We have investigated the hypothesis that different contractile agonists activate distinct catalytic subunits of phosphoinositide (PI) 3-kinase in smooth muscle cells. Endothelin (10⁷ 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 C₂ ceramide-induced and endothelin-induced PI 3-kinase activation and contraction. Preincubation with herbimycin A, an Src kinase inhibitor (3 µM), inhibited only C₂ ceramide-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 p110 and p110 indicated that both endothelin and C₂ ceramide interacted with the regulatory subunit p85; endothelin interacted with the catalytic subunits p110 and p110, whereas C₂ ceramide interacted only with the catalytic subunit p110. In summary, C₂ ceramide activated PI 3-kinase p110 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 p110 and p110 subunits (type IA and IB) of PI 3-kinase.

Src kinase; G proteins; tyrosine kinase

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