DYSREGULATION OF Ca2+ MOVEMENT IN PLATELETS FROM PATIENTS WITH ACUTE ISCHAEMIC STROKE

• 1 Platelets play a pivotal role during acute ischaemic stroke. An increase in cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) triggers intracellular signal transduction, leading to platelet aggregation and thrombosis. In the present study, we examined the differences between platelets from acute ischaemic stroke patients and at‐risk controls in terms of the increase in platelet [Ca²⁺]_i.
• 2 Thirty‐one patients with acute ischaemic stroke and 27 at‐risk controls were enrolled in the present study. Platelet [Ca²⁺]_i was measured using the fluorescent dye fura‐2 after stimulation with 100 µmol/L arachidonic acid (AA), 10 µmol/L ADP, 1 µmol/L platelet‐activation factor (PAF) and 0.1 U/mL thrombin.
• 3 Basal [Ca²⁺]_i was higher in the stroke group compared with at‐risk controls, irrespective of the presence or absence of extracellular Ca²⁺. In Ca²⁺‐containing medium, both PAF and ADP, but not AA and thrombin, significantly increased platelet [Ca²⁺]_i in the stroke group compared with the at‐risk controls. However, in Ca²⁺‐free medium, only PAF significantly increased platelet [Ca²⁺]_i in the stroke group compared with the at‐risk controls. Basal [Ca²⁺]_i and PAF‐induced platelet [Ca²⁺]_i increases were still higher in the stroke group at the subacute stage than in the at‐risk controls.
• 4 The results of the present study provide direct evidence that Ca²⁺ signalling in platelets from acute ischaemic stroke patients was altered in response to particular stimuli. The dysregulation of Ca²⁺ movement in platelets may persist up to the subacute stage of ischaemic stroke.

     

Functional roles of cyclic guanosine 3′,5′-monophosphate analogue in cerebral vasodilation

• 1.1. Vasodilating effects of cyclic nucleotides in cerebral vasculature were examined using membrane permeable cyclic nucleotide analogues, 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP) and 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP).
• 2.2. In isolated canine basilar artery (CBA), 8-Br-cGMP but not 8-Br-cAMP, significantly inhibited Ca²⁺-induced and agonist [serotonin(5-HT), prostaglandin(PG)F_2α or endothelin]-induced contraction, in a concentration-dependent manner.
• 3.3. When Ca²⁺ was depleted from intracellular store sites by pretreatment with A23187, 8-Br-cGMP but not 8-Br-cAMP strongly attenuated contractions induced by Ca²⁺-influx.
• 4.4. Neither 8-Br-cGMP nor 8-Br-cAMP modified contraction induced by caffeine which elicits Ca²⁺ release from intracellular Ca²⁺ store.
• 5.5. 8-Br-cGMP lowered the high K⁺-induced sustained [Ca²⁺] elevation.
• 6.6. These results suggest that, at least in CBA, cGMP exerts its inhibitory effect on the contraction induced by influx of Ca²⁺, by reducing the level of [Ca²⁺]_i and reducing [Ca²⁺]_i sensitivity of the contractile machinery.

     

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.     

Effects of P2 Purinoceptor Agonists on Membrane Potential and Intracellular Ca2+ of Human Cardiac Endothelial Cells

Abstract: Vasoactive agonists like adenosine-5′-triphosphate (ATP) increase intracellular Ca²⁺ ([Ca²⁺]_i) in vascular endothelial cells with an initial peak due to inositol 1,4,5-triphosphate-mediated Ca²⁺ release from intracellular stores followed by a sustained plateau that is dependent on the presence of extracellular Ca²⁺, thus leading to an increased synthesis and release of prostacyclin and nitric oxide. We studied the effects of nucleotides on membrane potential and [Ca²⁺]_i in confluent human microvascular cardiac endothelial cells obtained from patients with dilated cardiomyopathy. The whole-cell configuration of the patch-clamp technique and a confocal laser scanning microscope employing fluo-3 as a Ca²⁺ indicator were used. Both uridine-5′-triphosphate (UTP) and 2-methylthioadenosine-5′-triphosphate (2MeSATP) induced depolarizations in human microvascular cardiac endothelial cells and increased [Ca²⁺]_i with a rank order of potency 2MeSATP>ATP=UPP (EC₅₀ values (in μM) were 0.084 2MeSATP, 0.67 ATP and 1.1 UTP). This suggests that both P_2u and P_2y purinoceptors are present on human microvascular cardiac endothelial cells. Maximal [Ca²⁺]_i responses of confluent human microvascular cardiac endothelial cell monolayers to UTP were lower when compared to 2MeSATP. Nucleotide-induced increases in [Ca²⁺]_i consisted of a transient peak, which was also observed in the absence of extracellular Ca²⁺, and a sustained [Ca²⁺]_i plateau. This plateau, which was not observed in all monolayers studied, was not markedly influenced by increasing extracellular [K⁺]. Previous incubation with thapsigargin abolished ATP-induced increases of [Ca²⁺]_i. It is concluded that human microvascular cardiac endothelial cells express both P_2y and P_2u purinoceptors. P₂ purinoceptor agonists release Ca²⁺ from intracellular thapsigargin-sensitive stores and stimulate capacitative Ca²⁺ influx pathways. K⁺ efflux through Ca²⁺-dependent K⁺ (K_ca) channels does not play a major role in the regulation of nucleotide-induced Ca²⁺ influx in human microvascular cardiac endothelial cells, which might be related to an impaired function of the cells.     

