Calcitonin gene-related peptide elevates calcium and polarizes membrane potential in MG-63 cells by both cAMP-independent and -dependent mechanisms
Calcitonin gene-related peptide elevates calcium and polarizes membrane potential in MG-63 cells by both cAMP-independent and -dependent mechanisms
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DOI：
10.1152/ajpcell.00274.2003
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发表时间：
2004-08-01
影响因子：
5.5
通讯作者：
Kawase, T
中科院分区：
文献类型：
--
作者：
Burns, DM;Stehno-Bittel, L;Kawase, T
已发表的数据表明，神经肽降钙素基因相关肽（CGRP）可以刺激成骨细胞骨形成;然而，兴趣集中在成骨细胞中cAMP依赖性信号通路的激活，而没有充分考虑cAMP非依赖性信号的重要性。我们现在已经通过单细胞荧光共聚焦分析使用fluo 4-AM-fura red-AM和bis（1，3-dibarbituric acid）- trimethine oxanol [DiBAC（4）（3）] bis-oxonol测定来检查CGRP对前成骨细胞人MG-63细胞中细胞内Ca 2+浓度（[Ca 2 +]（int））和膜电位（E-m）的影响。CGRP引起[Ca 2 +]（int）的两阶段变化：快速的瞬时峰值和二次持续增加。两种反应均具有剂量依赖性，EC 50值接近0.30 nM，在20 nM时观察到最大效应（最初接近基础水平的3倍）。初始相对用毒胡萝卜素抑制Ca 2+动员敏感，而第二相仅通过用维拉帕米阻断跨膜Ca 2+内流或用腺苷3 '，5'-环硫代磷酸（Rp-cAMPS）的Rp异构体抑制cAMP依赖性信号传导来消除。这些数据表明，CGRP最初通过cAMP非依赖性机制刺激细胞内钙库的钙释放，随后通过cAMP依赖性机制刺激通过L型电压依赖性钙通道的钙内流。此外，CGRP剂量依赖性极化细胞Em，在20 nM时具有最大效应，EC 50为0.30 nM。这种作用被Charybdotoxin（-20%）或格列本脲（格列本脲;-80%）减弱，表明Em超极化是由Ca 2+激活和ATP敏感性K+通道诱导的。因此，CGRP信号强烈的cAMP依赖性和cAMP非依赖性信号通路在前成骨细胞的人MG-63细胞。
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 [DiBAC(4)(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 similar to0.30 nM, and the maximal effect (initially similar to3-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.