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 ( I_{Ca ,L}). RESULTS: The O₂^- donor pyrogallol (PYR, 100 μM) increased whole cell Ca²⁺ current I_Ca,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 I_Ca,L. O₂^--induced I_Ca,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 I_Ca,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 I_Ca,L. Similar inhibition of OO₂^--induced I_Ca,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.