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.     

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 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.     

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.     

Econazole‐induced Ca2+ fluxes and apoptosis in human oral cancer cells

The effect of econazole on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability was explored in human oral cancer cells (OC2), using the fluorescent dyes fura‐2 and WST‐1, respectively. Econazole at concentrations of >1 µM increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. The econazole‐induced Ca²⁺ influx was sensitive to blockade of aristolochic acid (phospholipase A₂ inhibitor) and GF109203X (PKC inhibitor). In Ca²⁺‐free medium, after treatment with 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), 30 µM econazole failed to induce a [Ca²⁺]_i rise. Inhibition of phospholipase C with 2 µM U73122 substantially suppressed econazole‐induced [Ca²⁺]_i rise. At concentrations of 5–70 µM econazole killed cells in a concentration‐dependent manner. The cytotoxic effect of 50 µM econazole was enhanced by prechelating cytosolic Ca²⁺ with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA). The ERK MAPK inhibitor, PD98059 (10 µM), also enhanced 20 µM econazole‐induced cell death. Propidium iodide staining data suggest that econazole induced apoptosis between concentrations of 10–70 µM. Collectively, in OC2 cells, econazole induced [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx from phospholipase A₂/PKC‐regulated Ca²⁺ channels. Furthermore, econazole caused cell death appeared to be regulated by ERK MAPK. Drug Dev Res 71: 240–248, 2010. © 2010 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.     

Cadmium‐induced Cytosolic Ca2+ Elevation and Subsequent Apoptosis in Renal Tubular Cells

Abstract: Cadmium (Cd²⁺) is an industrial and environmental metal. The effect of Cd²⁺ on intracellular free‐Ca²⁺ levels ([Ca²⁺]_i) and viability in Madin Darby canine kidney cells was explored. Cd²⁺increased [Ca²⁺]_i in a concentration‐dependent manner with an EC₅₀ of 85 µM. Cd²⁺‐induced Mn²⁺ entry demonstrated Ca²⁺ influx. Removal of extracellular Ca²⁺ decreased the [Ca²⁺]_i signal by 60%. The [Ca²⁺]_i signal was inhibited by La³⁺ but not by L‐type Ca²⁺ channel blockers. In Ca²⁺‐free medium, Cd²⁺‐induced [Ca²⁺]_i signal was abolished by pre‐treatment with 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺pump inhibitor) and 2 µM carbonylcyanide m‐chlorophenylhydrazone (CCCP; a mitochondrial uncoupler). Cd²⁺‐induced Ca²⁺ release was not altered by inhibition of phospholipase C. At concentrations between 10 and 100 µM, Cd²⁺killed cells in a concentration‐dependent manner. The cytotoxic effect of 100 µM Cd²⁺was reversed by pre‐chelating cytosolic Ca²⁺with BAPTA. Cd²⁺‐induced apoptosis was demonstrated by propidium iodide. Collectively, this study shows that Cd²⁺ induced a [Ca²⁺]_i increase in Madin Darby canine kidney cells via evoking Ca²⁺ entry through non‐selective Ca²⁺ channels, and releasing stored Ca²⁺ from endoplasmic reticulum and mitochondria in a phospholipase C‐independent manner.     

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.     

