Open AccessPKC-ζ Mediates Norepinephrine-Induced Phospholipase D Activation and Cell Proliferation in VSMC
Author(s) -
Jean-Hugues Parmentier,
Philip Smelcer,
Zoran Pavicevic,
Edin Basic,
Azra Idrizovic,
Anne M. Estes,
Kafait U. Malik
Publication year - 2003
Publication title -
hypertension
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.986
H-Index - 265
eISSN - 1524-4563
pISSN - 0194-911X
DOI - 10.1161/01.hyp.0000047873.76255.0b
Subject(s) - phosphatidylethanol , protein kinase c , phospholipase d , transfection , cell growth , endocrinology , medicine , vascular smooth muscle , biology , microbiology and biotechnology , cell culture , chemistry , phosphorylation , biochemistry , signal transduction , phospholipid , phosphatidic acid , genetics , membrane , smooth muscle
Norepinephrine (NE) stimulates phospholipase D (PLD) activity and cell proliferation in vascular smooth muscle cells (VSMCs). The objective of this study was to determine the contribution of PKC-ζ to NE-induced PLD activation and cell proliferation in VSMCs. PLD activity was measured by the formation of [3 H]phosphatidylethanol in VSMCs labeled with [3 H]oleic acid and exposed to ethanol. A high basal PLD activity was detected, and NE increased PLD activity over basal by 70%. This increase was abolished by the broad-range PKC inhibitor Ro 31-8220 (1 μmol/L, 30 minutes) and myristoylated PKC-ζ pseudosubstrate peptide inhibitor (25 μmol/L, 1 hour). Transfection of VSMCs with PKC-ζ antisense, but not sense, oligonucleotides, which reduced PKC-ζ protein level and basal PLD activity, caused a 92% decrease in NE-induced PLD activation. NE-induced increase in PLD activity was also reduced by 61% in cells transfected with kinase-deficient FLAG-T410A-PKC-ζ plasmid but not in those transfected with wild-type PKC-ζ. NE increased immunoprecipitable PKC-ζ activity and phosphorylation, reaching a maximum at 2 and 5 minutes, respectively. NE-induced increase in PKC-ζ activity was inhibited by Ro 31-8220 and by the pseudosubstrate inhibitor. Treatment of VSMCs for 48 hours with PKC-ζ antisense, but not sense, oligonucleotides also inhibited basal and NE-stimulated cell proliferation by 54% and 57%, respectively, as measured by [3 H]thymidine incorporation. The inhibitor of PLD activityn -butanol, but not its inactive analogtert -butanol, also reduced the basal and blocked NE-induced cell proliferation. These data suggest that PKC-ζ mediates PLD activation and cell proliferation elicited by NE in rabbit VSMCs.