The exploration of effect of terfenadine on Ca2+ signaling in renal tubular cells

Terfenadine, an antihistamine used for the treatment of allergic conditions, affected Ca²⁺-related physiological responses in various models. However, the effect of terfenadine on cytosolic free Ca²⁺ levels ([Ca²⁺]_i) and its related physiology in renal tubular cells is unknown. This study examined whether terfenadine altered Ca²⁺ signaling and caused cytotoxicity in Madin–Darby canine kidney (MDCK) renal tubular cells. The Ca²⁺-sensitive fluorescent dye fura-2 was used to measure [Ca²⁺]_i. Cell viability was measured by the fluorescent reagent 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] water soluble tetrazolium-1 (WST-1) assay. Terfenadine at concentrations of 100–1000?μM induced [Ca²⁺]_i rises concentration dependently. The response was reduced by approximately 35% by removing extracellular Ca²⁺. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) partly inhibited terfenadine-evoked [Ca²⁺]_i rises. Conversely, treatment with terfenadine abolished BHQ-evoked [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) with U73122 inhibited 95% of terfenadine-induced Ca²⁺ release. Terfenadine-induced Ca²⁺ entry was supported by Mn²⁺-caused quenching of fura-2 fluorescence. Terfenadine-induced Ca²⁺ entry was partly inhibited by an activator of protein kinase C (PKC), phorbol 12-myristate 13 acetate (PMA) and by three modulators of store-operated Ca²⁺ channels (nifedipine, econazole, and SKF96365). Terfenadine at 200–300?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). Together, in MDCK cells, terfenadine induced [Ca²⁺]_i rises by evoking PLC-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via PKC-sensitive store-operated Ca²⁺ entry. Furthermore, terfenadine caused cell death that was not triggered by preceding [Ca²⁺]_i rises.     

Effects of cryopreservation on the intracellular calcium concentration of human spermatozoa and its response to progesterone

The intracellular free calcium concentration [Ca²⁺]_i of sperm from 23 ejaculates was measured before and after cryopreservation using the fluorescent probe Fura-2. Spermatozoa were treated with 3.18 μM progesterone so that the regulation of [Ca²⁺]_i in a dynamic situation could be studied. [Ca²⁺]_i (nM) was 290 ± 13 in fresh spermatozoa vs. 550 ± 26 in cryopreserved samples (mean ± S.E.M. P < 0.0001 paired t-test). Progesterone at a dose of 3.18 μM stimulated a large and rapid increase in [Ca²⁺]_i to a peak value > 1 μM after 10–20 seconds. [Ca²⁺]_i then declined to a slightly raised basal level over the next 30–40 seconds. This phenomenon occurred in all the fresh samples, but about half the frozen thawed samples failed to respond. The peak [Ca²⁺] attained by frozen samples which did respond after the addition of progesterone was similar to that observed with fresh sperm. The calcium channel blocker verapamil (200 μM) completely inhibited the transient rise in [Ca²⁺]_i produced by progesterone, but 100 μM verapamil had only a partial effect. We conclude that (1) cryopreservation causes a substantial elevation of the [Ca²⁺]_i in human spermatozoa and (2) damage to the plasma membrane during cryopreservation may result in the loss of the progesterone receptor. Both factors may contribute to the loss of fertility after cryopreservation. © 1994 Wiley-Liss, Inc.     