INVOLVEMENT OF PROTEIN KINASE C IN THE REGULATION OF Na+/Ca2+ EXCHANGER IN BOVINE ADRENAL CHROMAFFIN CELLS

• 1 The Na⁺/Ca²⁺ exchanger (NCX) exchanges Na+ and Ca²⁺ bidirectionally through the forward mode (Ca²⁺ extrusion) or the reverse mode (Ca²⁺ influx). The present study was undertaken to clarify the role of protein kinase C (PKC) in the regulation of NCX in bovine adrenal chromaffin cells. The Na⁺‐loaded cells were prepared by treatment with 100 µmol/L ouabain and 50 µmol/L veratridine. Incubation of Na⁺‐loaded cells with Na⁺‐free solution in the presence of the Ca²⁺ channel blockers nicardipine (3 µmol/L) and ω‐conotoxin MVIIC (0.3 µmol/L) caused Ca²⁺ uptake and catecholamine release.
• 2 The Na⁺‐dependent Ca²⁺ uptake and catecholamine release were inhibited by 2‐[4‐[(2,5‐difluorophenyl)methoxy]phenoxy]‐5‐ethoxyaniline (SEA0400; 1 µmol/L) and 2‐[2‐[4‐(4‐nitrobenzyloxy)phenyl]isothiourea (KB‐R7943; 10 µmol/L), both NCX inhibitors. These results indicate that the Na⁺‐dependent responses are mostly due to activation of the NCX working in the reverse mode.
• 3 In addition, we examined the effects of PKC inhibitors and an activator on the NCX‐mediated Ca²⁺ uptake and catecholamine release. Bisindolylmaleimide I (0.3–10 µmol/L) and chelerythrine (3–100 µmol/L), both PKC inhibitors, inhibited NCX‐mediated responses. In contrast, phorbol 12,13‐dibutyrate (0.1–10 µmol/L), a PKC activator, enhanced the responses. Bisindolylmaleimide I and chelerythrine, at effective concentrations for inhibition of Na⁺‐dependent catecholamine release, had a little or no effect on high K⁺‐induced catecholamine release in intact cells or on Ca²⁺‐induced catecholamine release in β‐escin‐permeabilized cells.
• 4 These results suggest that PKC is involved in the activation of NCX in bovine adrenal chromaffin cells.

     

Maprotiline‐induced Ca2+ fluxes and apoptosis in human osteosarcoma cells

The effect of maprotiline on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and cell viability was explored in human osteosarcoma cells (MG63), using the fluorescent dyes fura‐2 and WST‐1, respectively. Maprotiline at concentrations of ≥20 µM increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca²⁺. The maprotiline‐induced Ca²⁺ influx was sensitive to inhibition by aristolochic acid (a phospholipase A₂ inhibitor). In Ca²⁺‐free medium, after treatment with 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), 200 µM maprotiline failed to induce a [Ca²⁺]_i rise. At concentrations of 50–100 µM maprotiline killed cells in a concentration‐dependent manner. The cytotoxic effect of 60 µM maprotiline was slightly enhanced by prechelating cytosolic Ca²⁺ with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA). Propidium iodide staining data suggested that maprotiline induced apoptosis between concentrations of 60–70 µM, which was enhanced by BAPTA. Collectively, in MG63 cells, maprotiline induced [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx from phospholipase A₂‐regulated Ca²⁺ channels. Furthermore, maprotiline caused apoptosis that was regulated by Ca²⁺. Drug Dev Res 71: 268–274, 2010. © 2010 Wiley‐Liss, Inc.     

Effect of Methoxychlor on Ca(2+) Movement and Viability in MDCK Renal Tubular Cells

The effect of the insecticide methoxychlor on the physiology of renal tubular cells is unknown. This study aimed to explore the effect of methoxychlor on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) in MDCK renal tubular cells using the Ca²⁺‐sensitive fluorescent dye fura‐2. Methoxychlor at 5–20 μM increased [Ca²⁺]_i in a concentration‐dependent manner. The signal was reduced by 80% by removing extracellular Ca²⁺. Methoxychlor‐induced Ca²⁺ entry was not affected by nifedipine and SK&F96365 but was inhibited by econazole and protein kinase C modulators. In Ca²⁺‐free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5‐di‐tert‐butylhydroquinone (BHQ) partly inhibited methoxychlor‐induced [Ca²⁺]_i rise. Incubation with methoxychlor also inhibited thapsigargin‐ or BHQ‐induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 nearly abolished methoxychlor‐induced [Ca²⁺]_i rise. At 5–15 μM, methoxychlor slightly increased cell viability, whereas at 20 μM, it decreased viability. The cytotoxic effect of methoxychlor was not reversed by chelating cytosolic Ca²⁺ with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N,N‐tetraacetic acid/AM (BAPTA/AM). Annexin V‐FITC data suggest that 10 μM methoxychlor inhibited apoptosis, while 20 μM methoxychlor enhanced apoptosis. Methoxychlor (10 and 20 μM) increased the production of reactive oxygen species. Together, in renal tubular cells, methoxychlor induced [Ca²⁺]_i rise by inducing phospholipase C‐dependent Ca²⁺ release from multiple stores and Ca²⁺ entry via protein kinase C‐ and econazole‐sensitive channels. Methoxychlor slightly enhanced or inhibited cell viability in a concentration‐dependent, Ca²⁺‐independent manner. Methoxychlor induced cell death that may involve apoptosis via mitochondrial pathways.     

