Inhibition of thrombin-induced Ca²⁺ influx in platelets by R59949, an inhibitor of diacylglycerol kinase

Objectives The aim of this study was to determine whether diacylglycerol kinase (DGK) is involved in transplasmalemmal Ca²⁺ influx of platelets. Methods Effects of R59949, an inhibitor of diacylglycerol kinase, on intracellular Ca²⁺ concentration ([Ca²⁺]_i) and mRNA expression of DGK isozymes were investigated using washed human platelet suspensions. Key findings Thrombin‐induced increase in [Ca²⁺]_i was significantly inhibited by pretreatment of platelets with R59949, while thapsigargin‐induced increase in [Ca²⁺]_i was comparable in platelets with and without R59949 pretreatment. Thapsigargin‐induced increase in [Ca²⁺]_i was markedly attenuated in the presence of SKF‐96365. In the presence of SKF‐96365, thrombin‐induced increase in [Ca²⁺]_i was significantly attenuated, and additional treatment with R59949 caused a further decrease in [Ca²⁺]_i. Pretreatment of platelets with 1‐butanol significantly attenuated thrombin‐induced increase in [Ca²⁺]_i, while thrombin‐induced increase in [Ca²⁺]_i was augmented in the presence of propranolol. mRNA expression of DGK‐α and DGK‐γ, which are known to be inhibited by R59949, in platelets was confirmed by RT‐PCR analysis. Conclusions R59949 inhibited a store‐depletion‐insensitive component of transplasmalemmal Ca²⁺ entry induced by thrombin, while store‐operated Ca²⁺ entry was not affected by R59949. The results of this study suggest that phosphatidic acid is involved in thrombin‐induced Ca²⁺ influx of platelets.     

Effects of MK‐886, a leukotriene synthesis inhibitor,on [Ca2+]i and apoptosis in MG63 human osteosarcoma cells

The effect of MK‐886 (3‐[1‐(p‐chlorobenzyl)‐5‐(isopropyl)‐3‐tert‐butylthioindol‐2‐yl]‐2, 2‐dimethylpropanoic acid), a compound widely used to inhibit leukotriene synthesis, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in osteosarcoma cells has not been explored. This study examined whether MK‐886 altered [Ca²⁺]_i levels in suspended MG63 human osteosarcoma cells using fura‐2. MK‐886 at 0.1 μM and above increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. MK‐886 induced Mn²⁺ quenching of fura‐2 fluorescence, implicating Ca²⁺ entry. MK‐886‐induced Ca²⁺ influx was inhibited by store‐operated Ca²⁺ entry inhibitors, nifedipine, econazole, and SKF96365; and by the protein kinase C modulators, phorbol 12‐myristate 13‐acetate (PMA) and GF109203X. In Ca²⁺‐free medium, after pretreatment with 5 μM MK‐886, 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor)‐induced [Ca²⁺]_i rises were abolished; conversely, thapsigargin pretreatment nearly abolished MK‐886‐induced [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 did not change MK‐886‐induced [Ca²⁺]_i rises. Collectively, in MG63 osteosarcoma cells, MK‐886 induced [Ca²⁺]_i rises by causing phospholipase C‐independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via protein kinase C‐regulated store‐operated Ca²⁺ entry. Drug Dev Res 69: 49–57, 2008. © 2008 Wiley‐Liss, Inc.     

