DIRECT EFFECT OF CALCIUM-CHANNEL ANTAGONISTS ON OSTEOCLAST FUNCTION -ALTERATIONS IN BONE-RESORPTION AND INTRACELLULAR CALCIUM CONCENTRATIONS

Osteoclasts attach to mineralized surfaces and resorb bone matrix, rel easing calcium into the area surrounding the osteoclast. The productio n of high levels of extracellular calcium increases intracellular calc ium concentrations ([Ca2+](i)), and bone resorption is decreased. To s tudy this mechanism, the dihydropyridine-sensitive L-type calcium chan nel antagonists PN 200-110, (-)202-791, and nifedipine were studied fo r their effects on bone resorption using the disaggregated osteoclast pit assay. Changes in [Ca2+](i) after treatment with these compounds w ere determined with the fluoroprobe fura2. In osteoclast-enriched cult ures, significant decreases in bone resorption were noted in the prese nce of PN 200-110 and (-)202-798. The decrease in bone resorption corr elated with an increase in [Ca2+](i). To determine whether the effects of these compounds on osteoclasts were mediated via osteoblasts, prol iferation and differentiation of rat osteoblast-like cells (ROS 17/2.8 ) were examined after the addition of these agents. There were no chan ges in osteoblast proliferation or differentiation, as determined by [ H-3]thymidine incorporation and specific activity of alkaline phosphat ase, after treatment with these compounds at concentrations that inhib ited bone resorption in the disaggregated pit assay. This lack of effe ct of calcium channel antagonists on osteoblast growth and differentia tion at concentrations used to inhibit osteoclast function suggests th at the effects of PN 200-110 and (-)202-791 on the osteoclast are not mediated via the osteoblast. In addition, conditioned medium recovered from ROS 17/2.8 cultures treated with PN 200-110 or (-)202-791 had no effect on pit formation compared to the conditioned medium from cell- free cultures. This lack of effect of calcium channel conditioned medi um on bone resorption provides additional evidence that PN 200-110 and (-)202-791 are decreasing bone resorption directly by altering osteoc last function, not through osteoblast-osteoclast interactions. The add ition of (-)202-791 or PN 200-110 to osteoclasts resulted in a dose-de pendent rise in [Ca2+](i). These data suggest that calcium channel ant agonists may bind to the calcium channel of the osteoclast and lock it in an open state, leading to increased [Ca2+](i) and decreased bone r esorption.