2-Benzyloxybenzaldehyde inhibits formyl peptide-stimulated increase in intracellular Ca2+ in neutrophils mainly by blocking Ca2+ entry

2-Benzyloxybenzaldehyde (CCY1a) inhibited the formyl-Met-Leu-Phe (fMLP)-induced elevation of cytosolic [Ca²⁺] ([Ca²⁺]_i) in rat neutrophils. The late plateau phase, but not the initial Ca²⁺ spike, of the fMLP-induced [Ca²⁺]_i change was inhibited by CCY1a. In the absence of external Ca²⁺, CCY1a had no appreciable effect on either the fMLP- or cyclopiazonic acid (CPA)-induced [Ca²⁺]_i elevation. CCY1a failed to inhibit [Ca²⁺]_i changes induced by N-ethylmaleimide, GEA3162, ionomycin or sphingosine, but slightly inhibited the Ca²⁺ signals elicited by ATP or interleukin-8 (IL-8). In a classical Ca²⁺ readdition protocol, addition of CCY1a after cell activation strongly inhibited the [Ca²⁺]_i response to fMLP, whilst that to CPA was only slightly reduced. CCY1a nearly abrogated the fMLP-stimulated Mn²⁺ influx but was less effective on the CPA-induced response. CCY1a attenuated the levels of tyrosine-phosphorylated bands in the 70–85 kDa molecular mass range. CCY1a had no effect on the basal [Ca²⁺]_i level, the pharmacologically isolated plasma membrane Ca²⁺-ATPase activity or on the mitochondrial membrane potential. Thus, CCY1a blocks fMLP-induced Ca²⁺ entry into neutrophils probably by blocking the relevant Ca²⁺ channel directly or, alternatively, indirectly through the attenuation of tyrosine phosphorylation of some cellular proteins.     

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

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

Stimulation of cellular free Ca2+ elevation and inhibition of store-operated Ca2+ entry by kazinol B in neutrophils

Kazinol B, a natural isoprenylated flavan, stimulated the [Ca²⁺]_i elevation in the presence or absence of Ca²⁺ in the medium. Treatment with chymotrypsin or phorbol 12-myristate 13-acetate to shedding of l-selectin had no effect on subsequent kazinol B-induced Ca²⁺ response. Upon initial cyclopiazonic acid (CPA) treatment in the absence of external Ca²⁺, the subsequent [Ca²⁺]_i rise followed by challenge with kazinol B was greatly diminished. The ryanodine receptor blockers, 8-bromo-cyclic ADP-ribose and ruthenium red did not affect kazinol B-evoked Ca²⁺ release from internal stores. However, the inhibitors of sphingosine kinase, dimethylsphingosine, but not dihydrosphingosine, inhibited kazinol B-induced Ca²⁺ release. Kazinol B-induced [Ca²⁺]_i rise was not affected by two nitric oxidase inhibitors, N-(3-aminomethyl)benzylacetamidine (1400W) and 7-nitroindazole, cytochalasin B and Na⁺-deprivation. This response was slightly attenuated by 2-aminoethyldiphenyl borate (2-APB), a d-myo-inositol 1,4,5-trisphosphate (IP₃) receptor blocker, and by genistein, a general tyrosine kinase inhibitor. However, the Ca²⁺ response was greatly diminished by two actin filament reorganizers, calyculin A and jasplakinolide, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002), an inhibitor of phosphoinositide 3-kinase, N-(3-aminomethyl)benzylacetamidine (SB 203580), the p38 mitogen-activated protein kinase inhibitor, 1-[6-[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), the inhibitor of phospholipase C-coupled processes, and by 0.3 mM La³⁺ or Ni²⁺. Kazinol B did not evoke any appreciable Ba²⁺ and Sr²⁺ entry into cells. The Ca²⁺ entry blockers, 1-[-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365), but not cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL-12,330A), inhibited a kazinol B-induced [Ca²⁺]_i rise. Kazinol B had no effect on the pharmacologically isolated plasma membrane Ca²⁺-ATPase activity. In a Ca²⁺-free medium, kazinol B inhibited the subsequent Ca²⁺ addition, resulting in robust entry in CPA- and formyl peptide-activated cells. Kazinol B produced a concentration-dependent reduction in the mitochondrial membrane potential. These results indicate that kazinol B stimulates Ca²⁺ release from internal Ca²⁺ store, probably through the sphingosine 1-phosphate and IP₃ signaling, and activates external Ca²⁺ influx mainly through a non-store-operated Ca²⁺ entry (non-SOCE) pathway. Inhibition of SOCE by kazinol B is probably attributable to a break in the Ca²⁺ driven force of mitochondria.     

