CELL SIGNALING AND ESTROGENS IN FEMALE RAT OSTEOBLASTS - A POSSIBLE INVOLVEMENT OF UNCONVENTIONAL NONNUCLEAR RECEPTORS

Estrogen deficiency is associated with bone loss, and estrogen replace ment is an effective treatment of this osteoporotic process. This stud y examines the early (5-120 s) effects of 17beta-estradiol on the intr acellular calcium and phospholipid metabolism in confluent female rat osteoblasts. The cytosolic free Ca2+ concentration ([Ca2+]i) was deter mined using fura-2/AM as Ca2+ probe. Cells were labeled with myo-[2-H- 3]inositol or [C-14]arachidonic acid for inositol or lipid determinati on. Inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) produc tion were determined by either mass measurement or anion-exchange chro matography or by thin-layer chromatography, respectively. 17beta-Estra diol (1 pM to 1 nM) increased [Ca2+]i in a biphasic manner within 10 s via Ca2+ influx from the extracellular milieu, as shown by the effect s of the calcium chelator EGTA and the Ca2+ Channel blockers nifedipin e and verapamil, and via Ca2+ mobilization from the endoplasmic reticu lum (ER), as shown by the effects of thapsigargin. 17beta-Estradiol (1 pM to 1 nM) induced a biphasic and concomitant increase in IP., and D AG formation. Estradiol immobilized on bovine serum albumin (BSA) [E-( O-carboxymethyl)oxime BSA] and its derivative (0-carboxymethyl)oxime r apidly increased ([Ca 2+]i, IP3, and DAG and were full agonists, altho ugh they were less potent than the free estradiol. They had the same a ction time course and acted via Ca2+ influx and Ca2+ mobilization from ER. Tamoxifen, a potent inhibitor of genomic steroid responses, did n ot block the rapid increase in Ca2+, IP3, and DAG induced by estradiol . Finally, inhibitor of phospholipase C (neomycin) and pertussis toxin abolished the effects of 17beta-estradiol on IP3 and DAG formation. T hese results suggest that female rat osteoblasts bear nongenomic uncon ventional cell surface receptors for estradiol, belonging to the class of the membrane receptors coupled to a phospholipase C vis a pertussi s toxin-sensitive G protein.