Published Online
on December 16, 2002

A more recent version of this article appeared on March 1, 2003

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Submitted on October 5, 2002
Revised on October 24, 2002

PKC- Mediates Norepinephrine-Induced Phospholipase D Activation and Cell Proliferation in VSMC

Jean-Hugues Parmentier; Philip Smelcer; Zoran Pavicevic; Edin Basic; Azra Idrizovic; Anne Estes; and Kafait U. Malik^*

From the Department of Pharmacology and Vascular Biology Center of Excellence, College of Medicine, The University of Tennessee Health Science Center, Memphis.

^* To whom correspondence should be addressed. E-mail: kmalik{at}utmem.edu.

Abstract—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- to NE-induced PLD activation and cell proliferation in VSMCs. PLD activity was measured by the formation of [³H]phosphatidylethanol in VSMCs labeled with [³H]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- pseudosubstrate peptide inhibitor (25 µmol/L, 1 hour). Transfection of VSMCs with PKC- antisense, but not sense, oligonucleotides, which reduced PKC- 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- plasmid but not in those transfected with wild-type PKC-. NE increased immunoprecipitable PKC- activity and phosphorylation, reaching a maximum at 2 and 5 minutes, respectively. NE-induced increase in PKC- activity was inhibited by Ro 31-8220 and by the pseudosubstrate inhibitor. Treatment of VSMCs for 48 hours with PKC- antisense, but not sense, oligonucleotides also inhibited basal and NE-stimulated cell proliferation by 54% and 57%, respectively, as measured by [³H]thymidine incorporation. The inhibitor of PLD activity n-butanol, but not its inactive analog tert-butanol, also reduced the basal and blocked NE-induced cell proliferation. These data suggest that PKC- mediates PLD activation and cell proliferation elicited by NE in rabbit VSMCs.

Key words: phospholipase D • norepinephrine • muscle, smooth, vascular • protein kinases • cell proliferation

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