Carvacrol-induced [Ca2+]i rise and apoptosis in human glioblastoma cells

AimsThis study examined whether the essential oil component carvacrol altered cytosolic free Ca²⁺ level ([Ca²⁺]_i) and viability in human glioblastoma cells.Main methodsThe Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Cell viability was measured by detecting reagent WST-1. Apoptosis and reactive oxygen species (ROS) were detected by flow cytometry.Key findingsCarvacrol at concentrations of 400–1000 μM induced a [Ca²⁺]_i rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca²⁺. Carvacrol-induced Ca²⁺ signal was not altered by nifedipine, econazole, SK&;F96365, and protein kinase C activator phorbol myristate acetate (PMA), but was inhibited by the protein kinase C inhibitor GF109203X. When extracellular Ca²⁺ was removed, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished carvacrol-induced [Ca²⁺]_i rise. Incubation with carvacrol also abolished thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished carvacrol-induced [Ca²⁺]_i rise. At concentrations of 200–800 μM, carvacrol killed cells in a concentration-dependent manner. This cytotoxic effect was not changed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N–-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that carvacrol (200, 400 and 600 μM) induced apoptosis in a concentration-dependent manner. At concentrations of 200, 400 and 600 μM, carvacrol induced production of ROS.SignificanceIn human glioblastoma cells, carvacrol induced a [Ca²⁺]_i rise by inducing phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via protein kinase C-sensitive, non store-operated Ca²⁺ channels. Carvacrol induced cell death that might involve ROS-mediated apoptosis.     

Methylmercury-Induced Elevations in Intrasynaptosomal Zinc Concentrations: An 19F-NMR Study

Abstract: Methylmercury (MeHg) increases the concentration of intracellular Ca²⁺ ([Ca²⁺]_i) and another endogenous polyvalent cation in both synaptosomes and NG108-15 cells. In synaptosomes, the elevation in [Ca²⁺]_i was strictly dependent on extracellular Ca²⁺ (Ca²⁺_e); similarly, in NG108-15 cells, a component of the elevations in [Ca²⁺]_i was Ca²⁺_e dependent. The MeHg-induced elevations in endogenous polyvalent cation concentration were independent of Ca²⁺_e in synaptosomes and NG108-15 cells. The pattern of alterations in fura-2 fluorescence suggested the endogenous polyvalent cation may be Zn²⁺. Using ¹⁹F-NMR spectroscopy of rat cortical synaptosomes loaded with the fluorinated chelator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N′,N′-tetraacetic acid (5F-BAPTA), we have determined unambiguously that MeHg increases the free intrasynaptosomal Zn²⁺ concentration ([Zn²⁺]_i). In buffer containing 200 µM EGTA to prevent the Ca²⁺_e-dependent elevations in [Ca²⁺]_i, the [Zn²⁺]_i was 1.37 ± 0.20 nM; following a 40-min exposure to MeHg-free buffer [Zn²⁺]_i was 1.88 ± 0.53 nM. Treatment of synaptosomes for 40 min with 125 µM MeHg yielded [Zn²⁺]_i of 2.69 ± 0.55 nM, whereas 250 µM MeHg significantly elevated [Zn²⁺]_i to 3.99 ± 0.68 nM. No Zn²⁺ peak was observed in synaptosomes treated with the cell-permeant heavy metal chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN, 100 µM) following 250 µM MeHg exposure. [Ca²⁺]_i in buffer containing 200 µM EGTA was 338 ± 26 nM and was 370 ± 64 nM following an additional 40-min exposure to MeHg-free buffer. [Ca²⁺]_i was 498 ± 28 or 492 ± 53 nM during a 40-min exposure to 125 or 250 µM MeHg, respectively. None of the values of [Ca²⁺]_i differed significantly from either pretreatment levels or buffer-treated controls.     

Econazole-evoked [Ca2+]i Rise and Non-Ca2+-triggered Cell Death in Rabbit Corneal Epithelial Cells (SIRC)

The effects of econazole, an antifungal drug applied for treatment of keratitis and mycotic corneal ulcer, on cytosolic-free Ca²⁺ concentrations ([Ca²⁺]_i) and viability of corneal cells was examined by using SIRC rabbit corneal epithelial cells as model. [Ca²⁺]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Econazole at concentrations ≥ 1 µM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. The econazole-induced Ca(2+) influx was insensitive to L-type Ca²⁺ channel blockers and protein kinase C modulators. In Ca²⁺-free medium, after pretreatment with 20 µM econazole, [Ca²⁺]_i rises induced by 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor) were abolished. Conversely, thapsigargin pretreatment also abolished econazole-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 µM U73122 did not change econazole-induced [Ca²⁺]_i rises. At concentrations between 10 and 80 µM, econazole killed cells in a concentration-dependent manner. The cytotoxic effect of 20 µM econazole was not reversed by prechelating cytosolic Ca²⁺ with BAPTA. This shows that in SIRC cells econazole induces [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx from unknown pathways. Econazole-caused cytotoxicity was independent from a preceding [Ca²⁺]_i rise.     

