Osteoblast-specific CGRP receptor -expression of osteoblastic differentiation-independent non-type I CGRP receptor-

1) CGRP对细胞质Ca^<2+>动员和膜电位的影响在人成骨MG63细胞中，CGRP （1-100 nM）诱导瞬时Ca^<2+>峰值，随后细胞质游离Ca^<2+>浓度缓慢增加（[Ca^<2+>]_i）。在大鼠成骨细胞UMR106中未观察到第二阶段。CGRP还能诱导质膜超极化。CGRP亚型I受体（CGRP- r1）拮抗剂（CGRP_<8-37>）仅使该作用减弱约50%。这些发现表明，这些CGRP的作用不仅是由已知的CGRP- r1介导的，而且还由其他非I亚型受体介导。2) CGRP诱导细胞反应的Schild图分析在MG63细胞中，CGRP诱导的细胞反应，如cAMP的产生、p38-MAPK的磷酸化和CREB的磷酸化，被鉴定为由相同的单一CGRP受体亚型介导，可能是CGRP- r1。相比之下，CGRP诱导的ERK去磷酸化可能是由CGRP受体的单一未知亚型介导的，也可能是由CGRP- r1和未知受体亚型的联合介导的。3)肾上腺髓质素、降钙素及其特异性拮抗剂对细胞内信号通路的影响除MG63细胞外，本研究还采用了人牙周韧带细胞。肾上腺髓质素（ADM）和降钙素（CT）在较高浓度（1 μM）下均能模拟CGRP的作用，但它们的特异性拮抗剂，如ADM_<22-52>和CT_<8-32>，不能显著阻断CGRP的作用。这些结果表明，我们观察到的CGRP作用不是由ADM受体或CT受体介导的，而是由几种CGRP特异性受体亚型介导的。

1) Effects of CGRP on cytoplasmic Ca^<2+> mobilization and membrane potentialIn human osteoblastic MG63 cells, CGRP (1-100 nM) induced a transient Ca^<2+> spike and a subsequent slow increment in cytoplasmic free Ca^<2+> concentrations ([Ca^<2+>]_i). The second phase was not observed in rat osteoblastic UMR106 cells. CGRP also induced plasma membrane hyperpolarization. This action was attenuated only approximately 50% by an antagonist of CGRP subtype I receptor (CGRP-R1), CGRP_<8-37>. These findings suggest that These CGRP actions are not solely mediated by known CGRP-R1 but also by other non-subtype I receptors.2) Schild plot analysis of CGRP-induced cellular responsesIn MG63 cells, the CGRP-induced cellular responses, such as cAMP production, p38-MAPK phosphorylation, and CREB phosphorylation, were identified to be mediated by the same single CGRP receptor subtype, probably CGRP-R1. In contrast, CGRP-induced ERK dephosphorylation was suggested to be mediated either by a single unknown subtype of CGRP receptor or by a combination of CGRP-R1 and a unknown receptor subtype(s).3) Effects of adrenomedullin, calcitonin, and their specific antagonists on intracellular signaling pathwaysIn addition to MG63 cells, human periodontal ligament cells were employed here. Both adrenomedullin (ADM) and calcitonin (CT) at higher concentrations (1 μM) mimicked CGRP action, but their specific antagonists, such as ADM_<22-52> and CT_<8-32>, failed to significantly block CGRP action. These findings suggest that CGRP actions we have observed are not mediated either by ADM receptor or CT receptor, but by several CGRP-specific receptor subtypes.