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 ([Ca2+] 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.