The investigation of minoxidil-induced [Ca2+]i rises and non-Ca2+-triggered cell death in PC3 human prostate cancer cells

Minoxidil is clinically used to prevent hair loss. However, its effect on Ca²⁺ homeostasis in prostate cancer cells is unclear. This study explored the effect of minoxidil on cytosolic-free Ca²⁺ levels ([Ca²⁺]_i) and cell viability in PC3 human prostate cancer cells. Minoxidil at concentrations between 200 and 800?μM evoked [Ca²⁺]_i rises in a concentration-dependent manner. This Ca²⁺ signal was inhibited by 60% by removal of extracellular Ca²⁺. Minoxidil-induced Ca²⁺ influx was confirmed by Mn²⁺-induced quench of fura-2 fluorescence. Pre-treatment with the protein kinase C (PKC) inhibitor GF109203X, PKC activator phorbol 12-myristate 13 acetate (PMA), nifedipine and SKF96365 inhibited minoxidil-induced Ca²⁺ signal in Ca²⁺ containing medium by 60%. Treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-ditert-butylhydroquinone (BHQ) in Ca²⁺-free medium abolished minoxidil-induced [Ca²⁺]_i rises. Conversely, treatment with minoxidil abolished BHQ-induced [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) with U73122 abolished minoxidil-evoked [Ca²⁺]_i rises. Overnight treatment with minoxidil killed cells at concentrations of 200–600?μM 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 prevent minoxidil’s cytotoxicity. Together, in PC3 cells, minoxidil induced [Ca²⁺]_i rises that involved Ca²⁺ entry through PKC-regulated store-operated Ca²⁺ channels and PLC-dependent Ca²⁺ release from the endoplasmic reticulum. Minoxidil-induced cytotoxicity in a Ca²⁺-independent manner.     

Mechanisms of resveratrol-induced changes in [Ca2+]i and cell viability in PC3 human prostate cancer cells

Abstract

Resveratrol is a natural compound that affects cellular Ca²⁺ homeostasis and viability in different cells. This study examined the effect of resveratrol on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells. The Ca²⁺-sensitive fluorescent dye fura-2 was used to measure [Ca²⁺]_i and WST-1 was used to measure viability. Resveratrol-evoked [Ca²⁺]_i rises concentration-dependently. The response was reduced by removing extracellular Ca²⁺. Resveratrol-evoked Ca²⁺ entry was not inhibited by nifedipine, econazole, SKF96365 and the protein kinase C inhibitor GF109203X, but was nearly abolished by the protein kinase C activator phorbol 12-myristate 13 acetate. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone decreased resveratrol-evoked rise in [Ca²⁺]_i. Conversely, treatment with resveratrol inhibited BHQ-evoked rise in [Ca²⁺]_i. Inhibition of phospholipase C with U73122 did not alter resveratrol-evoked rise in [Ca²⁺]_i. Previous studies showed that resveratrol between 10 and 100?µM induced cell death in various cancer cell types including PC3 cells. However, in this study, resveratrol (1–10?μM) increased cell viability, which was abolished by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid-acetoxymethyl ester (BAPTA/AM). Therefore, it is suggested that in PC3 cells, resveratrol had a dual effect on viability: at low concentrations (1–10?µM) it induced proliferation, whereas at higher concentrations it caused cell death. Collectively, our data suggest that in PC3 cells, resveratrol-induced rise in [Ca²⁺]_i by evoking phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry, via protein kinase C-regulated mechanisms. Resveratrol at 1–10?µM also caused Ca²⁺-dependent cell proliferation.     