Effect of MK‐886 on Ca2+ Level and Viability in PC3 Human Prostate Cancer Cells

Abstract: 3‐[1‐(p‐chlorobenzyl)‐5‐(isopropyl)‐3‐tert‐butylthioindol‐2‐yl]‐2, 2‐dimethylpropanoic acid (MK‐886) is widely used for inhibition of leucotriene synthesis in in vitro studies, however, many of its other effects have been reported. The present study investigated the effect of MK‐886 on cytosolic‐free Ca²⁺ concentrations ([Ca²⁺]_i) and viability in human PC3 prostate cancer cells. [Ca²⁺]_i in suspended cells was measured by using fura‐2. MK‐886 at concentrations of 1 µM and above increased [Ca²⁺]_i in a concentration‐dependent manner with an EC₅₀ value of 20 µM. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. MK‐886 evoked Mn²⁺ quenching of fura‐2 fluorescence, implicating Ca²⁺ entry. MK‐886‐induced Ca²⁺ influx was inhibited by store‐operated Ca²⁺ entry inhibitors nifedipine, econazole and SKF96365. In Ca²⁺‐free medium, after pre‐treatment with 10 µM MK‐886, 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor)‐induced [Ca²⁺]_i rises were abolished; and conversely, thapsigargin pre‐treatment abolished MK‐886‐induced [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 did not alter MK‐886‐induced [Ca²⁺]_i rises. MK‐886 at concentrations of 1–100 µM concentration‐dependently decreased cell viability with an IC₅₀ value of 60 µM. The cytotoxic effect of MK‐886 was not inhibited by pre‐chelating cytosolic Ca²⁺ with BAPTA/AM. Together, in PC3 cells, MK‐886 induced [Ca²⁺]_i rises by causing phospholipase C‐independent Ca²⁺ release from the endoplasmic reticulum; and Ca²⁺ influx via store‐operated Ca²⁺ channels. Independently, MK‐886 was cytotoxic to cells in a Ca²⁺‐independent manner.     

Effect of the Anti‐Breast Cancer Drug Tamoxifen on Ca2+ Movement in Human Osteosarcoma Cells

Abstract:The anti‐breast cancer drug tamoxifen has recently been shown to cause an increase in [Ca²⁺]_i in renal tubular cells, breast cells and bladder cells. Because tamoxifen is known to interact with oestrogens leading to modulation of bone metabolism, the present study was aimed at exploring whether tamoxifen could alter Ca²⁺ signaling in human osteoblast‐like MG63 cells. Cytosolic free Ca²⁺ levels were recorded by using the Ca²⁺‐sensitive dye fura‐2. Tamoxifen induced a sustained [Ca²⁺]_i increase at concentrations above 1 μM with an EC₅₀ of 8 μM. Removal of extracellular Ca²⁺ reduced the response by 40%, suggesting that tamoxifen induced both Ca²⁺ influx and store Ca²⁺ release. Tamoxifen‐induced Ca²⁺ influx was confirmed as tamoxifen caused Mn²⁺ influx‐induced quench of fura‐2 fluorescence. In Ca²⁺‐free medium, pretreatment with 10 μM tamoxifen abolished the [Ca²⁺]_i increase induced by 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), and by 2 μM carbonylcyanide m‐chlorophenylhydrazone (a mitochondrial uncoupler). Conversely, pretreatment with thapsigargin and carbonylcyanide m‐chlorophenylhydrazone only reduced 64% of tamoxifen‐induced [Ca²⁺]_i increases. Addition of 2 μM U73122 to inhibit phospholipase C activity abolished the [Ca²⁺]_i increase induced by 1 μM histamine, a phospholipase C‐dependent Ca²⁺ mobilizer, without affecting 10 μM tamoxifen‐induced Ca²⁺ release. The [Ca²⁺]_i increase induced by 10 μM tamoxifen was not altered by 10 μM of nifedipine, verapamil and diltiazem. Together, the data show that tamoxifen induced a lasting increase in [Ca²⁺]_i in human osteoblast‐like cells by causing Ca²⁺ influx and releasing Ca²⁺ from multiple stores in a phospholipase C‐independent manner.     

Intracellular calcium concentrations in human bladder tumor cells could be increased by NPC‐14686, a novel antiinflammatory agent

