M Kang1, G R Ross, H I Akbarali. 1. Department of Pharmacology and Toxicology, VCU Program in Enteric Neuromuscular Sciences, Virginia Commonwealth University, Richmond, VA 23298, USA.
Abstract
BACKGROUND AND PURPOSE: Excitation-transcriptional coupling involves communication between plasma membrane ion channels and gene expression in the nucleus. Calcium influx through L-type Ca(2+) channels induces phosphorylation of the transcription factor, cyclic-AMP response element binding protein (CREB) and downstream activation of the cyclic-AMP response element (CRE) promoter regions. Tyrosine nitration of Ca(2+) channels attenuates interactions with c-Src kinase, decreasing Ca(2+) channel currents and smooth muscle contraction during colonic inflammation. In this study we examined the effect of tyrosine nitration and colonic inflammation on Ca(2+) channel mediated phosphorylation of CREB and CRE activation. EXPERIMENTAL APPROACH: CREB and phospho-CREB were detected by Western blots and CRE activation measured by dual luciferase assay. Chinese hamster ovary (CHO) cells were transfected with hCa(v)1.2b and hCa(v)1.2b c-terminal mutants. Colonic inflammation was induced by intracolonic instillation of 2,4,6 trinitrobenzene sulphonic acid in mouse colon. KEY RESULTS: In hCa(v)1.2b transfected CHO cells and in native colonic smooth muscle, depolarization with 80 mM KCl induced CREB phosphorylation (pCREB). Treatment with peroxynitrite inhibited KCl-induced pCREB. Following experimental colitis, KCl-induced CREB phosphorylation was abolished in smooth muscle, concomitant with tyrosine nitration of Ca(2+) channels. Depolarization increased CRE activation in hCa(v)1.2b CHO cells by 2.35 fold which was blocked by nifedipine and by protein nitration of Ca(2+) channels with peroxynitrite. The Src-kinase inhibitor, PP2, blocked depolarization-induced CRE activation. Mutation of the C-terminus tyrosine residue, Y2134F, but not Y1861F, blocked CRE activation. CONCLUSIONS AND IMPLICATIONS: Post-translational modification of Ca(2+) channels due to tyrosine nitration modified excitation-transcriptional coupling in colonic inflammation.
BACKGROUND AND PURPOSE: Excitation-transcriptional coupling involves communication between plasma membrane ion channels and gene expression in the nucleus. Calcium influx through L-type Ca(2+) channels induces phosphorylation of the transcription factor, cyclic-AMP response element binding protein (CREB) and downstream activation of the cyclic-AMP response element (CRE) promoter regions. Tyrosine nitration of Ca(2+) channels attenuates interactions with c-Src kinase, decreasing Ca(2+) channel currents and smooth muscle contraction during colonic inflammation. In this study we examined the effect of tyrosine nitration and colonic inflammation on Ca(2+) channel mediated phosphorylation of CREB and CRE activation. EXPERIMENTAL APPROACH: CREB and phospho-CREB were detected by Western blots and CRE activation measured by dual luciferase assay. Chinese hamster ovary (CHO) cells were transfected with hCa(v)1.2b and hCa(v)1.2b c-terminal mutants. Colonic inflammation was induced by intracolonic instillation of 2,4,6 trinitrobenzene sulphonic acid in mouse colon. KEY RESULTS: In hCa(v)1.2b transfected CHO cells and in native colonic smooth muscle, depolarization with 80 mM KCl induced CREB phosphorylation (pCREB). Treatment with peroxynitrite inhibited KCl-induced pCREB. Following experimental colitis, KCl-induced CREB phosphorylation was abolished in smooth muscle, concomitant with tyrosine nitration of Ca(2+) channels. Depolarization increased CRE activation in hCa(v)1.2b CHO cells by 2.35 fold which was blocked by nifedipine and by protein nitration of Ca(2+) channels with peroxynitrite. The Src-kinase inhibitor, PP2, blocked depolarization-induced CRE activation. Mutation of the C-terminus tyrosine residue, Y2134F, but not Y1861F, blocked CRE activation. CONCLUSIONS AND IMPLICATIONS: Post-translational modification of Ca(2+) channels due to tyrosine nitration modified excitation-transcriptional coupling in colonic inflammation.
Authors: Alexander S Bayden; Vasily A Yakovlev; Paul R Graves; Ross B Mikkelsen; Glen E Kellogg Journal: Free Radic Biol Med Date: 2010-12-21 Impact factor: 7.376
Authors: Ketrija Touw; Saikat Chakraborty; Wenwu Zhang; Alexander G Obukhov; Johnathan D Tune; Susan J Gunst; B Paul Herring Journal: Am J Physiol Gastrointest Liver Physiol Date: 2011-10-06 Impact factor: 4.052
Authors: Robert J G Cardnell; Christopher S Rabender; Gracious R Ross; Chunqing Guo; Eric L Howlett; Asim Alam; Xiang-Yang Wang; Hamid I Akbarali; Ross B Mikkelsen Journal: J Pharmacol Exp Ther Date: 2013-08-02 Impact factor: 4.030