Ca2+/calmodulin‐dependent protein kinase IIδ orchestrates G‐protein‐coupled receptor and electric field stimulation‐induced cardiomyocyte hypertrophy

1. G‐Protein‐coupled receptors (GPCR) and electrical field stimulation (EFS) regulate cardiac function and pathological remodelling, including cardiac hypertrophy. Cardiac Ca²⁺/calmodulin‐dependent protein kinase (CaMK) IIδ expression and activity are altered in cardiac hypertrophy and heart failure. The aim of the present study was to determine the effects of CaMKIIδ isoforms on neonatal rat cardiomyocyte hypertrophy induced by GPCR and EFS. 2. Cardiac hypertrophy was induced by angiotensin II, phenylephrine or EFS and was confirmed by increases in cell volume, [³H]‐leucine incorporation, sarcomere assembly and mRNA expression of atrial natriuretic factor and β‐myosin heavy chain. The effects of the CaMKII inhibitors KN93 and autocamtide 2‐related inhibitory peptide (AIP) on cardiomyocyte hypertrophy were investigated, as was the effect of overexpression of dominate negative CaMKIIδ. 3. Cardiomyocyte hypertrophy was inhibited by the CaMKII inhibitors KN93 and AIP and by overexpression of dominate negative CaMKIIδ, but was potentiated by overexpression of wild‐type CaMKIIδB or CaMKIIδC. Activation of CaMKII by GPCR agonists or EFS was inhibited by the CaMKII inhibitors. 4. The GPCR agonists and EFS synergistically activated CaMKII and upregulated CaMKIIδB and CaMKIIδC mRNA expression and protein synthesis. All these effects were abolished by the CaMKII inhibitors. 5. The findings of the present study indicate that CaMKII orchestrates additive prohypertrophic factors between GPCR agonists and EFS. Thus, CaMKII may be a useful target in the clinical treatment of hypertrophy and cardiac remodelling.     

Ca2+/calmodulin dependent kinase II: A critical mediator in determining reperfusion outcomes in the heart?

Ischaemic heart disease is a major cause of death and disability in the Western world, and a substantial health burden. Cardiomyocyte Ca²⁺ overload is known to significantly contribute to contractile dysfunction and myocyte death in ischaemia and reperfusion, and significant advancements have been made in identifying the downstream mediators and cellular origins of this Ca²⁺ mismanagement. Ca²⁺/calmodulin‐dependent kinase II (CaMKII) is recognized as an important mediator linking pathological changes in subcellular environments to modifications in cardiomyocyte Ca²⁺ handling. Activated in response to fluctuations in cellular Ca²⁺ and to various post‐translational modifications, CaMKII targets numerous Ca²⁺ channels/transporters involved in Ca²⁺ handling and contractile function regulation. CaMKII is activated early in reperfusion, where it exacerbates Ca²⁺ leak from the sarcoplasmic reticulum and promotes the onset of ventricular arrhythmias. Inhibiting CaMKII can increase functional recovery in reperfusion and reduce apoptotic/necrotic death, at least partly through indirect and direct influences on mitochondrial Ca²⁺ levels and function. Yet, CaMKII can also have beneficial actions in ischaemia and reperfusion, in part by providing inotropic support for the stunned myocardium and contributing as an intermediate to cardioprotective preconditioning signalling cascades. There is considerable potential in targeting CaMKII as a part of a surgical reperfusion strategy, though further mechanistic understanding of the relationship between CaMKII activation status and the extent of ischaemia/reperfusion injury are required to fully establish an optimal pharmacological approach.     

The toxic effects of Bisphenol A on the mouse spermatocyte GC‐2 cell line: the role of the Ca2+‐calmodulin‐Ca2+/calmodulin‐dependent protein kinase II axis

Bisphenol A (BPA), an endocrine‐disrupting chemical (EDC), is known to induce male reproductive toxicity in rodents. However, its toxic effects on the germ cells are still poorly understood. It has been proposed that Ca²⁺ homeostasis and Ca²⁺ sensors, including calmodulin (CaM) and calmodulin‐dependent protein kinase II (CaMKII), play critical roles in spermatogenesis. Therefore, in the present study, we aimed to investigate whether a perturbation in Ca²⁺‐CaM‐CaMKII signaling was involved in the BPA‐induced injury to mouse spermatocyte GC‐2spd (ts) (GC‐2) cells. Our results showed that BPA (range from 0.2 to 20 μM) induced obvious GC‐2 cell injury, including decreased cell viability, the release of mitochondrial cytochrome c and the activation of caspase‐3. However, these processes could be partially abrogated by pretreatment with a Ca²⁺ chelator (BAPTA/AM), a CaM antagonist (W7) or a CaMKII inhibitor (KN93). These results, taken together, indicate that BPA exposure contributes to male germ cell injury, which may be partially mediated through a perturbation in Ca²⁺/CaM/CaMKII signaling and the mitochondrial apoptotic process. Copyright © 2015 John Wiley & Sons, Ltd.     