NPC‐14686 (Fmoc‐L‐homophenylalanine), a novel antiinflammatory agent, increases intracellular Ca²⁺ concentrations ([Ca²⁺]_i] in T24 bladder tumor cells. Using fura‐2 as a Ca²⁺ probe, NPC‐14686 (10–200 μM) increased [Ca²⁺]_i in a concentration‐dependent manner. The [Ca²⁺]_i increase comprised an initial slow rise and a plateau over a time period of 5 min. Ca²⁺ removal partly inhibited the Ca²⁺ signals. In Ca²⁺‐free medium, pretreatment with 100 μM NPC‐14686 abolished the [Ca²⁺]_i increases induced by 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor] and 2 μM carbonylcyanide m‐chlorophenylhydrazone (a mitochondrial uncoupler). However, 100 μM NPC‐14686 still slightly increased [Ca²⁺]_i after Ca²⁺ stores had been depleted by pretreating with 2 μM CCCP and 1 μM thapsigargin. These results suggest that NPC‐14686 released Ca²⁺ from multiple pools. Adding 3 mM Ca²⁺ increased [Ca²⁺]_i in cells pretreated with 100 μM NPC‐14686 in Ca²⁺‐free medium, indicating that NPC‐14686 activated capacitative Ca²⁺ entry. Inhibiting formation of inositol‐1,4,5‐trisphosphate (IP₃] by blocking phospholipase C with 2 μM U73122 had little effect on NPC‐14686‐induced Ca²⁺ release. Activating protein kinase C with phorbol 12‐myristate 13‐acetate (PMA) significantly potentiated NPC‐14686‐induced [Ca²⁺]_i increase. NPC‐14686 (100 μM) also increased [Ca²⁺]_i in MDCK renal cells, BFTC bladder tumor cells, and MS‐1 endothelial cells. Together, the findings suggest that in T24 bladder tumor cells NPC‐14686 induced Ca²⁺ release followed by Ca²⁺ entry. The Ca²⁺ release was unlinked to IP₃ and the [Ca²⁺]_i signal could be modulated by protein kinase C. Drug Dev. Res. 50:147–152, 2000. © 2000 Wiley‐Liss, Inc.     

3,3'-Diindolylmethane alters Ca2+ homeostasis and viability in MG63 human osteosarcoma cells

Abstract: The effect of the natural product 3,3′‐diindolylmethane (DIM) on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in MG63 human osteosarcoma cells was explored. The Ca²⁺‐sensitive fluorescent dye fura‐2 was applied to measure [Ca²⁺]_i. DIM at concentrations of 40–80 μM induced a [Ca²⁺]_i rise in a concentration‐dependent manner. The response was reduced partly by removing Ca²⁺. DIM‐evoked Ca²⁺ entry was suppressed by nifedipine, econazole, SK&F96365 and protein kinase C modulators. In the absence of extracellular Ca²⁺, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitors thapsigargin or 2,5‐di‐tert‐butylhydroquinone (BHQ) inhibited or abolished DIM‐induced [Ca²⁺]_i rise. Incubation with DIM also inhibited thapsigargin or BHQ‐induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished DIM‐induced [Ca²⁺]_i rise. At concentrations of 10–50 μM, DIM killed cells in a concentration‐dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca²⁺ with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA). Annexin V/propidium iodide staining data implicate that DIM (20 and 40 μM) induced apoptosis in a concentration‐dependent manner. In sum, in MG63 cells, DIM induced a [Ca²⁺]_i rise by evoking phospholipase C‐dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via protein kinase C‐sensitive store‐operated Ca²⁺ channels. DIM caused cell death that may involve apoptosis.     

Effect of clomiphene on [Ca2+]i rises and cell viability in rabbit corneal epithelial cells

The effect of clomiphene a first‐line therapy for WHO group II (eu‐estrogenic) infertility on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability has not been explored in rabbit corneal epithelial cells (SIRC). This study examined whether clomiphene altered [Ca²⁺]_i levels and caused cell death in SIRC cells. [Ca²⁺]_i and cell viability were measured using the fluorescent dyes fura‐2 and WST‐1, respectively. Clomiphene at concentrations ≥5 µM increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. The clomiphene‐induced Ca²⁺ influx was insensitive to blockade of L‐type Ca²⁺ channel blockers. In Ca²⁺‐free medium, after pretreatment with 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), clomiphene failed to increase [Ca²⁺]_i. Inhibition of phospholipase C with 2 µM U73122 did not change clomiphene‐induced [Ca²⁺]_i rises. At concentrations of 0.5–20 µM, clomiphene killed cells in a concentration‐dependent manner. The cytotoxic effect of 15 µM clomiphene was not reversed by prechelating cytosolic Ca²⁺ with BAPTA/AM. Collectively, in SIRC cells, clomiphene‐induced [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx from non‐L‐type Ca²⁺ channels. Clomiphene‐caused cytotoxicity was not mediated by a preceding [Ca²⁺]_i rise. Drug Dev Res 69:272–278, 2008 ©2008 Wiley‐Liss, Inc.     

