Ca2+ stores in primary cultures of cerebellar granule cells and suggest that these cells provide an excellent model neuronal system in which to study the relative functional roles of Ca2+ release from intracellular stores and Ca(2+)-entry in neuronal Ca2+ homeostasis.     

Effect of carvedilol on Ca2+ movement and cytotoxicity in human MG63 osteosarcoma cells

Carvedilol is a useful cardiovascular drug for treating heart failure, however, the in vitro effect on many cell types is unclear. In human MG63 osteosarcoma cells, the effect of carvedilol on intracellular Ca2+ concentrations ([Ca2+]i) and cytotoxicity was explored by using fura-2 and tetrazolium, respectively. Carvedilol at concentrations greater than 1 microM caused a rapid rise in [Ca2+]i in a concentration-dependent manner (EC50=15 microM). Carvedilol-induced [Ca2+]i rise was reduced by 60% by removal of extracellular Ca2+. Carvedilol-induced Mn2+-associated quench of intracellular fura-2 fluorescence also suggests that carvedilol induced extracellular Ca2+ influx. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of carvedilol on [Ca2+]i was inhibited by 50%. Conversely, pretreatment with carvedilol to deplete intracellular Ca2+ stores totally prevented thapsigargin from releasing more Ca2+. U73122, an inhibitor of phospholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca2+ mobilizer)-induced, but not carvedilol-induced, [Ca2+]i rise. Pretreatment with phorbol 12-myristate 13-acetate and forskolin to activate protein kinase C and adenylate cyclase, respectively, did not alter carvedilol-induced [Ca2+]i rise. Separately, overnight treatment with 0.1-30 microM carvedilol inhibited cell proliferation in a concentration-dependent manner. These findings suggest that in human MG63 osteosarcoma cells, carvedilol increases [Ca2+]i by stimulating extracellular Ca2+ influx and also by causing intracellular Ca2+ release from the endoplasmic reticulum and other stores via a phospholipase C-independent manner. Carvedilol may be cytotoxic to osteoblasts.     

Activation of phospholipase C in SH-SY5Y neuroblastoma cells by potassium-induced calcium entry.

1. We used SH-SY5Y human neuroblastoma cells to investigate whether depolarization with high K+ could stimulate inositol (1,4,5)trisphosphate (Ins(1,4,5)P3) formation and, if so, the mechanism involved. 2. Ins(1,4,5)P3 was measured by a specific radioreceptor mass assay, whilst [Ca2+]i was measured fluorimetrically with the Ca2+ indicator dye, Fura-2. 3. Depolarization with K+ caused a time- and dose-dependent increase in [Ca2+]i (peak at 27 s, EC50 of 50.0 +/- 9.0 mM) and Ins(1,4,5)P3 formation (peak at 30 s, EC50 of 47.4 +/- 1.1 mM). 4. Both the K(+)-induced Ins(1,4,5)P3 formation and increase in [Ca2+]i were inhibited dose-dependently by the L-type voltage-sensitive Ca2+ channel closer, (R+)-BayK8644, with IC50 values of 53.4 nM and 87.9 nM respectively. 5. These data show a close temporal and dose-response relationship between Ca2+ entry via L-type voltage-sensitive Ca2+ channels and Ins(1,4,5)P3 formation following depolarization with K+, indicating that Ca2+ influx can activate phospholipase C in SH-SY5Y cells.     

Quisqualate and carbachol-induced increases in intrasynaptosomal free calcium are mediated by different products of phospholipid hydrolysis

The mechanisms by which quisqualate and carbachol increase intrasynaptosomal free calcium ([Ca2+]i) were studied in rat cortical synaptosomes. Quisqualate (0.01-100 microM) and carbachol (100-1000 microM) increased [Ca2+]i in Fura-2 acetoxymethyl ester (Fura-2 AM)-loaded synaptosomes. The resting level of [Ca2+]i was 118 nM. The maximum increase (55%) was produced by 10 microM quisqualate which had an EC50 of 0.2 microM. The maximum increase (28%) elicited by carbachol occurred at 1000 microM and the EC50 was 30 microM. The stimulatory effects of quisqualate on [Ca2+]i were blocked by heparin (100 I.U.) but not by staurosporine (1 microM), nifedipine (1 microM) or omega-conotoxin fraction GVIA (omega-CgTx) (0.5 microM). On the other hand, the effects of carbachol on [Ca2+]i were abolished by staurosporine, nifedipine or omega-CgTx but not by heparin. Carbachol (100 microM) also significantly increased 45Ca accumulation into either resting or K+ (30 mM)-depolarised synaptosomes and these effects were inhibited by staurosporine and nifedipine. Quisqualate (10 microM) had no effect on 45Ca accumulation under resting or depolarised conditions. When quisqualate and carbachol were used in combination, there were apparently additive effects on [Ca2+]i but not on 45Ca accumulation. It is concluded that carbachol increases [Ca2+]i by facilitating Ca2+ entry through L-type Ca2+ channels via a 1,2-diacylglycerol (DAG)-protein kinase C (PKC)-dependent pathway while quisqualate mobilizes Ca2+ from inositol 1,4,5-trisphosphate (IP3)-sensitive stores.