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.     

Statins rise cytoplasmic calcium level [Ca2+]i in cultured endothelial cells

Recently, we have shown that some HMG-CoA reductase inhibitors (statins) induce immediate pleiotropic effects in vascular endothelium both in vivo and in vitro, to mention only PGI2-mediated thrombolysis in rats and NO-mediated endothelium-dependent vasodilation in guinea pig coronary circulation. Here we look whether immediate endothelial effect of statins is associated with mobilization of intracellular calcium ions [Ca2+]i in cultured bovine aortic endothelial cells (BAEC). We analyzed the effects of various statins (atorvastatin, cerivastatin, simvastatin, lovastatin and pravastatin at concentration of 10-30 microM) on [Ca2+]i in BAEC in comparison to responses induced by bradykinin (Bk) (10 nM), adenosine diphosphate (1 microM), acetylcholine (100 nM), adrenaline (10 microM), serotonin (10 microM) or calcium ionophore A 23187 (0.1 microM) using FURA-2 according to fluorimetric method of Grynkiewicz et al. Basal [Ca2+]i level in BAEC was between 60 and 100 nM. Bk was the most potent to induce [Ca2+]i response. Delta[Ca2+]i induced by Bk was 331.9 +/- 19.49 nM (n = 36). Delta[Ca2+]i induced by statins (30 microM), i.e. atorvastatin, cerivastatin, simvastatin, lovastatin and pravastatin were 66.4 +/- 7.38% (n = 6), 54.8 +/- 10.12% (n = 5), 58.8 +/- 13.9% (n = 8), 27.7 +/- 7.19% (n = 5) and 0% (n = 5) of the response induced by Bk (10 nM), respectively. In summary, all statins tested, except pravastatin, induce immediate increase in [Ca2+]i in endothelium. This pleiotropic activity of statins in endothelium, most likely not related to the inhibition of HMG-CoA reductase, may represent an intracellular correlate for the immediate release of NO and PGI2 by these drugs that was reported by us previously.     

The mechanism of sertraline-induced [Ca2+]i rise in human OC2 oral cancer cells

Effect of sertraline, an antidepressant, on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in human cancer cells is unclear. This study examined if sertraline altered basal [Ca(2+)](i) levels in suspended OC2 human oral cancer by using fura-2 as a Ca(2+)-sensitive fluorescent probe. At concentrations of 10-100 μM, sertraline induced a [Ca(2+)](i) rise in a concentration-dependent fashion. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+) indicating that Ca(2+) entry and release both contributed to the [Ca(2+)](i) rise. Sertraline induced Mn(2+) influx, leading to quench of fura-2 fluorescence suggesting Ca(2+) influx. This Ca(2+) influx was inhibited by suppression of phospholipase A2, inhibition of store-operated Ca(2+) channels or by modulation of protein kinase C activity. In Ca(2+)-free medium, pretreatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) nearly abolished sertraline-induced Ca(2+) release. Conversely, pretreatment with sertraline greatly reduced the inhibitor-induced [Ca(2+)](i) rise, suggesting that sertraline released Ca(2+) from the endoplasmic reticulum. Inhibition of phospholipase C did not change sertraline-induced [Ca(2+)](i) rise. Together, in human oral cancer cells, sertraline induced [Ca(2+)](i) rises by causing phospholipase C-independent Ca(2+) release from the endoplasmic reticulum and Ca(2+) influx via store-operated Ca(2+) channels.     

Lysophospholipids elevate [Ca2+]i and trigger exocytosis in bovine chromaffin cells

Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) are responsible for many physiological functions, including angiogenesis, neuronal survival, and immunity. However, little is known about their effects in modulating the stimulus-secretion coupling in bovine chromaffin cells. The result of PCR showed that at least two receptors (S1P(3) and LPA(1)) were expressed in bovine chromaffin cells. The elevation of [Ca(2+)](i) by S1P was fast and sustaining; but the elevation by LPA was slow and transient. The EC(50) for S1P and LPA in elevating the [Ca(2+)](i) were 0.55+/-0.01 and 0.54+/-0.40microM, respectively. This elevation could be totally blocked by thapsigargin, 2-APB, and U73122. Pertussis toxin pretreatment inhibited about half of the elevation in [Ca(2+)](i) suggesting the involvement of G(i) and other G-proteins. Repetitive [Ca(2+)](i) elevations elicited by S1P, but not LPA, were inhibited by ryanodine. S1P was more effective than LPA in triggering exocytosis as measured by the changes in membrane capacitance. The whole-cell Ca(2+) current was inhibited by both lysophospholipids but Na(+) current was inhibited by S1P only. These results suggest the differential effects of LPA and S1P in releasing Ca(2+) from the intracellular Ca(2+) stores and modulating the stimulus-secretion coupling in bovine chromaffin cells.     

