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2006 Abstracts: Superoxide Enhancement of L-Type Ca2+ Channels in Colonic Smooth Muscle is Dependent on Gβγ and PI3-Kinase
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Superoxide Enhancement of L-Type Ca2+ Channels in Colonic Smooth Muscle is Dependent on Gβγ and PI3-Kinase
Mandeep S. Saund2,1, Mahmood Zare3, Madhu Prasad2,1; 1Surgery, Harvard Medical School, Boston, MA; 2Surgery, Brigham and Women's Hospital, Boston, MA; 3Surgery, Boston University School of Medicine, Boston, MA

L-type Ca2+ (Ca2+L) channels govern action potential upstroke and Ca2+ influx, which underlie phasic contraction of colonic smooth muscle. In colitis, activated neutrophils generate large amounts of the highly toxic superoxide radical (O2-). This study tested the hypothesis that O2- produced by inflammatory cells contributes to markedly irregular motility patterns present in colitis by modulating Ca2+ channels in colonic smooth muscle cells (SMC). METHODS: Single SMCs prepared from intact tissues were placed in a recording chamber, superfused with HEPES-buffered physiological solution and studied using patch clamp techniques to record whole cell Ca2+ currents ( ICa ,L). RESULTS: The O2- donor pyrogallol (PYR, 100 μM) increased whole cell Ca2+ current ICa,L by 64 + 7 % (n =8). PYR-induced enhancement of Ca2+ channels was prevented by superoxide dismutase (100 U/ml), demonstrating mediation via O2-. The highly selective PI3-kinase (PI3K) inhibitors LY 294002 (20 μM) and wortmannin(WM) blocked O2- activation of ICa,L. O2--induced ICa,L was restored in WM-treated cells dialyzed with exogenous PI3K-γ, confirming the central role of PI3K in this pathway. Neither calphostin c (50 nM) which inhibits classical and novel protein kinase C (PKC) isoforms, nor the classical PKC antagonist Gö6976 (200 nM) prevented O2- activation of ICa,L. Anti-Gβγ antibodies also prevented activation of Ca2+L channels by O2-, suggesting a locus of action upstream to PI3K. The c-src antagonist PP-2 (10 μM) prevented O2- activation of ICa,L. Similar inhibition of OO2--induced ICa,L was found in cells dialyzed with anti-c-src antibodies. PYR depolarized smooth muscle cells by 11 + 2 mV (n=4) in intact strips of colon and induced spontaneous action potential spikes, effects that were abolished by the Ca2+L channel blocker nifedipine. CONCLUSIONS: Superoxide radical, a strongly reactive oxidant ubiquitous to sites of inflammation, markedly enhances the activity of Ca2+ channels in colonic smooth muscle. O2- activates Gβγ, leading to downstream enhancement of PI3K, c-Src kinase activation, and opening of Ca2+L channels. Though novel PKC is partly responsible for acetylcholine-induced Ca2+L channel opening, it plays no role in activation of this channel by O2-. O2--enhancement of Ca2+L channels increases electrical excitability of intact strips of colonic smooth muscle. Increased activity of Ca2+L channels in the presence of O2- would promote Ca2+ entry thereby altering the electrical and mechanical properties of colonic smooth muscle, actions that may in part explain the dysfunctional motility widely present in colitis.


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