L‐type Ca2+ channel opener BayK 8644‐induced Ca2+ influx and Ca2+ release in human oral cancer cells (OC2)

The effect of BayK 8644, a chemical widely used to activate L‐type Ca²⁺ channels, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in human oral cancer cells (OC2) has not been explored to date. The present study examined whether BayK 8644 altered basal [Ca²⁺]_i levels in suspended OC2 cells by using fura‐2. BayK 8644 (10 pM–10 µM) increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. BayK 8644‐induced Ca²⁺ influx was blocked by nifedipine, but was not altered by the store‐operated Ca²⁺ entry inhibitors, econazole and SKF96365; protein kinase C modulators phorbol 12‐myristate 13‐acetate (PMA) and GF109203X; the protein kinase A inhibitor H89; and the phospholipase A₂ inhibitor, aristolochic acid. In Ca²⁺‐free medium, after pretreatment with 1 µM BayK 8644, 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor)‐induced [Ca²⁺]_i rises were abolished; and conversely, thapsigargin pretreatment abolished BayK 8644‐induced [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 did not change BayK 8644‐induced [Ca²⁺]_i rises. Collectively, in OC2 cells, BayK 8644 induced [Ca²⁺]_i rises by causing phospholipase C‐independent Ca²⁺ release from the endoplasmic reticulum; and Ca²⁺ influx via L‐type Ca²⁺ channels. Drug Dev Res 69: 2008. © 2008 Wiley‐Liss, Inc.     

Calcium/calmodulin signaling elicits release of cytochrome c during 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced apoptosis in the human lymphoblastic T-cell line,L-MAT

We have reported previously that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces apoptosis in the human lymphoblastic T-cell line L-MAT, although these cells do not express the aromatic hydrocarbon receptor (AhR). The AhR-dependent pathway for the induction of immunotoxicity by TCDD has been studied extensively, but the AhR-independent pathway is not understood. Several studies have reported that TCDD elevates the concentration of free intracellular calcium ([Ca²⁺]_i) in various types of cells. However, the precise mechanism of the increase in [Ca²⁺]_i that occurs during apoptosis induced by TCDD has not been elucidated.     

Nonylphenol‐induced cytosolic Ca2+ elevation and death in renal tubular cells

Nonylphenol is an environmental endocrine disrupter. The effect of nonylphenol on intracellular free Ca²⁺ levels ([Ca²⁺]_i) and viability in Madin‐Darby canine kidney (MDCK) cells was explored. Nonylphenol increased [Ca²⁺]_i in a concentration‐dependent manner (EC₅₀～0.8 μM). Nonylphenol‐induced Mn²⁺ entry demonstrated Ca²⁺ influx and removal of extracellular Ca²⁺ partly decreased the [Ca²⁺]_i rise. The [Ca²⁺]_i rise was inhibited by the protein kinase C activator, phorbol 13‐myristate acetate (PMA) but not by L‐type Ca²⁺ channel blockers. In Ca²⁺‐free medium, nonylphenol‐induced [Ca²⁺]_i rise was partly inhibited by pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor). Conversely, nonylphenol pretreatment abolished thapsigargin‐induced Ca²⁺ release. Nonylphenol‐induced Ca²⁺ release was unaltered by inhibition of phospholipase C. At concentrations of 5–100 μM, nonylphenol killed cells in a concentration‐dependent manner. The cytotoxic effect of 100 μM nonylphenol was not affected by preventing [Ca²⁺]_i rises with BAPTA/AM. Collectively, this study shows that nonylphenol induced [Ca²⁺]_i increase in MDCK cells via evoking Ca²⁺ entry through protein kinase C‐regulated Ca²⁺ channels, and releasing Ca²⁺ from endoplasmic reticulum and other stores in a phospholipase C‐independent manner. Nonylphenol also killed cells in a Ca²⁺‐independent fashion. Drug Dev Res, 2009. © 2009 Wiley‐Liss, Inc.     