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.     

Effect of calmidazolium on [Ca2+]i and viability in human hepatoma cells

The effect of calmidazolium on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability has not been explored in human hepatoma cells. This study examined whether calmidazolium altered [Ca²⁺]_i and caused cell death in HA59T cells. [Ca²⁺]_i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Calmidazolium at concentrations ≥1 μM increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 1.5 μM. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Calmidazolium induced Mn²⁺ quench of fura-2 fluorescence implicating Ca²⁺ influx. The Ca²⁺ influx was insensitive to L-type Ca²⁺ entry blockers, but was inhibited partly by enhancing or inhibiting protein kinase C activity. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), calmidazolium-induced [Ca²⁺]_i rises were largely inhibited; and conversely, calmidazolium pretreatment totally suppressed thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 μM U73122 did not change calmidazolium-induced [Ca²⁺]_i rises. At concentrations between 1 and 15 μM, calmidazolium induced apoptosis-mediated cell death. Collectively, in HA59T hepatoma cells, calmidazolium induced [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca²⁺ influx via protein kinase C-regulated Ca²⁺ entry pathway. Calmidazolium caused cytotoxicity via apoptosis.     

Melittin-induced [Ca2+]i increases and subsequent death in canine renal tubular cells

The effect of melittin on cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and viability is largely unknown. This study examined whether melittin alters Ca(2+) levels and causes Ca(2+)-dependent cell death in Madin-Darby canine kidney (MDCK) cells. [Ca(2+)](i) and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. Melittin at concentrations above 0.5 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced by 75% by removing extracellular Ca(2+). The melittin-induced Ca(2+) influx was also implicated by melittin-caused Mn(2+) influx. After pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), melittin-induced Ca(2+) release was inhibited; and conversely, melittin pretreatment abolished thapsigargin-induced Ca(2+) release. At concentrations of 0.5-20 microM, melittin killed cells in a concentration-dependent manner. The cytotoxic effect of 0.5 microM melittin was nearly completely reversed by prechelating cytosolic Ca(2+) with BAPTA. Melittin at 0.5-2 microM caused apoptosis as assessed by flow cytometry of propidium iodide staining. Collectively, in MDCK cells, melittin induced a [Ca(2+)](i) rise by causing Ca(2+) release from endoplasmic reticulum and Ca(2+) influx from extracellular space. Furthermore, melittin can cause Ca(2+)-dependent cytotoxicity in a concentration-dependent manner.     

甲状旁腺素对大鼠心肌细胞内游离钙和细胞凋亡的影响

目的　研究甲状旁腺素 (PTH)对心肌细胞内游离钙以及细胞肥大和凋亡的影响。方法　利用培养的新生大鼠心肌细胞 ,以Fluo 3/AM负载 ,通过激光共聚焦显微镜(LSCM)测定细胞内游离钙浓度 ([Ca2 + ] i) ;以细胞面积和细胞蛋白含量作为心肌细胞肥大指标 ;采用电镜和流式细胞术观察细胞凋亡的变化。结果　PTH1～ 34 0 0 1和 0 1 μmol·L- 1 刺激 7d后 ,心肌细胞内钙荧光强度以及心肌细胞面积和蛋白含量、细胞凋亡率较对照组显著增加。而 0 1 μmol·L- 1 PTH1～ 34 刺激的同时分别加入 1、1 0 μmol·L- 1 硝苯地平 ,上述指标改善 ,但未能达正常。结论　PTH1～ 34 可显著增加心肌细胞 [Ca2 + ] i,诱导细胞肥大和凋亡 ,并呈浓度依赖性 ,电压依赖性钙通道开放引起的细胞外钙内流增加为其机制之一     

Effect of APGW-amide on [Ca2+]i in rat pheochromocytoma PC12 cells

In order to determine whether Ala-Pro-Gly-Try-NH2 (APGW-amide) could affect mammalian excitable cells, we investigated the effect of APGW-amide in PC12 cells. APGW-amide caused a rapid [Ca2+]i elevation, which was completely prevented by elimination of extracellular Ca2+ with EGTA and inhibited by two L-type Ca2+ channel blockers. [Ca2+]i elevation was also blocked by a specific PKC inhibitor and prolonged pretreatment of cells with PMA. These results indicate that APGW-amide elevates [Ca2+]i in PC12 cells, possibly by Ca2+ influx via L-type Ca2+ channel activated by PKC.     

