Simultaneous detection and functional response of testosterone and estradiol receptors in osteoblast plasma membranes.
Osteoblasts derived from the periosteal surfaces of two-three-week-old male broiler chicken tibias were cultured for eight days. The cells were then loaded with fura-2/AM ester to detect surges in intracellular Ca(2+). Treatment with 10(-7) M testosterone (T) or 17beta-estradiol (E) elicited a rapid (within seconds) response that was substantially reduced by introducing the calcium chelating agent EGTA or the calcium-channel blocker verapamil. The hormones were equally effective when covalently linked to bovine serum albumin (BSA), a procedure that ensures the hormone does not enter the cells. The rapid response to surface-bound steroids indicates that the responses were invoked through plasma-membrane receptors. The source of Ca(2+) was shown to be through entry from external sources, as well as from intracellular stores. Flow cytometry of fluorescein-tagged T-BSA and E-BSA revealed that osteoblasts derived from male chickens had similar and substantial levels of both receptors.
Armen
T A
TA
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Gay
C V
CV
D000818:Animals; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D002645:Chickens; D004533:Egtazic Acid / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology; D005434:Flow Cytometry; D066298:In Vitro Techniques; D008297:Male; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D011944:Receptors, Androgen / Q000378:metabolism*; D011959:Receptors, Estradiol / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013739:Testosterone / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
D002121:Calcium Channel Blockers; D002614:Chelating Agents; D011944:Receptors, Androgen; D011959:Receptors, Estradiol; C043090:estradiol-bovine serum albumin; C045036:testosterone-15 beta-bovine serum albumin conjugate; D012710:Serum Albumin, Bovine; D013739:Testosterone; D004958:Estradiol; D004533:Egtazic Acid; D014700:Verapamil