Impaired c‐src kinase regulation of muscle contraction during colonic inflammation is due to nitrosylation of Ca 2+ channels

Abstract

Basal levels of c-src kinase (pp60) are known to regulate smooth muscle Ca2+ channels. Colonic inflammation results in attenuated Ca2+ currents and muscle contraction. Here, we examined the role of pp60 in experimental colitis. Ca2+-influx induced contractions were measured by isometric tension recordings of mouse colonic longitudinal muscle strips depolarized by high K+. The Emax to CaCl2 was significantly less in inflamed tissues (38.4 ± 7.6%) than controls. The EC50 for CaCl2 was 1.2 × 10−4 M in controls and 2.4 × 10−4 M in inflamed tissues indicative of reduced Ca2+ influx. PP2, a selective pp60 inhibitor, significantly reduced the contractile amplitude and shifted the pD2 from 3.88 to 2.44 in controls while it was ineffective in inflamed tissues (3.66 vs 3.43). Following pretreatment with a SIN-1/peroxynitrite combination the maximal contraction to CaCl2 was reduced by 46 ± 7% in controls but unaffected in inflamed (13 ± 11%). Peroxynitrite also prevented the inhibitory effect of PP2 in control tissues. Neither the Ca2+ channel, Cav1.2b, gene expression (real –time PCR) nor the pp60 activity were altered by inflammation. Western analysis with 3-nitrotyrosine antibody revealed nitrotyrosylation of Ca2+ channel proteins by inflammation. These data suggest that posttranslational modification of Ca2+ channels, possibly nitrotyrosylation, prevents c-src kinase regulation resulting in decreased Ca2+ influx. Supported by NIDDK46367