Vasopressin-evoked [Ca2+]i responses in neonatal rat cardiomyocytes.

The presence of arginine vasopressin (AVP) V1 receptors on neonatal rat cardiomyocytes (NRCs) linked to processes capable of elevating intracellular free calcium ([Ca2+]i) is now firmly established. This study examined the sources and signaling involved in [Ca2+]i elevations evoked by AVP in NRCs. AVP promoted increases in both [Ca2+]i and 1,4,5-inositoltrisphosphate (IP3) levels in NRCs. The degree of [Ca2+]i elevation was less than that of angiotensin II, but greater than that of endothelin-1. Extracellular Mg2+ depletion led to diminution of the maximal [Ca2+]i response, with a rightward shift in the concentration-response curves to AVP. The phospholipase C inhibitors, D-609, NCDC, or U73122, and the IP3 receptor blocker, heparin, abolished the [Ca2+]i response to AVP. Neither cyclooxygenase inhibition with indomethacin nor PKC inhibition with staurosporine had any effect. Neither ryanodine nor caffeine, which deplete sarcoplasmic reticulum (SR) Ca2+ stores, nor ruthenium red, which inhibits both SR and mitochondrial Ca2+ stores, affected [Ca2+]i responses to AVP. The SR Ca2+ pump inhibitor, cyclopiazonic acid, abolished, and removal of extracellular Ca2+ attenuated, the response to AVP. These data indicate that activation of cardiac V1 receptors by AVP results in mobilization of Ca2+ from a distinct, non-SR, nonmitochondrial, intracellular Ca2+ pool that is Ca2+ pump replenished and IP3 sensitive. This process occurs secondary to phospholipase C (PLC)-mediated generation of IP3, requires the presence of Mg2+ and extracellular Ca2+, and occurs in a manner independent of PKC and cyclooxygenase activation. Such mechanisms of Ca2+ mobilization might indicate a distinct role for AVP in cardiac physiology and disease.     

DA-6034 Induces [Ca2+]i Increase in Epithelial Cells

DA-6034, a eupatilin derivative of flavonoid, has shown potent effects on the protection of gastric mucosa and induced the increases in fluid and glycoprotein secretion in human and rat corneal and conjunctival cells, suggesting that it might be considered as a drug for the treatment of dry eye. However, whether DA-6034 induces Ca²⁺ signaling and its underlying mechanism in epithelial cells are not known. In the present study, we investigated the mechanism for actions of DA-6034 in Ca²⁺ signaling pathways of the epithelial cells (conjunctival and corneal cells) from human donor eyes and mouse salivary gland epithelial cells. DA-6034 activated Ca²⁺-activated Cl^- channels (CaCCs) and increased intracellular calcium concentrations ([Ca²⁺]_i) in primary cultured human conjunctival cells. DA-6034 also increased [Ca²⁺]_i in mouse salivary gland cells and human corneal epithelial cells. [Ca²⁺]_i increase of DA-6034 was dependent on the Ca²⁺ entry from extracellular and Ca²⁺ release from internal Ca²⁺ stores. Interestingly, these effects of DA-6034 were related to ryanodine receptors (RyRs) but not phospholipase C/inositol 1,4,5-triphosphate (IP₃) pathway and lysosomal Ca²⁺ stores. These results suggest that DA-6034 induces Ca²⁺ signaling via extracellular Ca²⁺ entry and RyRs-sensitive Ca²⁺ release from internal Ca²⁺ stores in epithelial cells.     