STORE-OPERATED Ca2+ CHANNELS AND MICRODOMAINS OF Ca2+ IN LIVER CELLS

• 1 Oscillatory increases in the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) play essential roles in the hormonal regulation of liver cells. Increases in [Ca²⁺]_cyt require Ca²⁺ release from the endoplasmic reticulum (ER) and Ca²⁺ entry across the plasma membrane.
• 2 Store‐operated Ca²⁺ channels (SOCs), activated by a decrease in Ca²⁺ in the ER lumen, are responsible for maintaining adequate ER Ca²⁺. Experiments using patch‐clamp recording and the fluorescent Ca²⁺ reporter fura‐2 indicate there is only one type of SOC in rat liver cells. These SOCs have a high selectivity for Ca²⁺ and properties essentially indistinguishable from those of Ca²⁺ release‐activated Ca²⁺ (CRAC) channels.
• 3 Although Orai1, a CRAC channel pore protein, and stromal interaction molecule 1 (STIM1), a CRAC channel Ca²⁺ sensor, are components of liver cell SOCs, the mechanism of activation of SOCs, and in particular the role of subregions of the ER, are not well understood.
• 4 Recent experiments have used the transient receptor potential vanilloid 1 (TRPV1) non‐selective cation channel, ectopically expressed in liver cells, and a choleretic bile acid to deplete Ca²⁺ from different ER subregions. The results of these studies have provided evidence that only a small component of the ER is required for STIM1 redistribution and the activation of SOCs.
• 5 It is concluded that different Ca²⁺ microdomains in the ER and cytoplasmic space are important in both the activation of SOCs and in the signalling actions of Ca²⁺ in liver cells. Future experiments will investigate the nature of these microdomains further.

     

神经肽DGAVP和Org2766对神经细胞内游离Ca2+的影响

运用Ca²⁺指示剂Fura-2作为细胞内钙离子的荧光探针,利用AR—CM—MIC阳离子测定系统,检测了分离的神经细胞内游离钙及其变化,并观测了DGAVP和Org2766对蛋白质合成抑制剂茴香霉素(ANI)引起细胞内钙离子浓度([Ca²⁺]_i)变化的影响。结果表明茴香霉素可使[Ca²⁺]_i显著升高,且有量效关系;DGAVP本身并不引起[Ca²⁺]_i发生显著变化,但适当剂量的DGAVP可显著对抗一定剂量范围内ANI升高[Ca²⁺]_i的作用,提示DGAVP对抗ANI的蛋白质合成抑制效应可能是通过拮抗ANI升高[Ca²⁺]_i这一途径实现的,另一神经肽Org2766则可能不是通过这一机制发生作用。从细胞内Ca²⁺的角度看,这两种肽的作用机理显然是不同的。     

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.     

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.     

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.     

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 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.     

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.

