PT  - JOURNAL ARTICLE
AU  - Mubarack M. Muthalif
AU  - Jean-Hugues Parmentier
AU  - Ibrahim F. Benter
AU  - Nour Karzoun
AU  - Aftab Ahmed
AU  - Zinat Khandekar
AU  - Mohamed Z. Adl
AU  - Sylvain Bourgoin
AU  - Kafait U. Malik
TI  - Ras/Mitogen-Activated Protein Kinase Mediates Norepinephrine-Induced Phospholipase D Activation in Rabbit Aortic Smooth Muscle Cells by a Phosphorylation-Dependent Mechanism
DP  - 2000 Apr 01
TA  - Journal of Pharmacology and Experimental Therapeutics
PG  - 268--274
VI  - 293
IP  - 1
4099  - http://jpet.aspetjournals.org/content/293/1/268.short
4100  - http://jpet.aspetjournals.org/content/293/1/268.full
SO  - J Pharmacol Exp Ther2000 Apr 01; 293
AB  - Phospholipase D (PLD) activity is regulated by phosphatidylinositol 4,5-biphosphate, protein kinase C (PKC), ADP-ribosylation factor, and Rho. The present study was designed to investigate the mechanism of norepinephrine (NE)-mediated PLD activation in rabbit aortic vascular smooth muscle cells (VSMC). NE (10 μM) caused activation of PLD, as measured by the production of phosphatidylethanol in [3H]oleic acid-labeled cells. NE also increased PKC activity in VSMC. However, treatment of cells with bisindolylmaleimide, a PKC inhibitor, or long-term treatment with phorbol-12-myristate-13-acetate that depletes PKC did not decrease NE-induced activation of PLD. NE-stimulated PLD activity was attenuated by farnesyl transferase inhibitors (FPT III and SCH-56582), which reduce activation of both Ras and mitogen-activated protein (MAP) kinase. Moreover, transfection of VSMC with a dominant negative Ras resulted in inhibition of NE-stimulated MAP kinase and PLD activities. Treatment of cells with PD-98059, a MAP kinase kinase inhibitor, also reduced NE-stimulated PLD activity. These data suggest that NE-stimulated PLD activity is mediated via activation of Ras and MAP kinase in rabbit VSMC. To study the mechanism of activation of PLD by Ras/MAP kinase, NE-induced phosphorylation of PLD was examined. In VSMC, PLD of molecular mass 120 kDa was identified with polyclonal PLD antibody. Phosphorylation of PLD by NE, measured as 32P incorporation into PLD, was inhibited by PD-98059. Moreover, PLD immunoprecipitated from VSMC lysates was phosphorylated in vitro by MAP kinase. Collectively, these results show a novel pathway for activation of PLD that appears to be mediated through Ras/MAP kinase pathway by a mechanism involving phosphorylation. The American Society for Pharmacology and Experimental Therapeutics