Effect of thimerosal on Ca2+ movement and apoptosis in PC3 prostate cancer cells

The present study evaluated the effects of thimerosal, a vaccine preservative, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in human prostate cancer cells (PC3). Thimerosal (10–200 µM) increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Thimerosal‐induced Ca²⁺ influx was inhibited by econazole, SKF963656, the phospholipase A₂ inhibitor aristolochic acid, and protein kinase C modulators [phorbol 12‐myristate 13‐acetate (PMA) and GF109203X]. In Ca²⁺‐free medium, a 200‐µM thimerosal‐induced [Ca²⁺]_i rise was partly inhibited after pretreatment with 2,5‐di‐tert‐butylhydroquinone (BHQ) (an endoplasmic reticulum Ca²⁺ pump inhibitor). Thimerosal at 1–7 µM induced cell death in a concentration‐dependent manner that was not reversed when cytosolic Ca²⁺ was chelated with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA). Propidium iodide staining suggests that apoptosis played a role in the death. Collectively, in PC3 cells, thimerosal induced [Ca²⁺]_i rise by causing 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. Thimerosal also induced cell death in a Ca²⁺‐independent apoptotic manner. Drug Dev Res 72: 330–336, 2011. © 2011 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.     

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.     

Quinidine enhances intracellular Ca2+ accumulation during rapid stimulation

• 1.1. The quinidine-induced modification of intracellular Ca²⁺ concentration ([Ca²⁺]_i) was studied in guinea-pig myocardium using fura-2. Quinidine reduced the systolic fluorescence signal level for [Ca²⁺]_i and enhanced the end-diastolic signal level during a stimulation train.
• 2.2. The diastolic decay of [Ca²⁺]_i fitted 2 exponential curves. Quinidine distorted the stimulation frequency-dependent acceleration of rapid [Ca²⁺]_i decay, and prolonged the mean time constant of rapid decay after 2 Hz stimulation, from 154.4 to 205.3 msec (20 μM), and to 259.7 msec (60 μM quinidine). The time constant of slow recovery from [Ca²⁺]_i accumulation after the stimulation train was not affected by stimulation frequency, or by quinidine, or caffeine.
• 3.3. These results suggest that quinidine modulates [Ca²⁺]_i via a balance between the slowing of rapid [Ca²⁺]_i decay and the reduction of the systolic [Ca²⁺]_i. This effect may contribute to the anti-arrhythmic and pro-arrhythmic effects exerted by quinidine in some conditions.

     

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.     

Effect of capsazepine on [Ca2+]i in MDCK renal tubular cells

The current study explored whether capsazepine changed basal cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) levels in suspended Madin Darby canine kidney (MDCK) cells cells by using fura‐2 as a Ca²⁺‐selective fluorescent dye. At concentrations of 10–200 µM, capsazepine increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was partially reduced by 40% by removing extracellular Ca²⁺. Capsazepine induced Mn²⁺ quench of fura‐2 fluorescence, indirectly implicating Ca²⁺ entry. Capsazepine‐induced Ca²⁺ influx was unchanged by L‐type Ca²⁺ entry inhibitors and protein kinase C modulators [phorbol 12‐myristate 13‐acetate (PMA) and GF109203X]. In Ca²⁺‐free medium, 100 µM capsazepine‐induced Ca²⁺ release was substantially suppressed by pretreatment with thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor). Pretreatment with capsazepine nearly abolished thapsigargin‐induced Ca²⁺ release. Inhibition of phospholipase C with U73122 did not change capsazepine‐induced [Ca²⁺]_i rises. Collectively, in MDCK cells, capsazepine induced [Ca²⁺]_i rises by causing phospholipase C‐independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via non‐L‐type Ca²⁺ channels. Drug Dev Res 72: 323–329, 2011. © 2010 Wiley‐Liss, Inc.     

