Clomiphene, an ovulation-inducing agent, causes [Ca2+]i increases in human osteoblast-like cells.
The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca2+ levels ([Ca2+]i) in MG63 human osteosarcoma cells was explored by using fura-2 as a Ca2+ indicator. Clomiphene at concentrations between 5-75 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 of 50 microM. The [Ca2+]i signal consisted of an initial rise and a sustained phase. Ca2+ removal reduced the Ca2+ signal by 40+/-10%. The [Ca2+]i increase induced by 50 microM clomiphene was inhibited by 80+/-5% by 10 microM nifedipine, but was insensitive to 50 microM La3+ or 10 microM verapamil. In Ca2+-free medium, pretreatment with 50 microM brefeldin A (to disrupt the Golgi complex Ca2+ store), 1 microM thapsigargin (to inhibit the endoplasmic reticulum Ca2+ pump), and carbonylcyanide m-chlorophenylhydrazone (CCCP; to uncouple mitochondria) inhibited 51+/-3% of 50 microM clomiphene-induced Ca2+ release; conversely, pretreatment with 50 microM clomiphene abolished the [Ca2+]i increase induced by thapsigargin, CCCP, and brefeldin A. The Ca2+ release-induced by 50 pM clomiphene was unchanged by inhibition of phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Collectively, the results suggest that clomiphene increased [Ca2+]i, in osteoblast-like cells, by releasing intracellular Ca2+ in a phospholipase C-independent manner and by causing nifedipine-sensitive Ca2+ influx.
Chen
Y C
YC
Department of Orthopaedic Surgery, Chang-Gung Memorial General Hospital-Kaohsiung, Taiwan, ROC.
Wang
J L
JL
Liu
C P
CP
Cheng
J S
JS
Chang
H T
HT
Yuk-Keung
L
L
Su
W
W
Law
Y P
YP
Chen
W C
WC
Jan
C R
CR
D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D002996:Clomiphene / Q000494:pharmacology*; D004533:Egtazic Acid / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D005300:Fertility Agents, Female / Q000494:pharmacology*; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D009994:Osmolar Concentration; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
D002121:Calcium Channel Blockers; D002614:Chelating Agents; D004963:Estrenes; D005300:Fertility Agents, Female; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D002996:Clomiphene; D004533:Egtazic Acid; D010738:Type C Phospholipases; D002118:Calcium