title: Norepinephrine and endothelin activate diacylglycerol kinases in caveolae/rafts of rat mesenteric arteries: agonist-specific role of PI3-kinase.
abstract: The phosphatidylinositol (PI) signaling pathway mediates norepinephrine (NE)- and endothelin-1 (ET-1)-stimulated vascular smooth muscle contraction through an inositol-trisphosphate-induced rise in intracellular calcium and diacylglycerol (DG) activation of protein kinase C (PKC). Subsequent activation of DG kinases (DGKs) metabolizes DG to phosphatidic acid (PA), potentially regulating PKC activity. Because precise regulation and spatial restriction of the PI pathway is necessary for specificity, we have investigated whether this occurs within caveolae/rafts, specialized plasma membrane microdomains implicated in vascular smooth muscle contraction. We show that components of the PI signaling cascade-phosphatidylinositol 4,5-bisphosphate (PIP(2)), PA, and DGK-theta are present in caveolae/rafts prepared from rat mesenteric small arteries. Stimulation with NE or ET-1 induced [(33)P]PIP(2) hydrolysis solely within caveolae/rafts. NE stimulated an increase in DGK activity in caveolae/rafts alone, whereas ET-1 activated DGK in caveolae/rafts and noncaveolae/rafts; however, [(33)P]PA increased in all fractions with both agonists. Previously, we reported that NE activated DGK-theta in a phosphatidylinositol 3-kinase (PI3-kinase)-dependent manner; here, we describe PI3-kinase-dependent DGK activation and [(33)P]PA production in caveolae/rafts in response to NE but not ET-1. Additionally, PKB, a potential activator of DGK-theta, translocated to caveolae/rafts in response to NE but not ET-1, and PI3-kinase inhibition prevented this. Furthermore, PI3-kinase inhibition reduced the sensitivity of contraction to NE but not ET-1. Our study shows that caveolae/rafts are major sites of vasoconstrictor hormone activation of the PI pathway in intact small arteries and suggest a link between lipid signaling events within caveolae/rafts and contraction.
authors.affiliation: Cardiovascular Research Group, Division of Cardiovascular and Endocrine Sciences, Core Technology Facility, University of Manchester, 46 Grafton St., Manchester, UK.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Clarke
authors.forename: Vasken
authors.initials: V
authors.lastname: Ohanian
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Ohanian
mesh_terms: D000818:Animals; D021941:Caveolae / Q000378:metabolism*; D002478:Cells, Cultured; D003067:Coenzymes / Q000378:metabolism; D019852:Diacylglycerol Kinase / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D016232:Endothelins / Q000008:administration & dosage*; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D008638:Mesenteric Arteries / Q000378:metabolism* / Q000648:ultrastructure; D009638:Norepinephrine / Q000008:administration & dosage*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D003067:Coenzymes; D004338:Drug Combinations; D016232:Endothelins; D019869:Phosphatidylinositol 3-Kinases; D019852:Diacylglycerol Kinase; D009638:Norepinephrine