Characterization of a calcitonin gene-related peptide release assay in rat isolated distal colon

The release of calcitonin gene-related peptide (CGRP) plays a key role gastrointestinal tract homeostasis. We aimed to investigate mechanisms that mediate CGRP release from the rat colon in vitro. Colon segments were stimulated and the amount of CGRP released was measured using an enzyme immunoassay. Capsaicin and low pH induced significant increases in CGRP release which was shown to be mediated by TRPV1 activation as demonstrated with the TRPV1 antagonists CTPC and capsazepine. The mast cell degranulator, compound 48/80 significantly increased CGRP release an effect that was blocked in the presence of the mast cell stabilizer, ketotifen and the selective Gi inhibitor benzalkonium chloride. The addition of a mixture of inflammatory mediators containing pro-inflammatory cytokines, 5HT, bradykinin and PGE2 showed no effect at neutral pH but at low pH a significant additive effect was observed. We conclude that CGRP release in the rat distal colon occurs in response to mast cell degranulation, inflammatory mediators, low pH and capsaicin and describe a role for TRPV1 receptors in mediating the response.