Involvement of calcium/calmodulin‐dependent protein kinase II in methamphetamine‐induced neural damage

Methamphetamine (METH), an illicit drug, is widely abused in many parts of the world. Mounting evidence shows that METH exposure contributes to neurotoxicity, particularly for the monoaminergic neurons. However, to date, only a few studies have tried to unravel the mechanisms involved in METH‐induced non‐monoaminergic neural damage. Therefore, in the present study, we tried to explore the mechanisms for METH‐induced neural damage in cortical neurons. Our results showed that METH significantly increased intracellular [Ca²⁺]_i in Ca²⁺‐containing solution rather than Ca²⁺‐free solution. Moreover, METH also upregulated calmodulin (CaM) expression and activated CaM‐dependent protein kinase II (CaMKII). Significantly, METH‐induced neural damage can be partially retarded by CaM antagonist W7 as well as CaMKII blocker KN93. In addition, L‐type Ca²⁺ channel was also proved to be involved in METH‐induced cell damage, as nifedipine, the L‐type Ca²⁺ channel‐specific inhibitor, markedly attenuated METH‐induced neural damage. Collectively, our results suggest that Ca²⁺‐CaM‐CaMKII is involved in METH‐mediated neurotoxicity, and it might suggest a potential target for the development of therapeutic strategies for METH abuse. Copyright © 2016 John Wiley & Sons, Ltd.     

Cardiotoxicity of sorafenib is mediated through elevation of ROS level and CaMKII activity and dysregulation of calcium homoeostasis

Sorafenib, a multi‐kinase inhibitor, is recommended as a new standard therapy for advanced hepatocellular carcinoma (HCC); however, it also exhibits severe cardiotoxicity and the toxicity mechanisms are not completely elucidated. Recent studies suggested that sorafenib‐enhanced ROS may partially contribute to its anti‐HCC effect, which implies that redox mechanism might also be involved in sorafenib's cardiotoxicity. In this study, we aimed to investigate if sorafenib is able to induce oxidative stress and how this may impair cellular functions in cardiomyocyte, ultimately accounting for its cardiotoxicity. Our results showed that in isolated rat hearts, sorafenib caused ventricular arrhythmias and left ventricular dysfunction, which were alleviated by the antioxidant N‐(2‐mercaptopropionyl)‐glycine (MPG). In isolated ventricular myocytes, sorafenib increased diastolic intracellular Ca²⁺ levels, decreased Ca transients and the occurrence of Ca²⁺ waves. These changes were eliminated by MPG, CaMKII inhibitor KN‐93 and the mitochondrial permeability transition pore (mPTP)inhibitor cyclosporin A (CsA). Moreover, the levels of oxidized and phosphorylated CaMKII were significantly increased. Sorafenib elevated ROS levels, which was reversed by CsA and MPG; additionally, sorafenib reduced the activity of mitochondrial complex III and augmented mitochondrial ROS production. In vivo rats treated with sorafenib exhibited a reduction of antioxidant defence and abnormal histological alterations including hypertrophy, increased fibrosis, disordered myofibrils and damaged mitochondria, which were protected by MPG. We conclude that sorafenib induces the disruption of Ca²⁺ homoeostasis and cardiac injury via enhanced ROS potentially through inhibiting mitochondrial complex III, the opening of mPTP and overactivating CaMKII. These results provide a potential strategy for preventing or reducing cardiotoxicity of sorafenib.     

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.     