Effects of the Chinese herb component phellopterin on the increase in cytosolic free calcium in PC12 cells

The present study investigated the effects of the Chinese Herb component, phellopterin on high K⁺ and glutamate‐induced extracellular calcium influx and caffeine or cyclopiazonic acid (CPA)‐induced calcium release from internal stores in attached PC12 cells. Attached cells were loaded with the calcium fluorescent indicator Fluo‐3/AM with the final concentration of 5 µM for 50 min at 37°C and cytosolic free Ca²⁺ measured as fluorescent intensity (FI) (excitation: 488 nm; emission: 535 nm). When PC12 cells were exposed to extracellular Ca²⁺([Ca²⁺]₀) 2.0 mM, the FI for resting [Ca²⁺]_i was 1,188±163, high K⁺ (75 mM) and glutamate (10 mM) induced an increase in [Ca²⁺]_i with peak values of 4,270±982 and 3,096±402, respectively. Phellopterin (0.1–100 µM) had no apparent effect on resting [Ca²⁺]_i, but inhibited high K⁺ and glutamate induced the increase in [Ca²⁺]_i in a dose‐dependent manner. When PC12 cells were exposed to Ca²⁺‐free solution, the FI for resting [Ca²⁺]_i was 804±77. Caffeine (40 mM) and CPA (30 µM) stimulated Ca²⁺ release from caffeine‐ryanodine and inositol 1,4,5‐tris‐phosphate (InsP₃₎‐sensitive internal calcium stores, inducing an increase in [Ca²⁺]_i to 2,938±362 and 1,816±291, respectively. Phellopterin (0.1–100 µmol/L) inhibited caffeine and CPA stimulated intracellular calcium release in a dose‐dependent manner. In summary, phellopterin, a novel component isolated from Changii radix, inhibited Ca²⁺ influx induced by stimulation of voltage‐gated and receptor‐dependent calcium channels with a greater inhibition of receptor‐dependent calcium channels. It also inhibited Ca²⁺ release from caffeine‐ryanodine and InsP₃‐sensitive internal stores, being more potent for caffeine stimulation. Phellopterin may be a promising candidate for the development of new classes of calcium antagonists. Drug Dev Res 68:79–83, 2007. © 2007 Wiley‐Liss, Inc.     

Benzo[a]pyrene‐decreased gap junctional intercellular communication via calcium/calmodulin signaling increases apoptosis in TM4 cells

The mechanism of male reproductive toxicity induced by benzo[a]pyrene (BaP) is poorly understood. Gap junctional intercellular communication (GJIC) is known to play a critical role in maintaining spermatogenesis. The aim of the present study was to determine the toxic effects of BaP in Sertoli cells, and to explore the possibility and potential mechanisms of BaP‐induced changes in the level of GJIC, and the relationship between GJIC and BaP‐induced apoptosis. We treated mouse Sertoli cell lines (TM4) with different concentrations (0.1–100 μm ) of BaP for 1–48 hours, and found that GJIC exhibited a dose‐ and time‐dependent downregulation. Treatment with 10 μm BaP increased apoptosis, intracellular Ca²⁺ level ([Ca²⁺]_i) and calmodulin (CaM) protein expression, and decreased the protein level of connexin 43 (Cx43) (also known as gap junction α‐1 protein [GJA1]) in TM4 cells. However, BaP had no effect on the phosphorylation of Cx43 at Ser279/282, Ser255, Ser368 or Ser262. Downregulation of [Ca²⁺]_i by BAPTA‐AM significantly attenuated the BaP‐induced GJIC suppression, Cx43 protein decrease and CaM protein increase. Interestingly, inhibition of CaM expression by W7 partially recovered BaP‐induced GJIC inhibition, but had no effect on BaP‐induced Cx43 protein decrease. Pretreatment with the GJIC activator retinoic acid significantly mitigated BaP‐induced apoptosis. In conclusion, these results suggest that BaP can decrease GJIC via Ca²⁺/CaM signaling, and that BaP‐induced GJIC suppression increases apoptosis in TM4 cells.     

