Nitrotyrosylation of Ca2+ channels prevents c-Src kinase regulation of colonic smooth muscle contractility in experimental colitis.
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.
Ross
Gracious R
GR
Department of Pharmacology and Toxicology, Virginia Commonwealth University, 1112 E. Clay Street, Richmond, VA 23298, USA.
Kang
Minho
M
Shirwany
Najeeb
N
Malykhina
Anna P
AP
Drozd
Mary
M
Akbarali
Hamid I
HI
D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D002118:Calcium / Q000502:physiology; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D003092:Colitis / Q000473:pathology / Q000503:physiopathology; D003106:Colon / Q000502:physiology*; D004195:Disease Models, Animal; D004594:Electrophysiology; D006801:Humans; D007249:Inflammation; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009119:Muscle Contraction / Q000187:drug effects*; D009130:Muscle, Smooth / Q000502:physiology*; D017942:Nitric Acid / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D014443:Tyrosine / Q000031:analogs & derivatives*; D019061:src-Family Kinases
D015220:Calcium Channels; C002744:3-nitrotyrosine; D017942:Nitric Acid; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D002118:Calcium