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, releasing calcium into the area surrounding the osteoclast. The production of high levels of extracellular calcium increases intracellular calcium concentrations ([Ca2+]i), and bone resorption is decreased. To study this mechanism, the dihydropyridine-sensitive L-type calcium channel antagonists PN 200-110, (-)202-791, and nifedipine were studied for their effects on bone resorption using the disaggregated osteoclast pit assay. Changes in [Ca2+]i after treatment with these compounds were determined with the fluoroprobe fura2. In osteoclast-enriched cultures, significant decreases in bone resorption were noted in the presence of PN 200-110 and (-)202-791. The decrease in bone resorption correlated with an increase in [Ca2+]i. To determine whether the effects of these compounds on osteoclasts were mediated via osteoblasts, proliferation and differentiation of rat osteoblast-like cells (ROS 17/2.8) were examined after the addition of these agents. There were no changes in osteoblast proliferation or differentiation, as determined by [3H]thymidine incorporation and specific activity of alkaline phosphatase, after treatment with these compounds at concentrations that inhibited bone resorption in the disaggregated pit assay. This lack of effect of calcium channel antagonists on osteoblast growth and differentiation at concentrations used to inhibit osteoclast function suggests that 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 medium on bone resorption provides additional evidence that PN 200-110 and (-)202-791 are decreasing bone resorption directly by altering osteoclast function, not through osteoblast-osteoclast interactions. The addition of (-)202-791 or PN 200-110 to osteoclasts resulted in a dose-dependent rise in [Ca2+]i. These data suggest that calcium channel antagonists may bind to the calcium channel of the osteoclast and lock it in an open state, leading to increased [Ca2+]i and decreased bone resorption.
Ritchie
C K
CK
Department of Internal Medicine, Mayo Clinic, Rochester, Minnesota 55905.
Maercklein
P B
PB
Fitzpatrick
L A
LA
D000818:Animals; D001862:Bone Resorption; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002478:Cells, Cultured; D007425:Intracellular Membranes / Q000378:metabolism*; D007536:Isomerism; D017275:Isradipine / Q000494:pharmacology; D009539:Nicotinic Acids / Q000737:chemistry / Q000494:pharmacology; D009543:Nifedipine / Q000494:pharmacology; D009994:Osmolar Concentration; D010010:Osteoclasts / Q000187:drug effects* / Q000502:physiology; D010069:Oxadiazoles; D051381:Rats; D014907:Whales
D002121:Calcium Channel Blockers; D009539:Nicotinic Acids; D010069:Oxadiazoles; C049834:PN 202-791; D009543:Nifedipine; D002118:Calcium; D017275:Isradipine