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

Abstract 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 μM induced an immediate increase in [Ca 2+ ] i in a concentration-dependent manner with an EC 50 of 10 μM. Removing extracellular Ca 2+ reduced the Ca 2+ signal by 70%. Pretreatment with 50 μM La 3+ or 10 μM of nifedipine, verapamil and diltiazem did not change 20 μM DES-induced [Ca 2+ ] i increases. Addition of 3 mM Ca 2+ increased [Ca 2+ ] i in cells pretreated with 20 μM DES in Ca 2+ -free medium. Pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca 2+ pump inhibitor) to deplete the endoplasmic reticulum Ca 2+ store partly inhibited 20 μM 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 μM DES abrogated CCCP- and thapsigargin-induced Ca 2+ release. Inhibition of phospholipase C activity with 2 μM U73122 did not alter 20 μM DES-induced Ca2+ release. Another estrogen 17β-estradiol also increased [Ca 2+ ]i in a concentration-dependent manner with an EC50 of 7 μM. 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.