AGE-RELATED SYNAPTIC CHANGES IN THE CA1 STRATUM RADIATUM AND SPATIAL LEARNING IMPAIRMENT IN RATS

• 1 Age‐related impairments in hippocampus‐dependent spatial learning and memory are not associated with a loss of neurons, but may be related to synaptic changes. In the present study, we analysed the behavioural performance of adult, middle‐aged and old Wistar rats using the Morris water maze, as well as the structure of synapses and the expression of autophosphorylated Ca²⁺/calmodulin‐dependent protein kinase II at threonine 286 (pThr286‐αCaMKII), a key post‐synaptic protein in the CA1 stratum radiatum, in the same rats.
• 2 Old Wistar rats showed significant cognitive deficits. Synaptic density, the area of post‐synaptic densities and the total number of synapses in the CA1 stratum radiatum of old rats were significantly decreased compared with adult rats. The decrease in autophosphorylated pThr286‐αCaMKII was age dependent.
• 3 These findings reveal that age‐related impairments in learning and memory are associated with synaptic atrophy. The decreased expression of pThr286‐CaMKII may result in reduced synaptic function with ageing.

     

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.     

Depotentiation of intact rat cardiac muscle unmasks an Epac‐dependent increase in myofilament Ca2+ sensitivity

Recently, a family of guanine nucleotide exchange factors have been identified in many cell types as important effectors of cyclic adenosine 3′,5′‐monophospahte (cAMP) signalling that is independent of protein kinase A (PKA). In the heart, investigation of exchange protein directly activated by cAMP (Epac) has yielded conflicting results. Since cAMP is an important regulator of cardiac contractility, this study aimed to examine whether Epac activation modulates excitation–contraction coupling in ventricular preparations from rat hearts. The study used 8‐(4‐chlorophenylthio)‐2′‐O‐methyladenosine‐3′, 5′‐cyclic monophosphate (cpTOME), an analogue of cAMP that activates Epac, but not PKA. In isolated myocytes, cpTOME increased Ca²⁺ spark frequency from about 7 to 32/100 μm³/s (n = 10), P = 0.05 with a reduction in the peak amplitude of the sparks. Simultaneous measurements of intracellular Ca²⁺ and isometric force in multicellular trabeculae (n = 7, 1.5 mmol/L [Ca²⁺]_o) revealed no effect of Epac activation on either the amplitude of Ca²⁺ transients (Control 0.7 ± 0.1 vs cpTOME 0.7 ± 0.1; 340/380 fura‐2 ratio, P = 0.35) or on peak stress (Control 24 ± 5 mN/mm² vs cpTOME 23 ± 5 mN/mm², P = 0.20). However, an effect of Epac in trabeculae was unmasked by lowering extracellular [Ca²⁺]_o. In these depotentiated trabeculae, activation of the Epac pathway increased myofilament Ca²⁺ sensitivity, an effect that was blocked by addition of KN‐93, a Ca²⁺/calmodulin‐dependent protein kinase II (CaMK‐II) inhibitor. This study suggests that Epac activation may be a useful therapeutic target to increase the strength of contraction during low inotropic states.     

Involvement of Calmodulin Kinase II in the Action of Sulphur Mustard on the Contraction of Vascular Smooth Muscle

Abstract: Sulphur mustard (SM) is an alkylating agent whose mechanism is not fully understood. To investigate the early action of SM, we examined the effect of SM on contraction of vascular smooth muscles. Phenylephrine (PE)‐induced contraction was reduced by SM, but only marginally by 70 mM KCl^‐. Additional reduction was induced by nifedipine in SM‐treated arteries. In the absence of extracellular Ca²⁺, contraction of arteries by PE was reduced, which was fully recovered by addition of 2 mM Ca²⁺. However, recovery was attenuated by pre‐treatment with SM. The effect of SM on contraction by PE was not influenced by pre‐ and post‐treatment with Phorbol 12, 13‐dibutyrate. Calmodulin kinase II (CaMKII) was implicated as being responsible for the action of SM, because the contractile mechanisms of vascular smooth muscle via both Ca²⁺‐calmodulin‐myosin light chain kinase axis and protein kinase C‐proline‐rich tyrosine kinase axis were not related to the action of SM. Elevation of phosphorylated CaMKII level by Ionomycin or PE was attenuated by treatment of SM on western blot. CaMKII may be a candidate target molecule of SM in early stage contraction of vascular smooth muscle.     

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.     

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.     

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.     

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.     

Activation of cardiac M3 muscarinic acetylcholine receptors has cardioprotective effects against ischaemia-induced arrhythmias

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Bitter tastants induce relaxation of rat thoracic aorta precontracted with high K+

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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 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 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.