The effect of tyrosine nitration of L-type Ca2+ channels on excitation-transcription coupling in colonic inflammation.
OBJECTIVE
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.


METHODS
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.


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
Post-translational modification of Ca(2+) channels due to tyrosine nitration modified excitation-transcriptional coupling in colonic inflammation.
Department of Pharmacology and Toxicology, VCU Program in Enteric Neuromuscular Sciences, Virginia Commonwealth University, Richmond, VA 23298, USA.
M
M
Kang
G R
GR
Ross
H I
HI
Akbarali
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D003092:Colitis / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D056966:Excitation Contraction Coupling / Q000187:drug effects*; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008807:Mice, Inbred BALB C; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D030421:Peroxynitrous Acid / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D020218:Response Elements / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015533:Transcriptional Activation / Q000187:drug effects*; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors
D020746:Calcium Channels, L-Type; D017362:Cyclic AMP Response Element-Binding Protein; D047428:Protein Kinase Inhibitors; D030421:Peroxynitrous Acid; D014443:Tyrosine; D019061:src-Family Kinases