Diethylstilbestrol-Induced Estrogen Receptor-Dependent [Ca2+]i Rises and Apoptosis in Chinese Hamster Ovary (CHO) Cells

The effect of the synthetic estrogen diethylstilbestrol (DES) on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and cell viability was explored in Chinese hamster ovary (CHO-K1). [Ca²⁺]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. DES at concentrations ≥ 1∝ increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. In Ca²⁺-free medium, after pretreatment with 50∝ DES, 1∝ thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor)-induced [Ca²⁺]_i rises were abolished. Conversely, thapsigargin pretreatment abolished DES-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 did not alter DES-induced [Ca²⁺]_i rises. At a concentration of 5∝, DES increased cell viability. At concentrations of 100–200 μ M, DES decreased viability in a concentration-dependent manner. The effect of 5 and 100 μM DES on viability was partly reversed by prechelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′ -tetraacetic acid (BAPTA). DES-induced cell death was induced via apoptosis as demonstrated by propidium iodide staining. DES (100 μ M)-induced [Ca²⁺]_i rises were largely inhibited by pretreatment with the estrogen receptor antagonist ICI-182,780 (100 μ M). ICI-182,780 did not affect 5 μ M DES-induced increase in viability but partly reversed 100 μ M DES-induced cell death. Collectively, in CHO-K1 cells, DES induced [Ca²⁺]_i rises by stimulating estrogen receptors leading to Ca²⁺ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca²⁺ influx. DES-caused cytotoxicity was mediated by an estrogen receptor- and Ca²⁺-dependent pathway.     

Thrombin and histamine activate phospholipase C in human endothelial cells via a phorbol ester-sensitive pathway

The effects of phorbol esters and synthetic diglycerides on thrombin- and histamine-stimulated increases in inositol trisphosphate (IP₃) and cytosolic free calcium ([Ca²⁺]_i) were studied in cultured human umbilical vein endothelial cells (HEC). Thrombin (0.003–3.0 U/ml) and histamine (10^?7–10^?4 M) induced rapid increases in [Ca²⁺]_i in suspended cells as monitored with the fluorescent calcium indicator fura-2. In [³H]myoinositol-labeled cells, both thrombin (3 U/ml)- and histamine (10^?4 M)-induced IP₃ increases (195% ± 6% and 98% ± 4%, respectively) occurred in less than 15 sec and were temporally correlated with [Ca²⁺]_i increases. Brief incubations (5–60 min) with different protein kinase C activators [4-β-phorbol 12-myristate 13-acetate (1–100 nM), mezerein (100 nM), and sn-1,2 dioctanoylglycerol (0.1–10 μM)] attenuated agonist-induced increases in [Ca²⁺]_i. These compounds also inhibited thrombin- and histaminestimulated IP₃ formation, thus suggesting a tight coupling between phospholipase C activation and calcium flux in cultured HEC. Overall, these observations suggest that the pathway linking receptors to phospholipase C stimulation in human endothelial cells is sensitive to protein kinase C activation.     

Effect of Celecoxib on Ca2+ Fluxes and Proliferation in MDCK Renal Tubular Cells

The effect of celecoxib on renal tubular cells is largely unexplored. In Madin Darby canine kidney (MDCK) cells, the effect of celecoxib on intracellular Ca^{2 +} concentration ([Ca^{2 +}]_i) and proliferation was examined by using the Ca^{2 +}-sensitive fluorescent dye fura-2 and the viability detecting fluorescent dye tetrazolium, respectively. Celecoxib (≥1 μ M) caused an increase of [Ca^{2 +}]_i in a concentration-dependent manner. Celecoxib-induced [Ca^{2 +}]_i increase was partly reduced by removal of extracellular Ca^{2 +}. Celecoxib-induced Ca^{2 +} influx was independently suggested by Mn^{2 +} influx-induced fura-2 fluorescence quench. In Ca^{2 +}-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca^{2 +}-ATPase, caused a monophasic [Ca^{2 +}]_i increase, after which celecoxib only induced a tiny [Ca^{2 +}]_iincrease; conversely, pretreatment with celecoxib completely inhibited thapsigargin-induced [Ca^{2 +}]_i increases. U73122, an inhibitor of phospholipase C, abolished ATP (but not celecoxib)-induced [Ca^{2 +}]_i increases. Overnight incubation with 1 or 10 μ M celecoxib decreased cell viability by 80% and 100%, respectively. These data indicate that celecoxib evokes a [Ca^{2 +}]_i increase in renal tubular cells by stimulating both extracellular Ca^{2 +} influx and intracellular Ca^{2 +} release and is highly toxic to renal tubular cells in vitro.     

