Abstract
Basal levels of c-Src kinase are known to regulate smooth muscle Ca2+ channels. Colonic inflammation results in attenuated Ca2+ currents and muscle contraction. Here, we examined the regulation of calcium influx-dependent contractility by c-Src kinase 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, indicative of reduced Ca2+ 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 pD2 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 CaCl2 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 Ca2+ currents by 84.1 ± 3.9% in normal but only 36.2 ± 13% in inflamed tissues. Neither the Ca2+ channel Cav1.2b, gene expression, nor the c-Src kinase activity was altered by inflammation. Western blot analysis showed no change in the Ca2+ channel protein expression but increased nitrotyrosylated-Ca2+ channel proteins during inflammation. These data suggest that post-translational modification of Ca2+ channels during inflammation, possibly nitrotyrosylation, prevents c-Src kinase regulation resulting in decreased Ca2+ influx.
Footnotes
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This work was supported by National Institutes of Health Grant DK46367.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.123075.
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ABBREVIATIONS: DSS, dextran sulfate sodium; TNBS, trinitrobenzene sulfonic acid; SH, Src homology; ONOO–, peroxynitrite; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; iNOS, inducible nitric-oxide synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEK, human embryonic kidney; ANOVA, analysis of variance; IL, interleukin; ACh, acetylcholine; Ct, cycle threshold; IBD, inflammatory bowel disease; FAM, carboxyfluorescein; HEX, hexachlorofluorescein; BHQ, Black Hole Quencher.
- Received March 19, 2007.
- Accepted June 4, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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