Effect of calcitonin gene‐related peptide on nitric oxide production in osteoblasts: an experimental study

Abstract

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 [Ca 2+ ] i (intracellular Ca 2+ ). 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 Ca 2+ 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 Ca 2+ to stimulate the activity of eNOS in vitro .