Effects of timolol on Ca2+ handling and viability in human prostate cancer cells

Timolol is a medication used widely to treat glaucoma. Regarding Ca²⁺ signaling, timolol was shown to modulate Ca²⁺-related physiology in various cell types, however, the effect of timolol on Ca²⁺ homeostasis and cell viability has not been explored in human prostate cancer cells. The aim of this study was to explore the effect of timolol on intracellular Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells. Timolol at concentrations of 100–1000?μM induced [Ca²⁺]_i rises. The Ca²⁺ signal in Ca²⁺-containing medium was reduced by removal of extracellular Ca²⁺ by approximately 75%. Timolol (1000?μM) induced Mn²⁺ influx suggesting of Ca²⁺ entry. Timolol-induced Ca²⁺ entry was partially inhibited by three inhibitors of store-operated Ca²⁺ channels: nifedipine, econoazole and SKF96365, and by a protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate [PMA]) or an inhibitor (GF109203X). In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin abolished timolol-evoked [Ca²⁺]_i rises. Conversely, treatment with timolol abolished thapsigargin-evoked [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) with U73122 abolished timolol-induced [Ca²⁺]_i rises. Timolol at concentrations between 200 and 600?μM killed cells in a concentration-dependent fashion. Chelation of cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM) did not reverse cytotoxicity of timolol. Together, in PC3 cells, timolol induced [Ca²⁺]_i rises by evoking Ca²⁺release from the endoplasmic reticulum in a PLC-dependent manner, and Ca²⁺ influx via PKC-regulated store-operated Ca²⁺ entry. Timolol also caused cell death that was not linked to preceding [Ca²⁺]_i rises.     

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.     

Effect of celecoxib on Ca2+ handling and viability in human prostate cancer cells (PC3)

Celecoxib has been shown to have an antitumor effect in previous studies, but the mechanisms are unclear. Ca²⁺ is a key second messenger in most cells. The effect of celecoxib on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in human suspended PC3 prostate cancer cells was explored by using fura-2 as a fluorescent dye. Celecoxib at concentrations between 5 and 30 μM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Celecoxib-induced Ca²⁺ influx was not blocked by L-type Ca²⁺ entry inhibitors or protein kinase C/A modulators [phorbol 12-myristate 13-acetate (PMA), GF109203X, H-89], but was inhibited by the phospholipase A₂ inhibitor, aristolochic acid. In Ca²⁺-free medium, 30 μM of celecoxib failed to induce a [Ca²⁺]_i rise after pretreatment with thapsigargin (an endoplasmic reticulum [ER] Ca²⁺ pump inhibitor). Conversely, pretreatment with celecoxib inhibited thapsigargin-induced Ca²⁺ release. Inhibition of phospholipase C with U73122 did not change celecoxib-induced [Ca²⁺]_i rises. Celecoxib induced slight cell death in a concentration-dependent manner, which was enhanced by chelating cytosolic Ca²⁺ with BAPTA. Collectively, in PC3 cells, celecoxib induced [Ca²⁺]_i rises by causing phospholipase C–independent Ca²⁺ release from the ER and Ca²⁺ influx via non-L-type, phospholipase A₂-regulated Ca²⁺ channels. These data may contribute to the understanding of the effect of celecoxib on prostate cancer cells.     