N-(3-oxododecanoyl)-homoserine lactone regulates osteoblast apoptosis and differentiation by mediating intracellular calcium

Pseudomonas aeruginosa (P. aeruginosa) is associated with periapical periodontitis. The lesions are characterized by a disorder in osteoblast metabolism. Quorum sensing molecular N-(3-oxododecanoyl)-homoserine lactone (AHL) is secreted by P. aeruginosa and governs the expression of numerous virulence factors. AHL can trigger intracellular calcium ([Ca²⁺]_i) fluctuations in many host cells. However, it is unclear whether AHL can regulate osteoblast metabolism by affecting [Ca²⁺]_i changes or its spatial correlation. We explored AHL-induced apoptosis and differentiation in pre-osteoblastic MC3T3-E1 cells and evaluated [Ca²⁺]_i mobilization using several extraction methods. The spatial distribution pattern of [Ca²⁺]_i among cells was investigated by Moran's I, an index of spatial autocorrelation. We found that 30 μM and 50 μM AHL triggered opposing osteoblast fates. At 50 μM, AHL inhibited osteoblast differentiation by promoting mitochondrial-dependent apoptosis and negatively regulating osteogenic marker genes, including Runx2, Osterix, bone sialoprotein (Bsp), and osteocalcin (OCN). In contrast, prolonged treatment with 30 μM AHL promoted osteoblast differentiation concomitantly with cell apoptosis. The elevation of [Ca²⁺]_i levels in osteoblasts treated with 50 μM AHL was spatially autocorrelated, while no such phenomenon was observed in 30 μM AHL-treated osteoblasts. The blocking of cell-to-cell spatial autocorrelation in the osteoblasts provoked by 50 μM AHL significantly inhibited apoptosis and partially restored differentiation. Our observations suggest that AHL affects the fate of osteoblasts (apoptosis and differentiation) by affecting the spatial correlation of [Ca²⁺]_i changes. Thus, AHL acts as a double-edged sword for osteoblast function.     

Ketamine inhibition of cytoplasmic calcium signalling in rat pheochromocytoma (PC-12) cells

This study examines the mechanism of action of ketamine, a dissociative anesthetic, with a specific focus on its ability to inhibit changes in the concentration of intracellular free calcium, [Ca²⁺]_i, in PC-12 cells. The resting [Ca²⁺]_i as measured with the fluorescent probe Fura-2 AM in control cells is 184.8±8.6 nM (mean±SEM, n = 15). Changes in [Ca²⁺]_i via influx through voltage-gated calcium channels after membrane depolarization with potassium chloride were monitored in the absence and presence of various concentrations of ketamine. Potassium-depolarization caused a dose-dependent rapid increase in [Ca²⁺]_i, averaging 62±5%, 33±2% and 18±3% (n = 10 each) above control levels for 70 mM, 50 mM and 35 mM KCl, respectively. Ketamine, in the dosage range studied (5 – 500 μM), inhibited the increase in [Ca²⁺]_i stimulated by potassium-depolarization in a dose-dependent manner. The computer-fitted dose-response curve of the pooled data yielded a half maximal suppression concentration, ED₅₀, of 33 μM. In conclusion, this study demonstrates that ketamine inhibits Ca²⁺ influx through voltage-gated Ca²⁺ channels in PC-12 cells at clinically relevant doses, and may play a role in ketamine's action as a general anesthetic agent.     

