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

We have investigated the hypothesis that different contractile agonists act ivate distinct catalytic subunits of phosphoinositide (PI) 3-kinase in smoo th 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 mu M) resulted in a s ignificant inhibition of both C-2 ceramide-induced and endothelin-induced P I 3-kinase activation and contraction. Preincubation with herbimycin A, an Src kinase inhibitor (3 mu M), inhibited only C-2 ceramide-induced PI 3-kin ase activation and contraction. Western blotting using Src kinase antibody showed that C-2 ceramide, not endothelin, stimulated the phosphorylation of Src kinase. Western blotting and immunoprecipitation with PI 3-kinase anti bodies to the regulatory subunit p85 and the catalytic subunits p110 alpha and p110 gamma indicated that:hat both endothelin and C-2 ceramide interact ed with the regulatory subunit p85; endothelin interacted with the catalyti c subunits p110 alpha and p110 gamma, whereas C-2 ceramide interacted nly w ith the catalytic subunit p110 alpha. In summary, Cg ceramide activated PI 3-kinase p110 alpha subunit by a tyrosine kinase-mediated pathway, whereas endothelin-induced contraction, unlike C-2 ceramide, was not mediated by th e activation of Src kinase but was mediated by G protein activation of both p110 alpha and p110 gamma subunits (type IA and IB) of PI 3-kinase.