Endothelin-1 induced contraction of rat aorta: contributions made by Ca2+ influx and activation of contractile apparatus associated with no change in cytoplasmic Ca2+ level

Summary The modes by which Endothelin-1 (ET) induces Ca²⁺-influx and the relative functional importance of the different sources of Ca²⁺ for ET-induced contraction were studied using fura 2-loaded and unloaded rat aortic strips. ET caused an increase in the cytosolic free Ca²⁺ level ([Ca²⁺]_i) followed by a tonic contraction in Ca²⁺-containing solution, and produced a transient elevation of [Ca²⁺]_i followed by a small sustained contraction in Ca²⁺-free medium. ET also stimulated ⁴⁵Ca influx into La²⁺-inaccessible fraction significantly. With the same change of [Ca²⁺]_i, ET caused a larger tension than that induced by high K. ET-induced contraction and [Ca²⁺]_i elevation were not significantly inhibited by 0.1–0.3 M nicardipine which nearly abolished the contraction and [Ca⁺]_i elevation produced by high K. During treatment of the strips with high K, addition of ET induced further increases in [Ca²⁺]_i and muscle tension, and vice versa. In Ca²⁺-free medium, ET-induced contraction was influenced neither by ryanodine-treatment nor by high K-treatment, although the former attenuated and the latter potentiated the [Ca²⁺]_i transient induced by ET. Further, the ET-induced sustained contraction under Ca²⁺-free conditions began to develop after the [Ca²⁺]_i level returned to the baseline. Thus, it seems that the Ca²⁺ released from the ryanodine-sensitive and -insensitive Ca²⁺ stores by ET may provide only a minor or indirect contribution, if any, to the tension development. ET might cause a contraction mainly by stimulating Ca²⁺-influx through Ca²⁺ channel(s) other than voltage-dependent Ca²⁺ channels in character, and by increasing the sensitivity of the contractile filaments to Ca²⁺ or activating them Ca²⁺-independently.Visiting from Zun Yi Medical College, China Send offprint requests to I. Takayanagi at the above address     

Effect of bisphenol A on Ca2+ fluxes and viability in Madin-Darby canine renal tubular cells

The effect of the environmental contaminant, bisphenol A, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in Madin-Darby canine kidney (MDCK) cells is unclear. This study explored whether bisphenol A changed basal [Ca²⁺]_i levels in suspended MDCK cells by using fura-2 as a Ca²⁺-sensitive fluorescent dye. Bisphenol A, at concentrations between 50 and 300 µM, increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced, partly, by removing extracellular Ca²⁺. Bisphenol A induced Mn²⁺ influx, leading to quenching of fura-2 fluorescence, suggesting Ca²⁺ influx. This Ca²⁺ influx was inhibited by phospholipase A2 inhibitor aristolochic acid, store-operated Ca²⁺ channel blockers nifedipine and SK&F96365, and protein kinase C inhibitor GF109203X. In Ca²⁺-free medium, pretreatment with the mitochondrial uncoupler, carbonylcyanide m-chlorophenylhydrazone (CCCP), and the endoplasmic reticulum Ca²⁺ pump inhibitors, thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ), inhibited bisphenol A–induced Ca²⁺ release. Conversely, pretreatment with bisphenol A abolished thapsigargin (or BHQ)- and CCCP-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished bisphenol-induced [Ca²⁺]_i rise. Bisphenol A caused a concentration-dependent decrease in cell viability via apoptosis in a Ca²⁺-independent manner. Collectively, in MDCK cells, bisphenol A induced [Ca²⁺]_i rises by causing phospholipase C–dependent Ca²⁺ release from the endoplasmic reticulum and mitochondria and Ca²⁺ influx via phospholipase A2–, protein kinase C–sensitive, store-operated Ca²⁺ channels.     