Lysophosphatidylcholine induces Ca2+-independent cellular injury attenuated by d-propranolol in rat cardiomyocytes

In isolated rat cardiomyocytes, exogenous lysophosphatidylcholine (LPC) (15 μM) increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) from 72 ± 5 to 3042 ± 431 nM accompanied by cell injury as indicated by the hypercontracture of the cells and the increase in creatine phosphokinase (CPK) release. In order to understand whether the cell injury induced by LPC was a consequence of the elevation of [Ca²⁺]_i, the effect of LPC was examined in the Ca²⁺-free solution containing EGTA. Under the Ca²⁺ -free conditions, LPC did not increase [Ca²⁺]_i, whereas it still inflicted injury on the cells in terms of cell-shape change and CPK release to the same degree as that under the Ca²⁺-present condition. Addition of ryanodine (10 μM) failed to prevent the changes in cell-shape and CPK release induced by LPC under both Ca²⁺-free and Ca²⁺ -present conditions. Preincubation of the myocytes with d-propranolol (50 μM) inhibited the LPC-induced changes in cell-shape and CPK release under both Ca²⁺ -free and Ca²⁺ -present conditions (p < 0.05). Our study provides clear evidence that the cellular injury induced by LPC could be independent of the increase in [Ca²⁺]_i, and the Ca²⁺ -independent cellular injury induced by LPC could be attenuated by d-propranolol, although the mechanism remains unknown.     

Ketoconazole-Evoked [Ca2+]i Rises and Non-Ca2+-Triggered Cell Death in Rabbit Corneal Epithelial Cells (SIRC)

The effect of ketoconazole on cytosolic free Ca^{2 +} concentrations ([Ca^{2 +}]_i) and proliferation has not been explored in corneal cells. This study examined whether ketoconazole alters Ca^{2 +} levels and causes cell death in SIRC rabbit corneal epithelial cells. [Ca^{2 +}]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Ketoconazole at concentrations of 5 μ M and above increased [Ca^{2 +}]_i in a concentration-dependent manner. The Ca^{2 +} signal was reduced partly by removing extracellular Ca^{2 +}. The ketoconazole-induced Ca^{2 +} influx was insensitive to L-type Ca^{2 +} channel blockers and protein kinase C modulators. In Ca^{2 +}-free medium, after pretreatment with 50 μ M ketoconazole, thapsigargin-(1 μ M)-induced [Ca^{2 +}]_i rises were abolished; conversely, thapsigargin pretreatment nearly abolished ketoconazole-induced [Ca^{2 +}]_i rises. Inhibition of phospholipase C with 2 μ M U73122 did not change ketoconazole-induced [Ca^{2 +}]_i rises. At concentrations between 5 and 100 μ M, ketoconazole killed cells in a concentration-dependent manner. The cytotoxic effect of 50 μ M ketoconazole was not reversed by prechelating cytosolic Ca^{2 +} with BAPTA. In summary, in corneal cells, ketoconazole-induced [Ca^{2 +}]_i rises by causing Ca^{2 +} release from the endoplasmic reticulum and Ca^{2 +} influx from unknown pathways. Furthermore, the cytotoxicity induced by ketoconazole was not caused via a preceding [Ca^{2 +}]_i rise.     

Flufenamic acid is a tool for investigating TRPC6-mediated calcium signalling in human conditionally immortalised podocytes and HEK293 cells

Mutations in the cation channel TRPC6 result in a renal-specific phenotype of familial nephrotic syndrome, affecting intracellular calcium ([Ca²⁺]_i) signalling in the glomerular podocyte. Tools to study native TRPC6 activity are scarce, although there has been recent success with flufenamic acid (FFA). We confirm the specificity of FFA for TRPC6 both in an artificial expression system and in a human conditionally immortalised podocyte cell line (ciPod).Cells were loaded with fura-2AM and changes in intracellular calcium ([Ca²⁺]_i) were calculated. 200 μM FFA induced an increase in [Ca²⁺]_i in HEK293 cells with native TRPC6 expression, which was enhanced by overexpression of TRPC6 and completely blocked in the absence of extracellular calcium. Expressed TRPC7 did not significantly affect the response to FFA whereas expressed TRPC3 reduced it. FFA also induced an increase ciPod in [Ca²⁺]_i, which was inhibited using SKF96365 and 2-APB, but not indomethacin. In ciPod, adenovirus (Ad-v) wild type (WT) TRPC6 increased [Ca²⁺]_i activity to FFA compared to native TRPC6, whereas activity was significantly reduced with Ad-v dominant negative (DN) TRPC6. The niflumic acid (NFA) induced increase in [Ca²⁺]_i in ciPod was not affected by Ad-v TRPC6 DN, and in HEK293 cells was not affected by WT TRPC6.In conclusion, FFA activates TRPC6 [Ca²⁺]_i signalling in both ciPod and HEK293 cells independently of TRPC3 and TRPC7, and independently of properties of the fenamate family.     

