Cell signaling and estrogens in female rat osteoblasts: a possible involvement of unconventional nonnuclear receptors.
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 17 beta-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-[2-3H]inositol or [14C]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. 17 beta-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. 17 beta-Estradiol (1 pM to 1 nM) induced a biphasic and concomitant increase in IP3 and DAG formation. Estradiol immobilized on bovine serum albumin (BSA) [E-(O-carboxymethyl)oxime BSA] and its derivative (O-carboxymethyl)oxime rapidly increased ([Ca2+]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 17 beta-estradiol on IP3 and DAG formation. These results suggest that female rat osteoblasts bear non-genomic unconventional cell surface receptors for estradiol, belonging to the class of the membrane receptors coupled to a phospholipase C via a pertussis toxin-sensitive G protein.
Lieberherr
M
M
Centre National de la Recherche Scientifique, Université Paris V, France.
Grosse
B
B
Kachkache
M
M
Balsan
S
S
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000494:pharmacology; D002478:Cells, Cultured; D004533:Egtazic Acid / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology*; D005260:Female; D007295:Inositol Phosphates / Q000378:metabolism*; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin
D002121:Calcium Channel Blockers; D007295:Inositol Phosphates; D013729:Terpenes; D004958:Estradiol; D004533:Egtazic Acid; D019284:Thapsigargin; D000252:Calcium-Transporting ATPases; D002118:Calcium