Department of Oral and Maxillofacial Surgery, Xingqiao Hospital, Third Military Medical University, Chongqing, Peoples Republic of China.
Cell Biol Int. 2011 Aug;35(8):757-65. doi: 10.1042/CBI20100832.
The aim of this study was to investigate the in vitro effects and regulatory mechanism of CGRP (calcitonin gene-related peptide) on NO (nitric oxide) production in osteoblasts. MOB (primary human mandibular osteoblasts) and osteoblast-like cells (MG-63) were either cultured with CGRP or co-incubated with inhibitors targeting eNOS (endothelial nitric oxide synthase), iNOS (inducible nitric oxide synthase), nNOS (neuronal nitric oxide synthase) and [Ca2+]i (intracellular Ca2+). The NO concentration in cell culture supernatants was measured during the first 24 h using the Griess test; cellular NO was marked with the fluorescent marker DAF-FM, DA (3-amino, 4-aminomethyl-2',7'-difluorescein; diacetate) and measured by fluorescence microscopy from 1 to 4 h after treatment. eNOS and iNOS mRNA expression levels were measured by quantitative RT-PCR during the first 24 h after treatment. CGRP-induced NO production in the supernatants was high between 1 to 12 h, while cellular NO was highest between 1 to 2 h after treatment and returned to basal levels by 3 h. Both in MG-63 cells and MOBs, the most effective CGRP concentration was 10 nM with a peak time of 1 h. CGRP-induced NO production decreased when eNOS activity was inhibited or when voltage-dependent L-type Ca2+ channels were blocked at 4 h. CGRP was not able to induce changes in iNOS or eNOS mRNA levels and had no effect on the cytokine-induced increase of iNOS expression. Our results suggest that CGRP transiently induces NO production in osteoblasts by elevating intracellular Ca2+ to stimulate the activity of eNOS in vitro.
本研究旨在探讨 CGRP（降钙素基因相关肽）在体外对成骨细胞中 NO（一氧化氮）产生的影响及其调控机制。将 MOB（原代人下颌骨成骨细胞）和类成骨细胞（MG-63）与 CGRP 一起培养，或与针对 eNOS（内皮型一氧化氮合酶）、iNOS（诱导型一氧化氮合酶）、nNOS（神经元型一氧化氮合酶）和 [Ca2+]i（细胞内 Ca2+）的抑制剂共同孵育。在最初的 24 小时内，使用格里斯测试测量细胞培养上清液中的 NO 浓度；用荧光标记物 DAF-FM 标记细胞内 NO，在处理后 1 至 4 小时通过荧光显微镜进行测量。在处理后的最初 24 小时内，通过定量 RT-PCR 测量 eNOS 和 iNOS mRNA 表达水平。CGRP 在 1 至 12 小时之间诱导上清液中产生大量的 NO，而细胞内 NO 在处理后 1 至 2 小时之间最高，并在 3 小时时恢复到基础水平。在 MG-63 细胞和 MOB 中，最有效的 CGRP 浓度为 10 nM，峰值时间为 1 小时。在 4 小时时抑制 eNOS 活性或阻断电压依赖性 L 型 Ca2+通道时，CGRP 诱导的 NO 产生减少。CGRP 不能诱导 iNOS 或 eNOS mRNA 水平发生变化，也不能影响细胞因子诱导的 iNOS 表达增加。我们的结果表明，CGRP 通过升高细胞内 Ca2+来刺激 eNOS 活性，在体外短暂诱导成骨细胞中产生 NO。
