Regulation of osteoclasts by calcitonin and amphiphilic calcitonin conjugates: Role of cytosolic calcium

The peptide hormone calcitonin is a potent inhibitor of osteoclastic resorption, but it is unstable and poorly absorbed following oral administration. Conjugates of salmon calcitonin covalently linked to low-molecular-weight amphiphilic polymers show improved stability and absorption. The purpose of this study was to investigate the biological activity of these conjugates in vitro using rat osteoclasts and HEK-293 cells transfected with the C1a isoform of the calcitonin receptor. Salmon calcitonin or its conjugates (10 pM-10 nM) caused rapid arrest of osteoclast membrane ruffling and subsequent retraction. The same amphiphilic polymer attached to an unrelated protein had no effect on osteoclast morphology or motility. Since calcitonin-induced retraction of osteoclasts is thought to be mediated by Ca²⁺ signaling, we investigated the effects of calcitonin and its conjugates on cytosolic free Ca²⁺ concentration ([Ca ²⁺]_i). In HEK-293 cells transfected with the calcitonin receptor, these agents induced transient elevations of [Ca²⁺] _i. However, the rise of [Ca²⁺]_i in HEK-293 cells occurred at concentrations 100-1000-fold higher than those required to elicit osteoclast retraction. To investigate the role of Ca²⁺ in osteoclast retraction, we preloaded cells with BAPTA to buffer changes in [Ca²⁺]_i. BAPTA decreased the initial rate of calcitonin-induced osteoclast retraction, but it did not affect the degree of retraction 2-3 hours following calcitonin, indicating that retraction is mediated primarily by Ca²⁺-independent processes. We conclude that calcitonin conjugates cause osteoclast retraction and [Ca²⁺] _i signaling in a manner similar to that elicited by calcitonin. Thus, orally bioavailable calcitonin conjugates show potential for use as antiresorptive agents.