Regulation of [Ca2+]i rise activated by doxepin-sensitive H1-histamine receptors in jurkat cells,cloned human T lymphocytes

To clarify the presence of histamine receptor and its transmembrane mechanism in human T lymphocytes, we investigated the effects of agonists or antagonists of histamine receptor subtypes and bacterial toxins on intracellular concentration of Ca²⁺ [Ca²⁺]_i, [³H]pyrilamine binding and c-fos mRNA expression in Jurkat cells, cloned human T lymphocytes. H₁-agonists (histamine and 2-methyl-histamine) caused a transient rise of [Ca²⁺], and H₁-antagonists (pyrilamine and doxepin) inhibited the histamine-induced [Ca²⁺]_i rise more potently than the H₂-antagonist (cimetidine) on the H₃-antagonist (impromidine). Binding parameters of [³H]pyrilamine binding were K_d=5.53 nM and B_max = 2,647 sites/ cell. Pretreatment with B.pertussis, V. cholera. orC. botulinum toxin did not influence histamine-induced [Ca²⁺]_i rise. Western Blot analysis using antibodies against subunits of GTP-binding proteins indicated that G_q/G₁₁ richly existed in Jurkat cells. Histamine induced mRNA expression of an immediate early gene c-fos. Pretreatment with a protein kinase C activator, phorbol 12-myristate 13-acetate, caused almost complete inhibition of histamine-induced [Ca²⁺]_i rise, but did not do so by activators of cAMP- and cGMP-dependent protein kinases.     

Effect of protriptyline on [Ca2+]i and viability in MG63 human osteosarcoma cells

Tricyclic antidepressants (TCA) have been clinically prescribed in the auxiliary treatment of cancer patients. Although protriptyline, a type of TCA, was used primarily in the clinical treatment of mood disorders in cancer patients, the effect of protriptyline on physiology in human osteosarcoma is unknown. This study examined the effect of protriptyline on cytosolic free Ca^2+?concentrations ([Ca²⁺]_i) and viability in MG63 human osteosarcoma cells. Protriptyline between 50 and 250?μM evoked [Ca²⁺]_i rises concentration-dependently. Protriptyline induced influx of Mn²⁺, indirectly implicating Ca^2+?influx. Protriptyline-evoked Ca^2+?entry was inhibited by nifedipine by 20% but was not altered by econazole, SKF96365, GF109203X, and phorbol-12-myristate-13-acetate (PMA). In Ca²⁺-free medium, treatment with protriptyline inhibited the endoplasmic reticulum Ca^2+?pump inhibitor thapsigargin-evoked [Ca²⁺]_i rises. Conversely, treatment with thapsigargin inhibited 45% of protriptyline-evoked [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) with U73122 failed to alter protriptyline-evoked [Ca²⁺]_i rises. Protriptyline at 50–250?μM decreased cell viability, which was not reversed by pretreatment with the Ca^2+?chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Collectively, our data suggest that in MG63 cells, protriptyline induced [Ca²⁺]_i rises by evoking Ca^2+?release from the endoplasmic reticulum and other stores in a PLC-independent manner, and Ca^2+?entry via a nifedipine-sensitive Ca^2+?pathway. Protriptyline also caused Ca²⁺-independent cell death.     

在地昔帕明诱导胶质瘤C6细胞凋亡过程中caspase3基因表达和[Ca^2＋]i稳态^1

目的：研究抗抑郁药地昔帕明（Des)对胶质瘤C6细胞的凋亡诱导作用以及对凋亡关键效应分子caspase3和凋亡早期信号[Ca^2 ]i的调控作用。方法：采用流式细胞术（FCM）和凝胶电泳观察Des对C6细胞凋亡的DNA裂解作用,RT－PCR分析caspase3基因的表达以及激光扫描共聚焦显微镜测量单个活细胞[Ca^2 ]i浓度。结果：Des(10,20,40μmol/L)处理C6细胞24h后,FCM图的G1峰左侧出现凋亡特征性亚二倍体细胞峰,凋亡细胞百分率分别为5.2%,21.9%和41.9%。同时,凝胶电泳显示典型的DNA“梯带”.Des 20 μmol/L处理C6细胞24h可明显增强caspase 3基因的表达,而未经Des处理的C6细胞则检测不到caspase 3基因的表达。此外,Des 40μmol/L可使C6细胞[Ca^2 ]i迅速升高并维持超过28min,而钙螯合剂依他酸可显著降低C6细胞[Ca^2 ]i增高幅度,提示Des致C6细胞[Ca^2 ]i增高主要与细胞外钙内流有关。结论：Des诱导C6胶质瘤细胞凋亡可能与caspase 3基因表达的上调以及细胞内钙稳态的失衡有关。     

Group 1 mGlu receptors elevate [Ca2+]i in rat cultured cortical type 2 astrocytes: [Ca2+]i synergy with adenosine A1 receptors.

