Basal levels of
c-Src kinase are known to regulate smooth muscle Ca(2+) channels. Colonic
inflammation results in attenuated Ca(2+) currents and muscle contraction. Here, we examined the regulation of
calcium influx-dependent contractility by
c-Src kinase in experimental
colitis. Ca(2+)-influx induced contractions were measured by isometric tension recordings of mouse colonic longitudinal muscle strips depolarized by high K(+). The E(max) to CaCl(2) was significantly less in inflamed tissues (38.4 +/- 7.6%) than controls, indicative of reduced Ca(2+) influx. PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]
pyrimidine], a selective
Src kinase inhibitor, significantly reduced the contractile amplitude and shifted the pD(2) from 3.88 to 2.44 in controls, whereas it was ineffective in inflamed tissues (3.66 versus 3.43). After pretreatment with a SIN-1 (3-morpholinosydnonimine)/
peroxynitrite combination, the maximal contraction to CaCl(2) was reduced by 46 +/- 7% in controls but unaffected in inflamed tissues (13 +/- 11%).
Peroxynitrite also prevented the inhibitory effect of PP2 in control tissues. In colonic single smooth muscle cells, PP2 inhibited Ca(2+) currents by 84.1 +/- 3.9% in normal but only 36.2 +/- 13% in inflamed tissues. Neither the Ca(2+) channel Ca(v)1.2b, gene expression, nor the
c-Src kinase activity was altered by
inflammation. Western blot analysis showed no change in the Ca(2+) channel
protein expression but increased nitrotyrosylated-Ca(2+) channel
proteins during
inflammation. These data suggest that post-translational modification of Ca(2+) channels during
inflammation, possibly nitrotyrosylation, prevents
c-Src kinase regulation resulting in decreased Ca(2+) influx.