Yee-Ping Law

Tamoxifen-induced increases in cytoplasmic free Ca2+ levels in human breast cancer cells.

Tamoxifen has been shown to increase cytoplasmic free Ca2+ levels [Ca2+]i in renal tubular cells and bladder cancer cells, and to alter Ca2+ signaling in MCF-7 breast cancer cells. The present study examined the effect of tamoxifen on [Ca2+]i in ZR-75-1 human breast cancer cells using fura-2 as an indicator. Tamoxifen increased [Ca2+]i at a concentration above 2u2009μM with an EC50 of 5u2009μM. Removing extracellular Ca2+ reduced the response by 48u2009±u20092%. In Ca2+-free medium, after tamoxifen-induced [Ca2+]i increased had returned to baseline, adding 3u2009mM Ca2+ increased [Ca2+]i in a concentration-dependent manner. Further, pretreatment with 10u2009μM tamoxifen abolished the [Ca2+]i increase induced by 1u2009μM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor); and conversely, pretreatment with thapsigargin prevented tamoxifen from releasing more Ca2+. Tamoxifen (10u2009μM)-induced Ca2+ release was not changed by inhibiting phospholipase C activity with 2u2009μM U73122. Trypan blue exclusion assay revealed that tamoxifen (1–10u2009μM) did not alter viability after 1u2009min of incubation, but killed 10% of cells after 3–10u2009min of incubation. Together, this study shows that tamoxifen (>2u2009μM) induced a significant, immediate increase in [Ca2+]i in ZR-75-1 breast cancer cells. Tamoxifen acted by releasing Ca2+ from the endoplasmic reticulum Ca2+ stores in a manner independent of phospholipase C activity, and by inducing Ca2+ entry from extracellular medium. Tamoxifen may be of mild cytotoxicity after acute exposure.

Mechanisms of diethylstilbestrol-induced calcium movement in MG63 human osteosarcoma cells.

The effect of the estrogen diethylstilbestrol (DES) on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in MG63 human osteoblasts was explored by using fura-2 as a Ca(2+) indicator. DES at concentrations between 5--20 microM induced an immediate increase in [Ca(2+)](i) in a concentration-dependent manner with an EC(50) of 10 microM. Removing extracellular Ca(2+) reduced the Ca(2+) signal by 70%. Pretreatment with 50 microM La(3+) or 10 microM of nifedipine, verapamil and diltiazem did not change 20 microM DES-induced [Ca(2+)](i) increases. Addition of 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with 20 microM DES in Ca(2+)-free medium. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+) store partly inhibited 20 microM DES-induced Ca(2+) release, but addition of carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler) and thapsigargin together abolished DES-induced Ca(2+) release. Conversely, pretreatment with 20 microM DES abrogated CCCP- and thapsigargin-induced Ca(2+) release. Inhibition of phospholipase C activity with 2 microM U73122 did not alter 20 microM DES-induced Ca2+ release. Another estrogen 17beta-estradiol also increased [Ca(2+)](i) in a concentration-dependent manner with an EC50 of 7 microM. Together, the data indicate that in human osteoblasts, DES increased [Ca(2+)](i) via causing Ca(2+) release from both mitochondria and the endoplasmic reticulum in a phospholipase C-independent manner, and by causing Ca(2+) influx.

CP55,940 increases intracellular Ca2+ levels in Madin-Darby canine kidney cells

The effect of CP55,940, a presumed CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca2+ levels ([Ca2+]i) in Madin-Darby canine kidney cells was examined by using the fluorescent dye fura-2 as a Ca2+ indicator. CP55,940 (2-50 microM) increased [Ca2+]i concentration-dependently with an EC50 of 8 microM. The [Ca2+]i signal comprised an initial rise and a sustained phase. Extracellular Ca2+ removal decreased the maximum [Ca2+]i signals by 32+/-12%. CP55,940 (20 microM)-induced [Ca2+]i signal was not altered by 5 microM of two cannabinoid receptor antagonists, AM-251 and AM-281. CP55,940 (20 microM)-induced [Ca2+]i increase in Ca2+-free medium was inhibited by 86+/-3% by pretreatment with 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 20 microM CP55,940 in Ca2+-free medium for 6 min abolished thapsigargin-induced [Ca2+]i increases. CP55,940 (20 microM)-induced intracellular Ca2+ release was not inhibited when inositol 1,4,5-trisphosphate formation was abolished by suppressing phospholipase C with 2 microM U73122. Collectively, this study shows that CP,55940 induced significant [Ca2+]i increases in canine renal tubular cells by releasing stored Ca2+ from the thapsigargin-sensitive pools in an inositol 1,4,5-trisphosphate-independent manner, and also by causing extracellular Ca2+ entry. The CP55,940s action appears to be dissociated from stimulation of cannabinoid receptors.