INTERACTIONS OF Na+, H2O2 AND THE Na+-Ca2+ EXCHANGER STIMULATE Ca2+ RELEASE IN CK1.4 CELLS

1. The present study aimed to demonstrate that interactions of cations, hydrogen peroxide (H₂O₂) and the Na⁺-Ca²⁺exchanger stimulate Ca²⁺ release and oscillations of cytosolic Ca²⁺ [Ca²⁺]i in non-transfected Chinese Hamster Ovary (CHO) C1 cells and in transfected CHO (CK1.4) cells that contained an expression vector coding the Na⁺-Ca²⁺ exchanger sequence. 2. The [⁴⁵Ca²⁺] uptake assay, fura-2 fluorescence imaging and 2² and 2³ factorial orthogonal statistics provide comparative, direct, efficient, quantitative and transient methods to delineate the effects of such interactions on Ca²⁺ influx, Ca²⁺release and [Ca²⁺]i in C1 and CK1.4 cells. 3. In contrast to the control of either Na⁺-, Ca²⁺- or H₂O₂-free or CI cells, an elevated [⁴⁵Ca²⁺] uptake was induced by Ca²⁺, Na⁺ and H₂O₂ individually and in combination, intracellular Ca²⁺ release was activated by H₂O₂ and by combinations of either H₂O₂ and Na⁺, H₂O₂ and the Na⁺-Ca²⁺ exchanger, Na⁺ and the Na⁺-Ca²⁺ exchanger or by H₂O₂, Na⁺ and the Na⁺-Ca²⁺ exchanger and a rise in [Ca²⁺]i was triggered by H₂O₂, Na⁺ and a combination of Na⁺ and the Na⁺-Ca²⁺exchanger. 4. These results indicate that interactions between H₂O₂, Na⁺ and the Na⁺-Ca²⁺ exchanger stimulate intracellular Ca²⁺mobilization via Ca²⁺-induced Ca²⁺ release mechanisms, ATP-activated G-protein coupled P_2y-purinoceptor-sensitive pathways, Na⁺-Ca²⁺ exchanger-mediated Ca²⁺ influx and cation-π interaction (a strong non-covalent force between the cation and the π face of an aromatic structure in the transmembrane protein). 5. The present findings provide important clues for understanding Ca²⁺ signal transduction mechanisms from the plasma membrane to the endoplasmic reticulum.     

Epigallocatechin-3-gallate increases intracellular [Ca<Superscript>2+</Superscript>] in U87 cells mainly by influx of extracellular Ca<Superscript>2+</Superscript> and partly by release of intracellular stores

Green tea has been receiving considerable attention as a possible preventive agent against cancer and cardiovascular disease. Epigallocatechin-3-gallate (EGCG) is a major polyphenol component of green tea. Using digital calcium imaging and an assay for [³H]-inositol phosphates, we determined whether EGCG increases intracellular [Ca²⁺] ([Ca²⁺]_i) in non-excitable human astrocytoma U87 cells. EGCG induced concentration-dependent increases in [Ca²⁺]_i. The EGCG-induced [Ca²⁺]_i increases were reduced to 20.9% of control by removal of extracellular Ca²⁺. The increases were also inhibited markedly by treatment with the non-specific Ca²⁺ channel inhibitors cobalt (3 mM) for 3 min and lanthanum (1 mM) for 5 min. The increases were not significantly inhibited by treatment for 10 min with the L-type Ca²⁺ channel blocker nifedipine (100 nM). Treatment with the inhibitor of endoplasmic reticulum Ca²⁺-ATPase thapsigargin (1 µM) also significantly inhibited the EGCG-induced [Ca²⁺]_i increases. Treatment for 15 min with the phospholipase C (PLC) inhibitor neomycin (300 µM) attenuated the increases significantly, while the tyrosine kinase inhibitor genistein (30 µM) had no effect. EGCG increased [³H]-inositol phosphates formation via PLC activation. Treatment for 10 min with mefenamic acid (100 µM) and flufenamic acid (100 µM), derivatives of diphenylamine-2-carboxylate, blocked the EGCG-induced [Ca²⁺]_i increase in non-treated and thapsigargin-treated cells but indomethacin (100 µM) did not affect the increases. Collectively, these data suggest that EGCG increases [Ca²⁺]_i in non-excitable U87 cells mainly by eliciting influx of extracellular Ca²⁺ and partly by mobilizing intracellular Ca²⁺ stores by PLC activation. The EGCG-induced [Ca²⁺]_i influx is mediated mainly through channels sensitive to diphenylamine-2-carboxylate derivatives.     