ROLE OF EXTRACELLULAR Na+, Ca2+-ACTIVATED Cl- CHANNELS AND BK CHANNELS IN THE CONTRACTION OF Ca2+ STORE-DEPLETED TRACHEAL SMOOTH MUSCLE

• 1 In the present study, we investigated the series of events involved in the contraction of tracheal smooth muscle induced by the re‐addition of Ca²⁺ in an in vitro experimental model in which Ca²⁺ stores had been depleted and their refilling had been blocked by thapsigargin.
• 2 Mean (±SEM) contraction was diminished by: (i) inhibitors of store‐operated calcium channels (SOCC), namely 100  µ mol/L SKF‐96365 and 100  µ mol/L 1‐(2‐trifluoromethylphenyl) imidazole (to 66.3 ± 4.4 and 41.3 ± 5.2% of control, respectively); (ii) inhibitors of voltage‐gated Ca²⁺ channels Ca_V1.2 channels, namely 1  µ mol/L nifedipine and 10  µ mol/L verapamil (to 86.2 ± 3.4 and 76.9 ± 5.9% of control, respectively); and (iii) 20  µ mol/L niflumic acid, a non‐selective inhibitor of Ca²⁺‐dependent Cl^? channels (to 41.1 ± 9.8% of control). In contrast, contraction was increased 2.3‐fold by 100 nmol/L iberiotoxin, a blocker of the large‐conductance Ca²⁺‐activated K⁺ (BK) channels.
• 3 Furthermore, contraction was significantly inhibited when Na⁺ in the bathing solution was replaced by N‐methyl–d ‐glucamine (NMDG⁺) to 39.9 ± 7.2% of control, but not when it was replaced by Li⁺ (114.5 ± 24.4% of control). In addition, when Na⁺ had been replaced by NMDG⁺, contractions were further inhibited by both nifedipine and niflumic acid (to 3.0 ± 1.8 and 24.4 ± 8.1% of control, respectively). Nifedipine also reduced contractions when Na⁺ had been replaced by Li⁺ (to 10.7 ± 3.4% to control), the niflumic acid had no effect (116.0 ± 4.5% of control).
• 4 In conclusion, the data of the present study demonstrate the roles of SOCC, BK channels and Ca_V1.2 channels in the contractions induced by the re‐addition of Ca²⁺ to the solution bathing guinea‐pig tracheal rings under conditions of Ca²⁺‐depleted sacroplasmic reticulum and inhibition of sarcoplasmic/endoplasmic reticulum calcium ATPase. The contractions were highly dependent on extracellular Na⁺, suggesting a role for SOCC in mediating the Na⁺ influx.

     

Serotonin but not norepinephrine-induced calcium mobilization of platelets is enhanced in affective disorders

Intracellular calcium concentrations ([Ca⁺⁺]_i) in blood platelets from 11 depressed patients and 11 healthy controls were investigated. The resting [Ca⁺⁺]_i of platelets from depressed patients was 69.4±2.9 nM while that from controls was 74.6±4.0 nM. Serotonin (5-HT), at a concentration of 10 µM, increased [Ca⁺⁺]_i by 129.2±3.9 nM in platelets from depressed patients, which was significantly greater than that found in platelets from control subjects (105.2±6.0 nM). Norepinephrine (NE) 100 µM increased [Ca⁺⁺]_i by 46.1±7.1 nM in platelets from depressed patients, and by 38.6±6.1 nM in platelets from controls, respectively. These results indicate that 5-HT₂ receptor function in platelets of depressed patients is enhanced, and support the hypothesis of hypersensitivity of 5-HT₂ receptors in affective disorders.     

Potentiation by endothelin-1 of Ca2+ sensitivity of contractile elements depends on Ca2+ influx through L-type Ca2+ channels in the canine cerebral artery

• 1.1. Endothelin-1 (ET-1) contracted canine cerebral artery in a concentration-dependent manner with an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i), and at higher concentrations it produced a greater contraction with a smaller increase in [Ca²⁺]_i.
• 2.2. Ca²⁺ channel antagonist such as d-cis-diltiazem inhibited the tension more effectively than the [Ca²⁺]_i increased by ET-1.
• 3.3. In Ca²⁺-free solution containing 0.2 mM EGTA, ET-1 elicited a transient increase in [Ca²⁺]_i and tension.
• 4.4. In the Staphylococcus aureus α-toxin-permeabilized artery, ET-1 shifted the pCa-tension relationship leftwards in the presence of GTP.
• 5.5. These findings suggest that ET-I contracts the canine cerebral artery by increasing not only the Ca²⁺ influx through L-type Ca²⁺ channels, but also Ca²⁺ release from the intracellular storage sites, and also Ca²⁺ sensitivity of contractile elements. The degree of Ca²⁺ sensitivity is strongly affected by [Ca²⁺]_i which is increased by the Ca²⁺ influx through L-type Ca²⁺ channels.