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 Ca2+. Treatment with 10−7 M testosterone (T) or 17β‐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 Ca2+ 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. J. Cell. Biochem. 79:620–627, 2000. © 2000 Wiley‐Liss, Inc.