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Effect of calcitonin gene-related peptide on nitric oxide production in osteoblasts: an experimental study
Li Yan*†, Tan Yinghui*1, Yang Bo*, Zhang Gang*, Xiao Xian‡ and Zheng Lu§
*Department of Oral and Maxillofacial Surgery, Xingqiao Hospital, Third Military Medical University, Chongqing, Peoples Republic of China, †Department of Oral and Maxillofacial Surgery, Chengdu Army General Hospital, Chengdu, Peoples Republic of China, ‡The First Outpatient Department of Chengdu Military District Institution, Chengdu Army General Hospital, Chengdu, Peoples Republic of China, and §Department of Hepatobiliary Surgery, Xingqiao Hospital, Third Military Medical University, Chongqing, Peoples Republic of China
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.
Key words: calcitonin gene-related peptide, nitric oxide production, osteoblast, signalling pathway
Abbreviations: ALP, alkaline phosphatase, [Ca2+]i, intracellular Ca2+, cAMP, cyclic adenosine monophosphate, CGRP, calcitonin gene-related peptide, cNOS, constitutive nitric oxide synthase, DA, diacetate, DAF-FM, 3-amino, 4-aminomethyl-2′,7′-difluorescein, DMEM, Dulbecco's modified Eagle's medium, eNOS, endothelial nitric oxide synthase, FCS, fetal calf serum, GAPDH, glyceraldehyde-3-phosphate dehydrogenase, IFN-γ, interferon-gamma, IL, interleukin-1, iNOS, inducible nitric oxide synthase, l-NAME, l-arginine, NG-nitro-l-arginine methyl ester, MAPK, mitogen-activated protein kinase, MOB, primary human mandibular osteoblasts, nNOS, neuronal nitric oxide synthase, NO, nitric oxide, NO2, nitrite, NO3, nitrate, RQ%, relative quantity, TNF-α, tumour necrosis factor-alpha
1To whom correspondence should be addressed (email email@example.com).
Received 23 November 2010/19 January 2011; accepted 11 March 2011
Published as Cell Biology International Immediate Publication 11 March 2011, doi:10.1042/CBI20100832
© The Author(s)