Mechanisms of carvedilol-induced [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> rises and death in human hepatoma cells

The effect of the cardiovascular drug carvedilol on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability has not been explored in human hepatoma cells. This study examined whether carvedilol 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. Carvedilol at concentrations ≥1 μM 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⁺. Carvedilol induced Mn²⁺ quench of fura-2 fluorescence, implicating Ca²⁺ influx. The Ca²⁺ influx was sensitive to La³⁺, econazole, nifedipine, and SKF96365. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), carvedilol-induced [Ca²⁺]_i rises were abolished; and conversely, carvedilol pretreatment inhibited a major part of thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 μM U73122 did not change carvedilol-induced [Ca²⁺]_i rises. At concentrations between 1 and 50 μM, carvedilol killed cells in a concentration-dependent manner. The cytotoxic effect of 1 μM (but not 30 μM) carvedilol was fully reversed by prechelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Apoptosis was induced by 30 (but not 1) μM carvedilol. Collectively, in HA59T hepatoma cells, carvedilol induced [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum in a phospholipase-C-independent manner and Ca²⁺ influx via store-operated Ca²⁺ channels. Carvedilol-caused cytotoxicity was mediated by Ca²⁺ and apoptosis in a concentration-dependent manner.     

The TRPC channel blocker SKF 96365 inhibits glioblastoma cell growth by enhancing reverse mode of the Na+/Ca2+ exchanger and increasing intracellular Ca2+

BACKGROUND AND PURPOSE

SKF 96365 is well known for its suppressing effect on human glioblastoma growth by inhibiting pre-activated transient receptor potential canonical (TRPC) channels and Ca²⁺ influx. The effect of SKF 96363 on glioblastoma cells, however, may be multifaceted and this possibility has been largely ignored.

EXPERIMENTAL APPROACH

The effects of SKF 96365 on cell cycle and cell viability of cultured human glioblastoma cells were characterized. Western blot, Ca²⁺ imaging and patch clamp recordings were used to delineate cell death mechanisms. siRNA gene knockdown provided additional evidence.

KEY RESULTS

SKF 96365 repressed glioblastoma cell growth via increasing intracellular Ca²⁺ ([Ca²⁺]_i) irrespective of whether TRPC channels were blocked or not. The effect of SKF 96365 primarily resulted from enhanced reverse operation of the Na⁺/Ca²⁺ exchanger (NCX) with an EC₅₀ of 9.79 μM. SKF 96365 arrested the glioblastoma cells in the S and G₂ phases and activated p38-MAPK and JNK, which were all prevented by the Ca²⁺ chelator BAPTA-AM or EGTA. The expression of NCX in glioblastoma cells was significantly higher than in normal human astrocytes. Knockdown of the NCX1 isoforms diminished the effect of SKF 96365 on glioblastoma cells.

CONCLUSIONS AND IMPLICATIONS

At the same concentration, SKF 96365 blocks TRPC channels and enhances the reverse mode of the NCX causing [Ca²⁺]_i accumulation and cytotoxicity. This finding suggests an alternative pharmacological mechanism of SKF 96365. It also indicates that modulation of the NCX is an effective method to disrupt Ca²⁺ homeostasis and suppress human glioblastoma cells.     

