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 ([Ca24]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 Ca(2+)-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.
Komarova
S V
SV
CIHR Group in Skeletal Development and Remodeling, Department of Physiology and Pharmacology, Division of Oral Biology, Faculty of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada.
Shum
J B
JB
Paige
L A
LA
Sims
S M
SM
Dixon
S J
SJ
D000818:Animals; D000831:Animals, Newborn; D001711:Biotransformation; D001862:Bone Resorption; D002116:Calcitonin / Q000378:metabolism / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology; D003600:Cytosol / Q000187:drug effects* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D007668:Kidney / Q000187:drug effects / Q000473:pathology / Q000502:physiology; D018715:Microscopy, Video; D010010:Osteoclasts / Q000187:drug effects* / Q000473:pathology / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D018003:Receptors, Calcitonin / Q000235:genetics / Q000378:metabolism; D013501:Surface-Active Agents / Q000378:metabolism / Q000494:pharmacology*; D014162:Transfection
D018003:Receptors, Calcitonin; D013501:Surface-Active Agents; D004533:Egtazic Acid; C028815:salmon calcitonin; D002116:Calcitonin; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium