Activation of phospholipase D in porcine tracheal smooth muscle: role of phosphatidylinositol 3-kinase and RhoA activation.
Muscarinic receptor agonists transiently activate phospholipase D in tracheal smooth muscle. Muscarinic activation of phospholipase D in this tissue is dependent on activation of protein kinase C and an unidentified pathway that is not protein kinase C dependent. Cholinergic agents have also been shown to activate phospholipase D by pathways linked to the small G protein, RhoA. This study explores the relationship between muscarinic activation of phophatidylinositol 3-kinase and activation of RhoA, and examines whether phospholipase D activation is dependent on either pathway in tracheal smooth muscle. Wortmannin or 2-(4-morphonyl)-8-phenyl-4H-1-benzopyran-4-one (LY-294002), putative specific inhibitors of phophatidylinositol 3-kinase, significantly inhibit acetylcholine-induced formation of phosphatidylethanol and also block acetylcholine-induced translocation of RhoA to the membrane. In previous experiments calphostin C, a protein kinase C inhibitor, partially inhibited both acetylcholine-induced and phorbol-12-myristate-13-acetate (PMA)-induced phosphatidylethanol formation. In the present study calphostin C did not block acetylcholine-induced RhoA translocation to the membrane. However, the Rho kinase inhibitor, N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632), significantly inhibited acetylcholine-induced phosphatidylethanol formation, but had no effect on activation of phospholipase D by PMA. Acetylcholine treatment also stimulated the phosphorylation of the 110-kDa subunit of phosphatidylinositol 3-kinase. Phosphorylation of phosphatidylinositol 3-kinase 110-kDa subunit could be blocked by wortmannin in a concentration-dependent manner, and acetylcholine-induced phosphatidylinositol 3-kinase activity was significantly inhibited by wortmannin. LY-294002 also inhibited acetylcholine-induced phosphorylation of 110-kDa subunit and activation of phosphatidylinositol 3-kinase. These results suggest that acetylcholine stimulation translocates RhoA to the membrane by a phosphatidylinositol 3-kinase-dependent mechanism and acetylcholine-induced phospholipase D stimulation is at least partly mediated via phosphatidylinositol 3-kinase, however, protein kinase C appears to activate phospholipase D independent of phosphatidylinositol 3-kinase or RhoA activation in porcine tracheal smooth muscle.
Mamoon
A M
AM
Department of Pharmacology and Toxicology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216-4505, USA.
Baker
R C
RC
Farley
J M
JM
D000109:Acetylcholine / Q000494:pharmacology; D000577:Amides / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002867:Chromones / Q000494:pharmacology; D004789:Enzyme Activation; D066298:In Vitro Techniques; D008297:Male; D009025:Morpholines / Q000494:pharmacology; D009130:Muscle, Smooth / Q000201:enzymology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology*; D010739:Phospholipase D / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology; D013552:Swine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014132:Trachea / Q000201:enzymology*; D000077191:Wortmannin; D020742:rhoA GTP-Binding Protein / Q000502:physiology*
D000577:Amides; D000730:Androstadienes; D002867:Chromones; D009025:Morpholines; D011725:Pyridines; C108830:Y 27632; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D019869:Phosphatidylinositol 3-Kinases; D010739:Phospholipase D; D020742:rhoA GTP-Binding Protein; D000109:Acetylcholine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; D000077191:Wortmannin