Effect of the neuroprotective agent riluzole on intracellular Ca2+ levels in IMR32 neuroblastoma cells

Abstract. Riluzole is an effective neuroprotective drug. Its effect on intracellular free Ca2+ levels ([Ca2+]i) has not been explored. This study examined the effect of riluzole on [Ca2+]i in IMR32 neuroblastoma cells using fura-2 as a Ca2+ probe. Riluzole 0.1–1 mM increased [Ca2+]i in a concentration-dependent manner. Removal of extracellular Ca2+ inhibited the response by 52±5%. The [Ca2+]i increase induced by 0.2xa0mM riluzole was unaltered by 0.1xa0mM La3+ or 10xa0µM verapamil, but was inhibited by 51±4% by 10xa0µM nifedipine. In Ca2+-free medium, pretreatment with 1xa0µM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) reduced the 0.2xa0mM riluzole-induced Ca2+ release by 44±3%; this reduction was augmented to 66±5% by additionally depleting the Ca2+ stores in the Golgi complex with 50xa0µM brefeldin A. Inhibition of inositol 1,4,5-trisphosphate formation by 2xa0µM U73122, a phospholipase C inhibitor, did not affect Ca2+ release induced by 0.2xa0µM riluzole. It was concluded that the neuroprotective agent riluzole increased [Ca2+]i in IMR32 neuroblastoma cells concentration-dependently by releasing Ca2+ from multiple stores in an inositol 1,4,5-trisphosphate-independent manner and also by inducing nifedipine-sensitive Ca2+ influx.

Effect of oleamide on Ca2+ signaling in human bladder cancer cells

Abstract The effect of oleamide, a sleep-inducing endogenous lipid in animal models, on intracellular free levels of Ca 2+ ([Ca 2+ ] i ) in non-excitable and excitable cells was examined by using fura-2 as a fluorescent dye. [Ca 2+ ] i in pheochromocytoma cells, renal tubular cells, osteoblast-like cells, and bladder cancer cells were increased on stimulation of 50 μM oleamide. The response in human bladder cancer cells (T24) was the greatest and was further explored. Oleamide (10–100 μM) increased [Ca 2+ ] i in a concentration-dependent fashion with an EC 50 of 50 μM. The [Ca 2+ ] i signal comprised an initial rise and a sustained plateau and was reduced by removing extracellular Ca 2+ by 85 ± 5%. After pre-treatment with 10–100 μM oleamide in Ca 2+ -free medium, addition of 3 mM Ca 2+ increased [Ca 2+ ] i in a manner dependent on the concentration of oleamide. The [Ca 2+ ] i increase induced by 50 μM oleamide was reduced by 100 μM La 3+ by 40%, but was not altered by 10 μM nifedipine, 10 μM verapamil, and 50 μM Ni 2+ . In Ca 2+ -free medium, pre-treatment with thapsigargin (1 μM), an endoplasmic reticulum Ca 2+ pump inhibitor, abolished 50 μM oleamide-induced [Ca 2+ ] i increases; conversely, pretreatment with 50 μM oleamide reduced 1 μM thapsigargin-induced [Ca 2+ ] i increases by 50 ± 3%. Suppression of the activity of phospholipase C with 2 μM U73122 failed to alter 50 μM oleamide-induced Ca 2+ release. Linoleamide (10–100 μM), another sleep-inducing lipid with a structure similar to that of oleamide, also induced an increase in [Ca 2+ ] i . Together, it was shown that oleamide induced significant [Ca 2+ ] i increases in cells by a phospholipase C-independent release of Ca 2+ from thapsigargin-sensitive stores and by inducing Ca 2+ entry.

Fendiline mobilizes intracellular Ca2+ in Chang liver cells.

1. The effects of the antianginal drug fendiline (N‐[3,3‐diphenylpropyl]‐α‐methyl‐benzylamine) on intracellular free Ca2+ levels ([Ca2+]i) in Chang liver cells were evaluated using fura‐2 as a fluorescent Ca2+ indicator.

Contents Vol. 55, 2001

W.F. Blum, Bad Homburg J.-P. Bourguignon, Liège H.G. Burger, Melbourne P.G. Chatelain, Lyon G. Chiumello, Milan P.E. Clayton, Manchester G. Copinschi, Brussels H.J. Degenhart, Rotterdam M.G. Forest, Lyon J. Girard, Basel P.D. Gluckman, Auckland A. Grüters, Berlin Z. Hochberg, Haifa R.P. Kelch, Iowa City, Iowa P.J. Keller, Zurich S.W.J. Lamberts, Rotterdam F. Leidenberger, Hamburg C.J. Migeon, Baltimore, Md. E. Milgrom, Bicêtre J. Müller, Copenhagen (Book Reviews) O.H. Pescovitz, Indianapolis, Ind. D.A. Price, Manchester R.G. Rosenfeld, Portland, Ohio G. Saggese, Pisa M.O. Savage, London S.M. Shalet, Manchester T. Tanaka, Tokyo G. Van Vliet, Montreal R.J. Voutilainen, Kuopio G.A. Werther, Parkville, Australia J.-M. Wit, Leiden M. Zachmann, Zurich