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Vol. 293, Issue 1, 268-274, April 2000
Department of Pharmacology, College of Medicine, The University of
Tennessee, Memphis, Tennessee (M.M.M., J.-H.P., A.A., Z.K., M.Z.A.,
K.U.M.); Southern College of Optometry, Memphis, Tennessee (I.F.B.,
N.K.); and Centre de Recherche du CHUL, Rhumatologie et Immunologie,
Ste-Foy, Quebec, Canada (S.B.)
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.
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