Brain macroglia are known to express a diverse array of neurotransmitter receptors whose signal transduction pathways may be subject to heteroreceptor 'cross-talk'. In the current study we have examined group 1 mGlu receptor-evoked [Ca2+]i signalling, and possible heteroreceptor cross-talk, in cultured type 2 astrocytes. The selective group 1 metabotropic glutamate (mGlu) receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG) elevated [Ca2+]i (EC50 = 1.7 +/- 0.6 microM); an effect reversed by the selective mGlu receptor antagonist (S)-alpha-methyl-4-carboxyphenylglycine (IC50 = 52.7 +/- 8.7 microM). DHPG-evoked [Ca2+]i responses were abolished by (1) thapsigargin (100 nM), implicating the involvement of internal Ca2+ stores in group 1 mGlu [Ca2+]i responses and (2) the removal of extracellular Ca2+. When applied alone, the selective adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA, 100 nM) failed to influence [Ca2+]i. However, in the presence of 1 microM DHPG, CPA potently (EC50 = 12.3 +/- 1.9 nM) increased [Ca2+]i responses. In the presence of 100 nM CPA, the efficacy of DHPG was doubled without any significant change in the DHPG EC50 value. This effect was reversed by either the selective adenosine A1 receptor antagonist, 8-cyclopentyltheophylline (IC50 = 50.3 +/- 19.9 nM) or overnight incubation with Pertussis toxin (100 ng/ml). We conclude that (1) type 2 astrocytes contain group 1 mGlu receptors coupled to [Ca2+]i signalling and (2) co-activation of adenosine A1 receptors enhances group 1 mGlu-evoked [Ca2+]i responses in these cells via a Gi/o G protein-mediated mechanism.     

Modulation of carbachol-induced [Ca2+]i oscillations by Ca2+ influx in single intestinal smooth muscle cells.

1. Oscillations of cytosolic Ca2+ concentration ([Ca2+]i) evoked by carbachol (CCh; 2 microM), a muscarinic agonist, were detected as oscillatory changes of muscarinic receptor-coupled cationic current (Icat) in guinea-pig ileal smooth muscle cells by the whole cell patch-clamp technique. 2. Reduction of extracellular Ca2+ from 2 mM to 0.2 or 0.05 mM, during CCh-induced Icat oscillations, caused them to disappear or to decrease markedly in frequency. A return to 2 mM Ca2+ concentration restored the initial Icat oscillations. 3. Application of nifedipine (1-3 microM) or D600 (2-5 microM) to block the voltage-gated Ca2+ channel (VGCC) decreased the frequency of the ongoing Icat oscillations in the cells held at -20 mV, but it was without effect in cells held at -60 mV. 4. Displacement of the holding potential of -20 mV to -60 mV to deactivate VGCC produced a decrease, an increase or no noticeable change in the frequency of the Icat oscillations in different cells. Displacement to 20 mV to inactivate VGCC invariably produced a decrease in the frequency. In nifedipine-treated cells, the Icat oscillations varied in frequency voltage-dependently in a reverse and linear way within the range -80 to 40 mV. 5. Application of thapsigargin (1 or 2 microM), an inhibitor of Ca(2+)-ATPase in the membrane of internal Ca2+ stores, caused CCh-induced Icat oscillations to disappear with a progressing phase during which their amplitude, but not frequency, declined. 6. The results suggest that membrane Ca2+ entry has a crucial role to play in regulation of the frequency of CCh-induced [Ca2+]i oscillations in addition to persistence of their generation, and that the effect is brought about by a potential mechanism independent of Ca2+ store replenishment. They also provide evidence that two types of Ca2+ permeant channels, VGCC and an as yet unidentified channel, are involved in the Ca2+ entry responsible for modulation of [Ca2+]i oscillations.     

