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

The effect of the estrogen diethylstilbestrol (DES) on cytosolic free Ca2levels ([Ca2+](i)) in MG63 human osteoblasts was explored by using fura-2 a s a Ca2+ indicator. DES at concentrations between 5-20 muM induced an immed iate increase in [Ca2+](i) in a concentration-dependent manner with an EC50 of 10 muM. Removing extracellular Ca2+ reduced the Ca2+ signal by 70%. Pre treatment with 50 muM La3+ or 10 muM of nifedipine. verapamil and diltiazem did not change 20 muM DES-induced [Ca2+](i) increases. Addition of 3 mM Ca 2+ increased [Ca2+](i) in cells pretreated with 20 AM DES in Ca2+-free medi um. Pretreatment with 1 muM thapsigargin (an endoplasmic reticulum Ca2+ pum p inhibitor) to deplete the endoplasmic reticulum Ca2+ store partly inhibit ed 20 muM DES-induced Ca2+ release, but addition of carbonylcyanide m-chlor ophenylhydrazone (CCCP; a mitochondrial uncoupler) and thapsigargin togethe r abolished DES-induced Ca2+ release. Conversely, pretreatment with 20 muM DES abrogated CCCP-and thapsigargin-induced Ca2+ release. Inhibition of pho spholipase C activity with 2 muM U73122 did not alter 20 muM DES-induced Ca 2+ release. Another estrogen 17 beta -estradiol also increased [Ca2+](i) in a concentration-dependent manner with an EC50 of 7 muM. Together, the data indicate that in human osteoblasts, DES increased [Ca2+](i) via causing Ca 2+ release from both mitochondria and the endoplasmic reticulum in a phosph olipase C-independent manner, and by causing Ca2+ influx. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.