Differential activation of phosphoinositide 3-kinase by endothelin and ceramide in colonic smooth muscle cells.

We have investigated the hypothesis that different contractile agonists activate distinct catalytic subunits of phosphoinositide (PI) 3-kinase in smooth muscle cells. Endothelin (10-7 M) induced a sustained increase in PI 3-kinase activity at both 30 s and 4 min of stimulation (151.5 ± 8.5% at 30 s and 175.8 ± 8.7% at 4 min, P < 0.005). Preincubation of smooth muscle cells with the tyrosine kinase inhibitor genistein (3 μM) resulted in a significant inhibition of both C2ceramide-induced and endothelin-induced PI 3-kinase activation and contraction. Preincubation with herbimycin A, an Src kinase inhibitor (3 μM), inhibited only C2ceramide-induced PI 3-kinase activation and contraction. Western blotting using Src kinase antibody showed that C2 ceramide, not endothelin, stimulated the phosphorylation of Src kinase. Western blotting and immunoprecipitation with PI 3-kinase antibodies to the regulatory subunit p85 and the catalytic subunits p110α and p110γ indicated that both endothelin and C2ceramide interacted with the regulatory subunit p85; endothelin interacted with the catalytic subunits p110α and p110γ, whereas C2 ceramide interacted only with the catalytic subunit p110α. In summary, C2 ceramide activated PI 3-kinase p110α subunit by a tyrosine kinase-mediated pathway, whereas endothelin-induced contraction, unlike C2 ceramide, was not mediated by the activation of Src kinase but was mediated by G protein activation of both p110α and p110γ subunits (type IA and IB) of PI 3-kinase.