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 microM) 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 microM), 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 p110alpha and p110gamma indicated that both endothelin and C2 ceramide interacted with the regulatory subunit p85; endothelin interacted with the catalytic subunits p110alpha and p110gamma, whereas C2 ceramide interacted only with the catalytic subunit p110alpha. In summary, C2 ceramide activated PI 3-kinase p110alpha 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 p110alpha and p110gamma subunits (type IA and IB) of PI 3-kinase.
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Department of Pediatrics, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0656, USA.
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D000818:Animals; D002118:Calcium / Q000378:metabolism; D002122:Calcium Chloride / Q000494:pharmacology; D002478:Cells, Cultured; D003106:Colon / Q000201:enzymology / Q000502:physiology*; D004533:Egtazic Acid / Q000494:pharmacology; D019332:Endothelin-1 / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D019833:Genistein / Q000494:pharmacology; D007700:Kinetics; D008954:Models, Biological; D009130:Muscle, Smooth / Q000201:enzymology / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011817:Rabbits; D015398:Signal Transduction / Q000187:drug effects; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013997:Time Factors
D019332:Endothelin-1; C064769:N-acetylsphingosine; D004533:Egtazic Acid; D019833:Genistein; D019869:Phosphatidylinositol 3-Kinases; D002122:Calcium Chloride; D013110:Sphingosine; D002118:Calcium