Novel effects of a sleep‐inducing lipid,oleamide, on Ca2+ signaling in renal tubular cells

The effect of oleamide, a sleep‐inducing endogenous lipid in animal models, on intracellular free levels of Ca²⁺ ([Ca²⁺]_i) in Madin‐Darby renal tubular cells was examined using fura‐2 as a fluorescent dye. Oleamide (5–50 μM) increased [Ca²⁺]_i in a concentration‐dependent fashion with an EC₅₀ value of 20 μM. The [Ca²⁺]_i signal comprised an initial rise and an elevated phase and was reduced by removing extracellular Ca²⁺ by 50%. After pretreatment with 5–50 μM oleamide in Ca²⁺‐free medium, addition of 3 mM Ca²⁺ increased [Ca²⁺]_i in a manner dependent on the concentration of oleamide. In Ca²⁺‐free medium, pretreatment with thapsigargin (1 μM), an endoplasmic reticulum Ca²⁺ pump inhibitor, abolished [Ca²⁺]_i increases induced by 20 μM oleamide; conversely, pretreatment with 20 μM oleamide reduced 1 μM thapsigargin‐induced [Ca²⁺]_i increases by 50%. Suppression of the activity of phospholipase C with 2 μM U73122 abolished 20 μM oleamide‐induced Ca²⁺ release. Collectively, these data demonstrate that oleamide induced significant [Ca²⁺]_i increases in renal tubular cells by a phospholipase C‐dependent release of Ca²⁺ from thapsigargin‐sensitive stores and by inducing Ca²⁺ entry via store‐operated Ca²⁺ entry. Drug Dev. Res. 54:40–44, 2001. © 2001 Wiley‐Liss, Inc.     

Lindane (γ‐Hexachlorocyclohexane) Induces Internal Ca2+ Release and Capacitative Ca2+ Entry in Madin‐Darby Canine Kidney Cells

The effect of lindane (γ‐hexachlorocyclohexane), an organochlorine pesticide, on Ca²⁺ mobilization in Madin‐Darby canine kidney cells was examined by fluorimetry using fura‐2 as a Ca²⁺ indicator. Lindane (5–200 μM) increased [Ca²⁺]_i concentration‐dependently. The [Ca²⁺]_i signal comprised an immediate initial rise followed by a persistent phase. Ca²⁺ removal inhibited the [Ca²⁺]_i signal by reducing both the initial rise and the sustained phase. This implies lindane‐triggered Ca²⁺ influx and Ca²⁺ release. In Ca²⁺‐free medium, 0.15 mM lindane increased [Ca²⁺]_i after pretreatment with carbonylcyanide m‐chlorophenylhydrazone (CCCP; 2 μM), a mitochondrial uncoupler, and two endoplasmic reticulum Ca²⁺ pump inhibitors, thapsigargin and cyclopiazonic acid. Conversely, pretreatment with lindane abolished CCCP‐ and thapsigargin‐induced Ca²⁺ release. This suggests that 0.15 mM lindane released Ca²⁺ from the endoplasmic reticulum, mitochondria and other stores. La³⁺ (1 mM) partly inhibited 0.1 mM lindane‐induced [Ca²⁺]_i increase, confirming that lindane induced Ca²⁺ influx. Addition of 3 mM Ca²⁺ increased [Ca²⁺]_i after pretreatment with 0.15 mM lindane for 750 sec. in Ca²⁺‐free medium, which indicates lindane‐induced capacitative Ca²⁺ entry. Lindane (0.15 mM)‐induced Ca²⁺ release was not reduced by inhibiting phospholipase C with 2 μM U73122, but was inhibited by 70% by the phospholipase A₂ inhibitor aristolochic acid (40 μM).     

