Regulation of Avian Osteoclastic H+-ATPase and Bone Resorption by Tamoxifen and Calmodulin Antagonists

We used highly purified avian osteoclasts and isolated membranes from osteoclasts to study effects of tamoxifen, 4-hydroxytamoxifen, calmodulin antagonists, estrogen, diethylstilbestrol, and the anti-estrogen ICI 182780 on cellular degradation of 3H-labeled bone in vitro and on membrane HCl transport. Bone resorption was reversibly inhibited by tamoxifen, 4-hydroxytamoxifen, and trifluoperazine with IC50 values of ∼1 μM. Diethylstilbestrol and 17-β-estradiol had no effects on bone resorption at receptor-saturating concentrations, while ICI 182780 inhibited bone resorption at concentrations greater than 1 μM. At these concentrations ICI 182780, like tamoxifen, inhibits calmodulin-stimulated cyclic nucleotide phosphodiesterase activity. Membrane HCl transport, assessed by ATP-dependent acridine orange uptake, was unaffected by 17-β-estradiol and diethylstilbestrol at concentrations up to 10 μM, while ICI 182780 inhibited HCl transport at concentrations greater than 1 μM. In contrast HCl transport was inhibited by tamoxifen, 4-hydroxytamoxifen, and the calmodulin antagonists, trifluoperazine and calmidazolium, with IC50 values of 0.25-1.5 μM. These results suggested the presence of a membrane-associated non-steroid receptor for tamoxifen in osteoclasts. Tamoxifen binding studies demonstrated saturable binding in the osteoclast particulate fraction, but not in the nuclear or cytosolic fractions. Membranes enriched in ruffled border by differential centrifugation following nitrogen cavitation showed binding consistent with one site, Kd∼1 μM. Our findings 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 hypothesis of a direct effect on osteoclasts by these compounds in this species.