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RECEPTORS AND SIGNAL TRANSDUCTION

Calcitonin gene-related peptide elevates calcium and polarizes membrane potential in MG-63 cells by both cAMP-independent and -dependent mechanisms

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 (E_m) 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 E_m, 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 E_m 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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