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RECEPTORS AND SIGNAL TRANSDUCTION
1Medical Research Service, Kansas City Department of Veterans Affairs Medical Center, and 2Department of Biochemistry and Molecular Biology, 3Department of Physical Therapy Education and Rehabilitation, and 4Department of Anatomy and Cell Biology University of Kansas Medical Center, Kansas City, Missouri 64128; and 5Division of Cellular Pharmacology, Department of Signal Transduction, Graduate School of Medical and Dental Sciences, Niigata University, Niigata 951-8514, Japan
Submitted 1 July 2003 ; accepted in final form 21 March 2004
Published data suggest that the neuropeptide calcitonin gene-related peptide (CGRP) can stimulate osteoblastic bone formation; however, interest has focused on activation of cAMP-dependent signaling pathways in osteogenic cells without full consideration of the importance of cAMP-independent signaling. We have now examined the effects of CGRP on intracellular Ca2+ concentration ([Ca2+]int) and membrane potential (Em) in preosteoblastic human MG-63 cells by single-cell fluorescent confocal analysis using fluo 4-AM-fura red-AM and bis(1,3-dibarbituric acid)-trimethine oxanol [DiBAC4(3)] bis-oxonol assays. CGRP produced a two-stage change in [Ca2+]int: a rapid transient peak and a secondary sustained increase. Both responses were dose dependent with an EC50 of 
0.30 nM, and the maximal effect (initially 3-fold over basal levels) was observed at 20 nM. The initial phase was sensitive to inhibition of Ca2+ mobilization with thapsigargin, whereas the secondary phase was eliminated only by blocking transmembrane Ca2+ influx with verapamil or inhibiting cAMP-dependent signaling with the Rp isomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS). These data suggest that CGRP initially stimulates Ca2+ discharge from intracellular stores by a cAMP-independent mechanism and subsequently stimulates Ca2+ influx through L-type voltage-dependent Ca2+ channels by a cAMP-dependent mechanism. In addition, CGRP dose-dependently polarized cellular Em, with maximal effect at 20 nM and an EC50 of 0.30 nM. This effect was attenuated with charybdotoxin (–20%) or glyburide (glibenclamide; –80%), suggesting that Em hyperpolarization is induced by both Ca2+-activated and ATP-sensitive K+ channels. Thus CGRP signals strongly by both cAMP-dependent and cAMP-independent signaling pathways in preosteoblastic human MG-63 cells.
osteoblastic cells; calcium; membrane potential; potassium channels; adenosine 3',5'-cyclic monophosphate
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