Amiodarone and desethylamiodarone increase intrasynaptosomal free calcium through receptor mediated channel

Summary Long term amiodarone (AM) therapy has been associated with several side effects including neurotoxicity. Since AM alters Ca²⁺ regulated events, we have studied its effects on the compartmentation of free Ca²⁺ in the synaptosomes as an attempt to understand the mechanism of AM and its metabolite, desethylamiodarone (DEA)-induced neurotoxicity. Intact brain synaptosomes were prepared from male Sprague-Dawley rats. Both AM and DEA produced a concentration dependent increase in intrasynaptosomal free Ca²⁺ concentration ([Ca²⁺]_i) to micromolar levels. The increase in [Ca²⁺]_i was not transient and a steady rise was observed with time. Omission of Ca²⁺ from the external medium prevented the AM- and DEA-induced rise in [Ca²⁺]_i suggesting that AM and DEA increased the intracellular [Ca²⁺]_i due to increased influx of Ca²⁺ from external medium. AM- and DEA-induced increase in intrasynaptosomal [Ca²⁺]_i was neither inhibited by a calcium channel blocker, verapamil, nor with a Na⁺ channel blocker, tetrodotoxin. However, the blockade of [Ca²⁺]_i rise by AM and DEA was observed with MK-801, a receptor antagonist indicating that AM and DEA induced rise in [Ca²⁺]_i is through receptor mediated channel. Both AM and DEA also inhibited N-methyl-D-aspartic acid (NMDA)-receptor binding in synaptic membranes in a concentration dependent manner, DEA being more effective, indicating that AM and DEA compete for the same site as that of NMDA and confirm the observation that these drugs increase intrasynaptosomal [Ca²⁺]_i through receptor mediated channel. ⁴⁵Ca accumulation into brain microsomes and mitochondria was significantly inhibited by AM and DEA, but without any effect on the Ca²⁺ release from these intracellular organelles. Also, both these drugs did not interfere with inositol 1,4,5-trisphosphate induced Ca²⁺ release from microsomes even at 10 M concentration. These results clearly indicate that both AM and DEA increase intrasynaptosomal [Ca²⁺]_i by an action on receptor mediated channel in plasma membrane, but not due to the release of Ca²⁺ from intracellular storage sites. This initial rise in [Ca²⁺]_i, together with other changes in Ca²⁺ homeostasis, might be responsible for AM and DEA-induced neurotoxicity. Send offprint requests to D. Desaiah at the above address     

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.     

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.     

Palytoxin-induced increase in endothelial Ca2+ concentration in the rabbit aortic valve

Palytoxin (PTX) is one of the most potent toxins isolated from marine coelenterates of the genus Palythoa. It induces depolarization in various types of cells by increasing the permeability for monovalent cations. It has been reported that PTX induces endothelium-dependent relaxation of vascular smooth muscle. In this study, we examined the effect of PTX on the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in the endothelium of rabbit aortic valves loaded with fluorescent Ca²⁺ indicators, fura-PE3 or fluo-3. PTX (10pM-300nM) irreversibly increased endothelial [Ca²⁺]_i in a concentration-dependent manner. ATP and thapsigargin also increased [Ca²⁺]_i. Imaging of [Ca²⁺]_i with a confocal microscope revealed that PTX increased [Ca²⁺]_i in all endothelial cells studied (n=13). An inorganic Ca²⁺ entry blocker, La³⁺ (30μM), had no effect on the increase in [Ca²⁺]_i induced by PTX whereas it inhibited the sustained phase of the increase in [Ca²⁺]_i induced by ATP or thapsigargin. The PTX-induced increase in [Ca²⁺]_i was partially inhibited by ouabain and was abolished by removal of external Ca²⁺ although decrease of Na⁺ concentration in the incubation medium was ineffective. Activation of protein kinase C by 1μM 12-deoxyphorbol 13-isobutyrate or inhibition of phosphatase by 10nM calyculin-A had no effect on the increase in [Ca²⁺]_i induced by PTX, whereas both agents inhibited the sustained phase of the increase in [Ca²⁺]_i induced by ATP or thapsigargin. Mn²⁺ influx, measured by the quenching of fura-PE3 fluorescence, was accelerated by ATP or thapsigargin, but not by PTX. These results suggest that PTX increases [Ca²⁺]_i in the endothelium of the rabbit aortic valve by increasing Ca²⁺ influx through a pathway which is different from that activated by ATP or thapsigargin. Received: 28 February 1997     

