Cell Signaling And Estrogens In Female Rat Osteoblasts - A Possible Involvement Of Unconventional Nonnuclear Receptors
JOURNAL OF BONE AND MINERAL RESEARCH(1993)
摘要
Estrogen deficiency is associated with bone loss, and estrogen replacement is an effective treatment of this osteoporotic process. This study examines the early (5-120 s) effects of 17beta-estradiol on the intracellular calcium and phospholipid metabolism in confluent female rat osteoblasts. The cytosolic free Ca2+ concentration ([Ca2+]i) was determined 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 determination. Inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) production were determined by either mass measurement or anion-exchange chromatography or by thin-layer chromatography, respectively. 17beta-Estradiol (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 effects of the calcium chelator EGTA and the Ca2+ Channel blockers nifedipine and verapamil, and via Ca2+ mobilization from the endoplasmic reticulum (ER), as shown by the effects of thapsigargin. 17beta-Estradiol (1 pM to 1 nM) induced a biphasic and concomitant increase in IP., and DAG formation. Estradiol immobilized on bovine serum albumin (BSA) [E-(O-carboxymethyl)oxime BSA] and its derivative (0-carboxymethyl)oxime rapidly increased ([Ca 2+]i, IP3, and DAG and were full agonists, although they were less potent than the free estradiol. They had the same action time course and acted via Ca2+ influx and Ca2+ mobilization from ER. Tamoxifen, a potent inhibitor of genomic steroid responses, did not 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. These results suggest that female rat osteoblasts bear nongenomic unconventional cell surface receptors for estradiol, belonging to the class of the membrane receptors coupled to a phospholipase C vis a pertussis toxin-sensitive G protein.
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