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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+]ilevels in suspended MG63 human osteosarcoma cells using fura‐2. MK‐886 at 0.1 μM and above increased [Ca2+]iin 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+]irises were abolished; conversely, thapsigargin pretreatment nearly abolished MK‐886‐induced [Ca2+]irises. Inhibition of phospholipase C with U73122 did not change MK‐886‐induced [Ca2+]irises. Collectively, in MG63 osteosarcoma cells, MK‐886 induced [Ca2+]irises 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.

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

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+]iin a concentration‐dependent fashion with an EC50value of 20 μM. The [Ca2+]isignal 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+]iin 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+]iincreases induced by 20 μM oleamide; conversely, pretreatment with 20 μM oleamide reduced 1 μM thapsigargin‐induced [Ca2+]iincreases 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+]iincreases 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.