Effect of the antidepressant paroxetine on Ca2+ movement in PC3 human prostate cancer cells

The effect of the antidepressant paroxetine on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in PC3 human prostate cancer cells is unclear. This study explored whether paroxetine changed basal [Ca²⁺]_i levels in suspended PC3 cells by using fura‐2 as a Ca²⁺‐sensitive fluorescent dye. Paroxetine at concentrations between 10–150 µM increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was reduced by 55% by removing extracellular Ca²⁺. Paroxetine‐induced Ca²⁺ influx was inhibited by the store‐operated Ca²⁺ channel blockers econazole and SK&F96365, the phospholipase A2 inhibitor aristolochic acid, and protein kinase C modulators. In Ca²⁺‐free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitors thapsigargin, 2,5‐di‐tert‐butylhydroquinone (BHQ), or cyclopiazonic acid (CPA) all abolished paroxetine‐induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 inhibited paroxetine‐induced [Ca²⁺]_i rise by 80%. Collectively, in PC3 cells, paroxetine induced [Ca²⁺]_i rise by causing phospholipase C‐dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via store‐operated Ca²⁺ channels in a manner regulated by protein kinase C and phospholipase A2. Drug Dev Res, 2009. © 2009 Wiley‐Liss, Inc.     

Effect of capsaicin on Ca2+ fluxes in Madin‐Darby canine renal tubular cells

The effect of capsaicin, a transient receptor potential vanniloid‐1 (TRPV1) receptor agonist, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in Madin Darby canine kidney (MDCK) cells is unclear. This study explored whether capsaicin changed basal [Ca²⁺]_i levels in suspended MDCK cells by using fura‐2 as a Ca²⁺‐sensitive fluorescent dye. Capsaicin at concentrations between 10–100 µM increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was reduced by 80% by removing extracellular Ca²⁺. Capsacin induced Mn²⁺ influx, leading to quench of fura‐2 fluorescence suggesting Ca²⁺ influx. This Ca²⁺ influx was inhibited by phospholipase A2 inhibitor aristolochic acid and the non‐selective Ca²⁺ entry blocker La³⁺, but not by store‐operated Ca²⁺ channel blockers nifedipine, econazole, and SK&F96365, and protein kinase C/A modulators. In Ca²⁺‐free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin abolished capsaicin‐induced Ca²⁺ release. Conversely, pretreatment with capsaicin partly reduced thapsigargin‐induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 did not alter capsaicin‐induced [Ca²⁺]_i rise. The TRPV1 receptor antagonist capsazepine also induced significant Ca²⁺ entry and Ca²⁺ release. Collectively, in MDCK cells, capsaicin induced [Ca²⁺]_i rises by causing phospholipase C‐independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via phospholipase A2‐regulated, La³⁺‐sensitive Ca²⁺ channels in a manner dissociated from stimulation of TRPV1 receptors. Drug Dev Res, 2009. © 2009 Wiley‐Liss, Inc.     

Modulation of cytosolic free calcium concentration by α1-adrenoceptors in rat atrial cells

Summary The effects of ₁-adrenoceptor stimulation by phenylephrine (PE) and -adrenoceptor stimulation by isoprenaline (ISO) on Ca²⁺ current (I_Ca) and free intracellular Ca²⁺ concentration ([Ca²⁺]_i) were studied in isolated atrial myocytes from rat hearts. PE did not significantly affect the magnitude of I_Ca, whereas large increases of peak I_Ca were observed in response to ISO. In electrically driven cells, PE evoked a concentration-dependent, gradual increase in diastolic [Ca²⁺]_i and, initially, an increase in the height of peak [Ca²⁺]_i transients. When the diastolic [Ca²⁺]_i was increased to a greater extent, the amplitude of [Ca²⁺]_i transients was decreased. Simultaneous measurements of [Ca²⁺]_i and membrane potential showed that the increase in diastolic [Ca²⁺]_i was associated with a depolarization of the membrane, and the greater amplitude of [Ca²⁺]_i transients with a prolongation of the action potential (AP). The PE-induced increase in diastolic [Ca²⁺]_i was eliminated when the cells were voltage-clamped at the original resting membrane potential (RP); under these conditions, an increase in [Ca²⁺]_i transients was observed in response to PE. ISO usually caused larger increases in the amplitude of [Ca²⁺]_i transients with only minor changes in diastolic [Ca²⁺]_i. These results suggest that PE and ISO increase the amplitude of [Ca²⁺]_i transients in rat atrium in different ways. The increase in [Ca²⁺]_i transients in response to -adrenoceptor stimulation is commonly thought to be mediated by a greater conductance of voltage-dependent Ca²⁺ channels causing a greater Ca²⁺ influx and a release of more Ca²⁺ from the sarcoplasmic reticulum during the AP. The increase in diastolic [Ca²⁺]_i in response to PE is probably a consequence of the depolarization of the membrane, possibly involving the voltage-dependent Na⁺-Ca²⁺ exchange mechanism. The increase in the amplitude of the [Ca²⁺]_i transients in response to PE may be ascribed both to the initial increase in diastolic [Ca²⁺]_i and the prolongation of the AP. Send offprint requests to H. Nawrath at the above address     