Effect of diindolylmethane on Ca2+ homeostasis and viability in PC3 human prostate cancer cells

The effect of the natural product diindolylmethane on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells was explored. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Diindolylmethane at concentrations of 20–50 µM induced [Ca²⁺]_i rise in a concentration-dependent manner. The response was reduced partly by removing Ca²⁺. Diindolylmethane-evoked Ca²⁺ entry was suppressed by nifedipine, econazole, SK&F96365, protein kinase C modulators and aristolochic acid. In the absence of extracellular Ca²⁺, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca²⁺]_i rise. Incubation with diindolylmethane also inhibited thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca²⁺]_i rise. At concentrations of 50–100 µM, diindolylmethane 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/PI staining data implicate that diindolylmethane (50 and 100 µM) induced apoptosis in a concentration-dependent manner. In conclusion, diindolylmethane induced a [Ca²⁺]_i rise in PC3 cells by evoking phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via phospholipase A₂-sensitive store-operated Ca²⁺ channels. Diindolylmethane caused cell death in which apoptosis may participate.     

Trifluoperazine enhancement of Ca2+-dependent inactivation of L-type Ca2+ currents inHelix aspersa neurons

The effects of trifluoperazine hydrochloride (TFP), a calmodulin antagonist, on L-type Ca²⁺ currents (L-type ICa²⁺) and their Ca²⁺-dependent inactivation, were studied in identifiedHelix aspersa neurons, using two microelectrode voltage clamp. Changes in [Ca²⁺]_i were measured in unclamped fura-2 loaded neurons. Bath applied TFP produced a reversible and dose-dependent reduction in amplitude of L-type ICa²⁺ (IC₅₀=28 μM). Using a double-pulse protocol, we found that TFP enhances the efficacy of Ca²⁺-dependent inactivation of L-type ICa²⁺. Trifluoperazine sulfoxide (50 μM), a TFP derivative with low calmodulin-antagonist activity, did not have any effects on either amplitude or inactivation of L-type ICa²⁺. TFP (20 μM) increased basal [Ca²⁺]_i from 147±37 nM to 650±40nM (N=7). The increase in [Ca²⁺]_i was prevented by removal of external Ca²⁺ and curtailed by depletion of caffeine-sensitive intracellular Ca²⁺ stores. Since TFP may also block protein kinase C (PKC), we tested the effect of a PKC activator (12-O-tetradecanoyl-phorbol-13-acetate) on L-type Ca²⁺ currents. This compound produced an increase in L-type ICa²⁺ without enhancing Ca²⁺-dependent inactivation. The results show that 1) TFP reduces L-type ICa²⁺ while enhancing the efficacy of Ca²⁺-dependent inactivation. 2) TFP produces an increase in basal [Ca²⁺]_i which may contribute to the enhancement of Ca²⁺-dependent inactivation. 3) PKC up-regulates L-type ICa²⁺ without altering the efficacy of Ca²⁺ dependent inactivation. 4) The TFP effects cannot be attributed to its action as PKC blocker.     

