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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL

Nitrotyrosylation of Ca²⁺ Channels Prevents c-Src Kinase Regulation of Colonic Smooth Muscle Contractility in Experimental Colitis

Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia (G.R.R., M.K., M.D., H.I.A.); and Department of Physiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma (N.S., A.P.M.)

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 E_max 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 Ca_v1.2b, 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.

Address correspondence to: Dr. Hamid I. Akbarali, Professor, Department of Pharmacology and Toxicology, McGuire Hall, 316, P.O. Box 980524, Virginia Commonwealth University, 1112 E. Clay Street, Richmond, VA 23298. E-mail: hiakbarali{at}vcu.edu

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