Effects of hypoxia on stimulus-release coupling mechanisms in cultured bovine adrenal chromaffin cells

Summary To clarify the effects of hypoxia on stimulus-release coupling, we have examined the effects of hypoxia on nicotine-induced catecholamine (noradrenaline and adrenaline) release from, and ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in, cultured bovine adrenal chromaffin cells. Experiments were carried out in media pre-equilibrated with 21% O₂/79% N₂ (control) or with 0% O₂/100% N₂ (hypoxia). Cells were stimulated with either nicotine (activating nicotinic acetylcholine (ACh) receptors) or a high K⁺ concentration (55 mmol/1 KCI; directly activating voltage-dependent Ca²⁺ channels). Hypoxia reduced both nicotine- and high K⁺-induced catecholamine releases from the cells, but the reduction of the former (to about 30% of the control value) was more pronounced than that of the latter (to about 40% of the control value). Nicotine-induced ²²Na⁺ influx, which is considered to reflect the function of nicotinic ACh receptors, was inhibited by hypoxia. Both nicotine- and high K⁺-induced ⁴⁵Ca²⁺ influx into the cells were reduced by hypoxia, but the reduction of the former was more pronounced than that of the latter. Nicotine- and high K⁺-induced increases in [Ca²⁺]_i were reduced by hypoxia to about 30% and 40% of the control values, respectively. These results suggest that hypoxia reduces cation influxes (Na⁺ and Ca²⁺) through both the ligand-gated cation channels of the nicotinic ACh receptor and the voltage-dependent Ca²⁺ channels. Correspondence to K. Lee at the present address     

Functional comparison of the reverse mode of Na+/Ca2+ exchangers NCX1.1 and NCX1.5 expressed in CHO cells

Aim:

To investigate the reverse mode function of Na⁺/Ca²⁺ exchangers NCX1.1 and NCX1.5 expressed in CHO cells as well as their modulations by PKC and PKA.

Methods:

CHO-K1 cells were transfected with pcDNA3.1 (+) plasmid carrying cDNA of rat cardiac NCX1.1 and brain NCX1.5. The expression of NCX1.1 and NCX1.5 was examined using Western blot analysis. The intracellular Ca²⁺ level ([Ca²⁺]_i) was measured using Ca²⁺ imaging. Whole-cell NCX currents were recorded using patch-clamp technique. Reverse mode NCX activity was elicited by perfusion with Na⁺-free medium. Ca²⁺ paradox was induced by Ca²⁺-free EBSS medium, followed by Ca²⁺-containing solution (1.8 or 3.8 mmol/L CaCl₂).

Results:

The protein levels of NCX1.1 and NCX1.5 expressed in CHO cells had no significant difference. The reverse modes of NCX1.1 and NCX1.5 in CHO cells exhibited a transient increase of [Ca²⁺]_i, which was followed by a Ca²⁺ level plateau at higher external Ca²⁺ concentrations. In contrast, the wild type CHO cells showed a steady increase of [Ca²⁺]_i at higher external Ca²⁺ concentrations. The PKC activator PMA (0.3-10 μmol/L) and PKA activator 8-Br-cAMP (10-100 μmol/L) significantly enhanced the reverse mode activity of NCX1.1 and NCX1.5 in CHO cells. NCX1.1 was 2.4-fold more sensitive to PKC activation than NCX1.5, whereas the sensitivity of the two NCX isoforms to PKA activation had no difference. Both PKC- and PKA-enhanced NCX reverse mode activities in CHO cells were suppressed by NCX inhibitor KB-R7943 (30 μmol/L).

