REGULATION OF AVIAN OSTEOCLASTIC H-ATPASE AND BONE-RESORPTION BY TAMOXIFEN AND CALMODULIN ANTAGONISTS - EFFECTS INDEPENDENT OF STEROID-RECEPTORS()

We used highly purified avian osteoclasts and isolated membranes from osteoclasts to study effects of tamoxifen, 4-hydroxytamoxifen, calmodu lin antagonists, estrogen, diethylstilbestrol, and the anti-estrogen I CI 182780 on cellular degradation of H-3-labeled bone in vitro and on membrane HCl transport. Bone resorption was reversibly inhibited by ta moxifen, 4-hydroxytamoxifen, and trifluoperazine with IC50 values of s imilar to 1 mu M. Diethylstilbestrol and 17-beta-estradiol had no effe cts on bone resorption at receptor-saturating concentrations, while IC I 182780 inhibited bone resorption at concentrations greater than 1 mu M. At these concentrations ICI 182780, like tamoxifen, inhibits calmo dulin stimulated cyclic nucleotide phosphodiesterase activity. Membran e HCl transport, assessed by ATP-dependent acridine orange uptake, was unaffected by 17-beta-estradiol and diethylstilbestrol at concentrati ons up to 10 mu M, while ICI 182780 inhibited HCl transport at concent rations greater than 1 mu M. In contrast HCl transport was inhibited b y tamoxifen, 4-hydroxytamoxifen, and the calmodulin antagonists, trifl uoperazine and calmidazolium, with IC50 values of 0.25-1.5 mu M. These results suggested the presence of a membrane-associated non steroid r eceptor for tamoxifen in osteoclasts. Tamoxifen binding studies demons trated saturable binding in the osteoclast particulate fraction, but n ot in the nuclear or cytosolic fractions. Membranes enriched in ruffle d border by differential centrifugation following nitrogen cavitation showed binding consistent with one site, K-d similar to 1 mu M. Our fi ndings indicate that tamoxifen inhibits osteoclastic HCl transport by binding membrane-associated target(s), probably similar or related to calmodulin antagonist targets. Further, effects of estrogens or highly specific anti-estrogens on bone turnover do not support the hypothesi s of a direct effect on osteoclasts by these compounds in this species .