Role of Calcineurin-Mediated Dephosphorylation in Modulation of an Inwardly Rectifying K+ Channel in Human Proximal Tubule Cells

Activity of an inwardly rectifying K⁺ channel with inward conductance of about 40 pS in cultured human renal proximal tubule epithelial cells (RPTECs) is regulated at least in part by protein phosphorylation and dephosphorylation. In this study, we examined involvement of calcineurin (CaN), a Ca²⁺/calmodulin (CaM)–dependent phosphatase, in modulating K⁺ channel activity. In cell-attached mode of the patch-clamp technique, application of a CaN inhibitor, cyclosporin A (CsA, 5 μM) or FK520 (5 μM), significantly suppressed channel activity. Intracellular Ca²⁺ concentration ([Ca²⁺] _i ) estimated by fura-2 imaging was elevated by these inhibitors. Since inhibition of CaN attenuates some dephosphorylation with increase in [Ca²⁺] _i , we speculated that inhibiting CaN enhances Ca²⁺-dependent phosphorylation, which might result in channel suppression. To verify this hypothesis, we examined effects of inhibitors of PKC and Ca²⁺/CaM-dependent protein kinase-II (CaMKII) on CsA-induced channel suppression. Although the PKC inhibitor GF109203X (500 nM) did not influence the CsA-induced channel suppression, the CaMKII inhibitor KN62 (20 μM) prevented channel suppression, suggesting that the channel suppression resulted from CaMKII-dependent processes. Indeed, Western blot analysis showed that CsA increased phospho-CaMKII (Thr286), an activated CaMKII in inside–out patches, application of CaM (0.6 μM) and CaMKII (0.15 U/ml) to the bath at 10^?6 M Ca²⁺ significantly suppressed channel activity, which was reactivated by subsequent application of CaN (800 U/ml). These results suggest that CaN plays an important role in supporting K⁺ channel activity in RPTECs by preventing CaMKII-dependent phosphorylation.     

Tamoxifen-Induced [Ca2+]i Rises and Ca2+-Independent Cell Death in Human Oral Cancer Cells

The purpose of this study was to explore the effect of tamoxifen on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and cell viability in OC2 human oral cancer cells. [Ca²⁺]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Tamoxifen at concentrations above 2 μM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. The tamoxifen-induced Ca²⁺ influx was sensitive to blockade of L-type Ca²⁺ channel blockers but insensitive to the estrogen receptor antagonist ICI 182,780 and protein kinase C modulators. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), tamoxifen-induced [Ca²⁺]_i rises were substantially inhibited; and conversely, tamoxifen pretreatment inhibited a part of thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 μM U73122 did not change tamoxifen-induced [Ca²⁺]_i rises. At concentrations between 10 and 50 μM tamoxifen killed cells in a concentration-dependent manner. The cytotoxic effect of 23 μM tamoxifen was not reversed by prechelating cytosolic Ca²⁺ with BAPTA. Collectively, in OC2 cells, tamoxifen induced [Ca²⁺]_i rises, in a nongenomic manner, by causing Ca²⁺ release from the endoplasmic reticulum, and Ca²⁺ influx from L-type Ca²⁺ channels. Furthermore, tamoxifen-caused cytotoxicity was not via a preceding [Ca²⁺]_i rise.     

Stimulation of Ca2+ influx in rat pituitary cells under exposure to a 50 Hz magnetic field

The effect of exposure of single rat pituitary cells to 50 Hz sine wave magnetic fields of various strengths on the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) was studied by using dual-emission microfluorimetry, using indo-1 as probe. A 30 min exposure of the cells to vertical 50 μT peak magnetic field triggered a long-lasting increase in [Ca²⁺]_i from a basal value of about 185 ± 4 nM to 326 ± 41 nM (S.E.; n = 150). The vertical and horizontal components of the static magnetic field were 57 and 15 μT, respectively. The 50 Hz ambient magnetic field was always below 0.1 μT rms. The effect was observed both at 25 ± 2 °C and at 37 ± 2 °C. Responsive cells, for which [Ca²⁺]_i rose to values above 309 nM, were identified as lactotrophs and represented 29% of the total pituitaries. [Ca²⁺]_i increase, for the most part, was due to Ca²⁺ influx through voltage-dependent dihydropiridine-sensitive calcium channels inhibited by PN 200-110. However, neither Ca²⁺ channel blockers nor removal of Ca²⁺ from the external medium during exposure completely prevented the field-induced [Ca²⁺]_i increase. Additional experiments using an MTT colorimetric assay showed that alteration of Ca²⁺ homeostasis of lactotrophs was associated with impairment of some mitochondrial processes. © Wiley-Liss, Inc.