Conclusion:

Both NCX1.1 and NCX1.5 are functional in regulating and maintaining stable [Ca²⁺]_i in CHO cells and differentially regulated by PKA and PKC. The two NCX isoforms might be useful drug targets for heart and brain protection.     

Inhibition of Na+-pump enhances carbachol-induced influx of 45Ca2+ and secretion of catecholamines by elevation of cellular accumulation of 22Na+ in cultured bovine adrenal medullary cells

Summary In bovine adrenal medullary cells, we reported that ²²Na⁺ influx via nicotinic receptor-associated Na⁺ channels is involved in ⁴⁵Ca²⁺ influx, a requisite for initiating the secretion of catecholamines (Wada et al. 1984, 1985b).In the present study, we investigated whether the inhibition of Na⁺-pump modulates carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion in cultured bovine adrenal medullary cells. We also measured ⁸⁶Rb⁺ uptake by the cells to estimate the activity of Na⁺, K⁺-ATPase. (1) Ouabain and extracellular K⁺ deprivation remarkably potentiated carbachol-induced ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and catecholamine secretion; this potentiation of carbachol-induced ⁴⁵Ca²⁺ influx and catecholamine secretion was not observed in Na⁺ free medium. (2) Carbachol increased the uptake of ⁸⁶Rb⁺; this increase was inhibited by hexamethonium and d-tubocurarine. In Na⁺ free medium, carbachol failed to increase ⁸⁶Rb⁺ uptake. (3) Ouabain inhibited carbachol-induced ⁸⁶Rb⁺ uptake in a concentration-dependent manner, as it increased the accumulation of cellular ²²Na⁺. These results suggest that Na⁺ influx via nicotinic receptor-associated Na⁺ channels increases the activity of Na⁺, K⁺-ATPase and the inhibition of Na⁺, K⁺-ATPase augmented carbachol-induced Ca²⁺ influx and catecholamine secretion by potentiating cellular accumulation of Na⁺. It seems that nicotinic receptor-associated Na⁺ channels and Na⁺, K⁺-ATPase, both modulate the influx of Ca²⁺ and secretion of catecholamines by accomodating cellular concentration of Na⁺.     

Forskolin Changes the Relationship between Cytosolic Ca2+ and Contraction in Guinea Pig Ileum

This study was designed to clarify the mechanism of the inhibitory effect of forskolin on contraction, cytosolic Ca²⁺ level ([Ca²⁺]_i), and Ca²⁺ sensitivity in guinea pig ileum. Forskolin (0.1 nM~10 µM) inhibited high K⁺ (25 mM and 40 mM)- or histamine (3 µM)-evoked contractions in a concentration-dependent manner. Histamine-evoked contractions were more sensitive to forskolin than high K⁺-evoked contractions. Spontaneous changes in [Ca²⁺]_i and contractions were inhibited by forskolin (1 µM) without changing the resting [Ca²⁺]_i. Forskoln (10 µM) inhibited muscle tension more strongly than [Ca²⁺]_i stimulated by high K⁺, and thus shifted the [Ca²⁺]_i-tension relationship to the lower-right. In histamine-stimulated contractions, forskolin (1 µM) inhibited both [Ca²⁺]_i and muscle tension without changing the [Ca²⁺]_i-tension relationship. In α-toxin-permeabilized tissues, forskolin (10 µM) inhibited the 0.3 µM Ca²⁺-evoked contractions in the presence of 0.1 mM GTP, but showed no effect on the Ca²⁺-tension relationship. We conclude that forskolin inhibits smooth muscle contractions by the following two mechanisms: a decrease in Ca²⁺ sensitivity of contractile elements in high K⁺-stimulated muscle and a decrease in [Ca²⁺]_i in histamine-stimulated muscle.