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

• Published in: American journal of physiology: Gastrointestinal and liver physiology 1999-04, Vol.276 (4), p.G853-G861
• Main Authors: Su, Xuehui, Wang, Pinglang, Ibitayo, Adenike, Bitar, Khalil N.
• Format: Article
• Language: English
• ISSN: 0193-1857; 1522-1547
• DOI: 10.1152/ajpgi.1999.276.4.G853

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 C 2 ceramide-induced and endothelin-induced PI 3-kinase activation and contraction. Preincubation with herbimycin A, an Src kinase inhibitor (3 μM), inhibited only C 2 ceramide-induced PI 3-kinase 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 antibodies to the regulatory subunit p85 and the catalytic subunits p110α and p110γ indicated that both endothelin and C 2 ceramide interacted with the regulatory subunit p85; endothelin interacted with the catalytic subunits p110α and p110γ, whereas C 2 ceramide interacted only with the catalytic subunit p110α. In summary, C 2 ceramide activated PI 3-kinase p110α subunit by a tyrosine kinase-mediated pathway, whereas endothelin-induced contraction, unlike C 2 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.