Calcitonin gene-related peptide-enhanced nitric oxide release and inducible NOS activity and mRNA expression in LPS-stimulated mouse peritoneal macrophages

Abstract

Previously we have shown that calcitonin gene-related peptide (CGRP), a neuropeptide increases lipopolysaccharide- (LPS) induced nitric oxide (NO) production in mouse peritoneal macrophages by using the Griess method. In this study we further examined whether CGRP could modulate inducible NO synthase (iNOS) protein and mRNA expression from mouse peritoneal macrophages. Macrophages were obtained from the peritoneal exude of male Balb/c mouse. The cells were plated on culture dishes at a density of 5 x 10(5) cells per well and were allowed to adhere for 2 h. After incubation for 24 h, the macrophages were cultured with 0.01 to 1 microg/mL LPS with or without CGRP (1-1,000 nM) for 24 h. The results showed that CGRP markedly enhanced 0.5 microg/mL LPS-induced NO release as compared with that of lower doses of LPS, such as 0.01 and 0.1 microg/mL LPS. NO was increased from 19.8 +/- 2.6 to the highest level of 31.5 +/- 4.2 microM in 5 x 10(5) cells by 10 nM CGRP in 0.5 microg/mL LPS-stimulated macrophages. The cGMP level in macrophages was augmented when CGRP was added with LPS. However, when using higher dose (1.0 microg/mL) of LPS to stimulate the macrophages, CGRP had no effect at all on NO release. CGRP had no direct effect on NO and cGMP production. CGRP increased the expression of inducible NOS protein in LPS-stimulated macrophages shown by immunocytochemistry method. The activity of iNOS was also enhanced by CGRP as compared with LPS-stimulation alone by detecting the 3H-L-citruline formation from 3H-L-arginine. We found that CGRP also increased the LPS-induced iNOS mRNA levels by using reverse transcriptase-PCR method. These data suggest that CGRP enhances LPS-induced NO release, iNOS activity, and iNOS mRNA in mouse peritoneal macrophages.