     

Endothelium-derived bradykinin is responsible for the increase in calcium produced by angiotensin-converting enzyme inhibitors in human endothelial cells

Summary The effects of angiotensin-converting enzyme (ACE) inhibitors on intracellular calcium concentration ([Ca²⁺]_i) were examined under resting conditions and after stimulation with bradykinin in cultured human umbilical vein endothelial cells. The ACE inhibitors ramiprilat and enalaprilat (0.3 M) enhanced the increase in [Ca²⁺]_i elicited by bradykinin (3 nM) and also caused an increase in resting [Ca²⁺]_i when given alone. This increase in resting [Ca²⁺]_i was long-lasting and accompanied by an increased formation of nitric oxide, as assessed by a N^G-nitro-l-arginine-sensitive cyclic GMP accumulation in the cells. Both increases in resting [Ca²⁺]_i and nitric oxide production by ACE inhibitors were inhibited by preincubation of the cells with the B₂-receptor antagonist Hoe 140. These data indicate that ACE inhibitors are able to unmask a release of bradykinin from cultured human endothelial cells. This endothelium-derived bradykinin can exert an autocrine function by stimulating endothelial B₂-receptors with a subsequent increase in [Ca²⁺]_i and nitric oxide formation. Send offprint requests to R. Busse at the above address     

Aryl hydrocarbon receptor-independent up-regulation of intracellular calcium concentration by environmental polycyclic aromatic hydrocarbons in human endothelial HMEC-1 cells

Polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (B(a)P) constitute a major family of widely‐distributed environmental toxic contaminants, known as potent ligands of the aryl hydrocarbon receptor (AhR). B(a)P has been recently shown to trigger an early and transient increase of intracellular calcium concentration ([Ca²⁺]_i), involved in AhR‐related up‐regulation of target genes by B(a)P. This study was designed to determine whether AhR may play a role in [Ca²⁺]_i induction provoked by B(a)P. We demonstrated that, in addition to B(a)P, various PAHs, including pyrene and benzo(e)pyrene, known to not or only very poorly interact with AhR, similarly up‐regulated [Ca²⁺]_i in human endothelial HMEC‐1 cells. Moreover, α‐naphthoflavone, a flavonoïd antagonist of AhR, was also able to induce [Ca²⁺]_i. Knocking‐down AhR expression in HMEC‐1 cells through transfection of siRNAs, was finally demonstrated to not prevent B(a)P‐mediated induction of [Ca²⁺]_i, whereas it efficiently counteracted B(a)P‐mediated induction of the referent AhR target gene cytochrome P‐450 1B1. Taken together, these data demonstrate that environmental PAHs trigger [Ca²⁺]_i induction in an AhR‐independent manner. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

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.     

Differential inhibition and potentiation by cell-permeant analogues of cyclic AMP and cyclic GMP and NO-containing compounds of exocytosis in human neutrophils

Summary The chemoattractants, N-formyl-L-methio-nyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), complement C5a and platelet-activating factor (PAF), induce ß-glucuronidase release and aggregation and an increase in cytosolic Ca²⁺ [Ca²⁺]_i in human neutrophils. We studied the roles of cAMP and cGMP in neutrophil avtivation, using their cell-permeant analogues, N⁶,2-O-dibutyryl adenosine 3:5-cyclic monophosphate (Bt2cAMP) and N² ,2-O-dibutyryl guanosine 3:5-cyclic monophosphate (Bt₂cGMP) and the NO-containing compounds, sodium nitroprusside (SNP), 3-morpholino-sydnonimine (SIN-1) and its prodrug, molsidomine (SIN-10). Bt₂cAMP, Bt₂cGMP, SIN-1 and SIN-10 but not SNP inhibited exocytosis induced by fMet-Leu-Phe. Superoxide dismutase potentiated the inhibitory effect of SIN-1. Bt₂cGMP and SNP potentiated C5a-induced ß-glucuronidase release, Bt₂cAMP, KCN, SIN-1 and SIN-10 being ineffective. KCN partially reversed the stimulatory effect of SNP, and in the presence of superoxide dismutase, SIN-1 potentiated C5a-induced exocytosis. PAF-induced ß-glucuronidase release was not affected by Bt₂cAMP, Bt₂cGMP, SNP and SIN-1. Bt₂cGMP was more effective than Bt2cAMP to inhibit aggregation and the increase in [Ca²⁺]_i induced by fet-Leu-Phe at submaximally effective concentrations. C5a-induced rises in [Ca²⁺]_i were not affected by Bt₂cAMP and Bt₂cGMP. Bt₂cAMP but not Bt₂cGMP inhibited the effect of PAF at submaximally effective concentrations on [Ca²⁺]_i. Our data suggest (I) that Bt2cGMP and Bt2cAMP differentially modulate neutrophil activation, that (II) NO-containing compounds partially mimick the effects of Bt₂cGMP on exocytosis and that (III) cGMP plays an inhibitory role in fMet-Leu-Phe- and a stimulatory role in C5a-induced ß-glucuronidase release. Send offprint requests to R. Seifert at the above address     

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.     

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.     

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 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.