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

Abstract

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 Ca(2+) concentration ([Ca(2+)](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 [DiBAC(4)(3)] bis-oxonol assays. CGRP produced a two-stage change in [Ca(2+)](int): a rapid transient peak and a secondary sustained increase. Both responses were dose dependent with an EC(50) of approximately 0.30 nM, and the maximal effect (initially approximately 3-fold over basal levels) was observed at 20 nM. The initial phase was sensitive to inhibition of Ca(2+) mobilization with thapsigargin, whereas the secondary phase was eliminated only by blocking transmembrane Ca(2+) 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 Ca(2+) discharge from intracellular stores by a cAMP-independent mechanism and subsequently stimulates Ca(2+) influx through L-type voltage-dependent Ca(2+) channels by a cAMP-dependent mechanism. In addition, CGRP dose-dependently polarized cellular E(m), with maximal effect at 20 nM and an EC(50) of 0.30 nM. This effect was attenuated with charybdotoxin (-20%) or glyburide (glibenclamide; -80%), suggesting that E(m) hyperpolarization is induced by both Ca(2+)-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.