Effect of melamine on [Ca2+]i and viability in PC3 human prostate cancer cells

Melamine is thought to be an endocrine disrupter that affects physiology in cells. This study examined the effect of melamine on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells. Melamine evoked [Ca²⁺]_i rises concentration-dependently. Melamine-evoked Ca²⁺ entry was inhibited by nifedipine, econazole, SKF96365, GF109203X and phorbol 12-myristate 13 acetate. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin inhibited melamine-evoked [Ca²⁺]_i rise. Conversely, treatment with melamine abolished thapsigargin-evoked [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 did not alter melamine-evoked [Ca²⁺]_i rise. Melamine at 500–800 μM decreased cell viability, which was not reversed by pretreatment with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Collectively, our data suggest that in PC3 cells, melamine induced [Ca²⁺]_i rises by evoking phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum, and Ca²⁺ entry via protein kinase C-regulated store-operated Ca²⁺ entry. Melamine also caused Ca²⁺-independent cell death.     

化学性缺氧致大鼠脑细胞[Ca^2＋]i和脑红蛋白表达变化及阿司匹林干预研究

目的：观察化学性缺氧状况下大鼠脑细胞[Ca^2＋]i和脑红蛋白（NGB）表达的变化及阿司匹林（ASA）对其变化的干预,并初步探讨其作用机制。方法：连二亚硫酸钠致原代培养大鼠脑细胞缺氧,用荧光探针Flou-3负载和NGB荧光免疫组化技术双标,激光共聚焦显微镜动态观察。结果：[Ca^2＋]i和NGB表达与缺氧持续时间有关,随缺氧时间的延长,荧光强度随之增高;经ASA预防性干预,两种荧光强度增高缓慢,其增高程度明显低于单纯缺氧组;治疗性干预,用药早期两种荧光强度增高明显,其增高程度与单纯缺氧组变化相近,后期则增高缓慢,其增高程度明显低于单纯缺氧组。[Ca^2＋]i浓度和NGB表达呈正相关。结论：ASA对原代培养大鼠脑细胞化学性缺氧损伤具有保护作用,其作用可能与ASA抑制细胞内Ca2^＋超载有关;NGB的表达可能是受到Ca2^＋-CaM复合物的调节;ASA预防性用药的早期保护作用明显优于治疗性用药。     

Effects of Antrodia camphorata on viability,apoptosis, [Ca2+]i,and MAPKs phosphorylation in MG63 human osteosarcoma cells

The present study explored the effect of Antrodia camphorata (AC) on viability, apoptosis, mitogen‐activated protein kinases (MAPKs) phosphorylation, and Ca²⁺ regulation in MG63 human osteosarcoma cells. AC (25–50 µg/ml) did not affect cell viability, but at 100–200 µg/ml decreased viability and induced apoptosis in a concentration‐dependent manner. AC at concentrations of 25–200 µg/ml did not alter basal [Ca²⁺]_i, but at 25 µg/ml decreased [Ca²⁺]_i increases induced by ATP, bradykinin, histamine, and thapsigargin. ATP, bradykinin, and histamine increased cell viability while thapsigargin decreased it. AC (25 µg/ml) pretreatment failed to alter bradykinin‐ and thapsigargin‐induced effects on viability, but potentiated ATP‐ and histamine‐induced increases in viability. Immunoblotting showed that MG63 cells did not have background phospho‐JNK and phospho‐p38 mitogen‐activated protein kinases (MAPKs); and AC did not induce the phosphorylation of these two MAPKs. Conversely, the cells had significant background phospho‐ERK MAPK that was inhibited by 200 µg/ml AC. The ERK‐specific inhibitor PD98059 also induced cell death. Collectively, in MG63 cells, AC exerted multiple effects on viability and [Ca²⁺]_i, caused apoptosis probably via inhibition of ERK MAPK phosphorylation. Drug Dev Res 68:71–78, 2007. © 2007 Wiley‐Liss, Inc.     

Opioid receptor antagonists increase [Ca^2＋]i in rat arterial smooth muscle cells in hemorrhagic shock

INTRODUCTION Circulation hypotension induced by hemorrhagicshock is one of the main causes of death after severewounds or trauma. When the hemorrhagic shock hasdeveloped to a decompensatory stage, it is difficult toreverse the hypotension using usual vasoconstrictor,such as norepinephrine. One of the explanations ofhypotension is due to the hyposensitivity of arterialsmooth muscle cells (SMC) to vascular constrictorstimuli[1,2]. Previous studies showed that the vascularhyporesponse was r…