ROLE OF CYCLIC NUCLEOTIDES IN THE CONTROL OF CYTOSOLIC Ca2+ LEVELS IN VASCULAR ENDOTHELIAL CELLS

• 1 Endothelial cells have a key role in the cardiovascular system. Most endothelial cell functions depend on changes in cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) to some extent and Ca²⁺ signalling acts to link external stimuli with the synthesis and release of regulatory factors in endothelial cells. The [Ca²⁺]_i is maintained by a well‐balanced Ca²⁺ flux across the endoplasmic reticulum and plasma membrane.
• 2 Cyclic nucleotides, such as cAMP and cGMP, are very important second messengers. The cyclic nucleotides can affect [Ca²⁺]_i directly or indirectly (via the actions of protein kinase (PK) A or PKG‐mediated phosphorylation) by regulating Ca²⁺ mobilization and Ca²⁺ influx. Fine‐tuning of [Ca²⁺]_i is also fundamental to protect endothelial cells against damaged caused by the excessive accumulation of Ca²⁺.
• 3 Therapeutic agents that control cAMP and cGMP levels have been used to treat various cardiovascular diseases.
• 4 The aim of the present review is to discuss: (i) the functions of endothelial cells; (ii) the importance of [Ca²⁺]_i in endothelial cells; (iii) the impact of excessive [Ca²⁺]_i in endothelial cells; and (iv) the balanced control of [Ca²⁺]_i in endothelial cells via involvement of cyclic nucleotides (cAMP and cGMP) and their general effectors.

     

Effect of nortriptyline on cytosolic Ca2+ regulation and viability in PC3 human prostate cancer cells

The effect of nortriptyline, a tricyclic antidepressant, on Ca²⁺ regulation and viability in human prostate cancer cells (PC3) is unclear. The present study examined whether nortriptyline altered basal [Ca²⁺]_i levels in suspended PC3 cells using fura‐2 as a Ca²⁺‐sensitive fluorescent probe. Nortriptyline (50–500 µM) increased [Ca²⁺]_i in a concentration‐dependent fashion. The Ca²⁺ signal was partially reduced by removing extracellular Ca²⁺, indicating that Ca²⁺ entry and release both contributed to the [Ca²⁺]_i rise. Nortriptyline induced Mn²⁺ influx, leading to quench of fura‐2 fluorescence, suggesting Ca²⁺ influx. This Ca²⁺ influx was inhibited by activation of protein kinase C, but not by inhibition of L‐type Ca²⁺ channels. In Ca²⁺‐free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitor, thapsigargin nearly abolished nortriptyline‐induced Ca²⁺ release. Conversely, pretreatment with nortriptyline greatly reduced the inhibitor‐induced [Ca²⁺]_i rise, suggesting that nortriptyline released Ca²⁺ from the endoplasmic reticulum. Inhibition of phospholipase C did not change the nortriptyline‐induced [Ca²⁺]_i rise. Nortriptyline at a concentration of 10 µM increased viability in a Ca²⁺‐independent manner. At 50 µM, nortriptyline killed 45% of cells. Nortriptyline at 10 µM did not induce apoptosis, but at 50 µM induced significant apoptosis measured by propidium iodide staining. Together, in PC3 cells, nortriptyline induced [Ca²⁺]_i rises by causing the phospholipase C‐independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via the protein kinase C‐sensitive pathway. Nortriptyline also induced both cell proliferation and death in a concentration‐dependent manner. Apoptosis was involved in the cell death. Drug Dev Res 71:323–330, 2010. © 2010 Wiley‐Liss, Inc.