title: PKC-zeta mediates norepinephrine-induced phospholipase D activation and cell proliferation in VSMC.
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-zeta 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 micromol/L, 30 minutes) and myristoylated PKC-zeta pseudosubstrate peptide inhibitor (25 micromol/L, 1 hour). Transfection of VSMCs with PKC-zeta antisense, but not sense, oligonucleotides, which reduced PKC-zeta 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-zeta plasmid but not in those transfected with wild-type PKC-zeta. NE increased immunoprecipitable PKC-zeta activity and phosphorylation, reaching a maximum at 2 and 5 minutes, respectively. NE-induced increase in PKC-zeta activity was inhibited by Ro 31-8220 and by the pseudosubstrate inhibitor. Treatment of VSMCs for 48 hours with PKC-zeta 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-zeta mediates PLD activation and cell proliferation elicited by NE in rabbit VSMCs.
authors.affiliation: Department of Pharmacology and Vascular Biology Center of Excellence, College of Medicine, The University of Tennessee Health Science Center, Memphis, USA.
authors.forename: Jean-Hugues
authors.initials: JH
authors.lastname: Parmentier
authors.forename: Philip
authors.initials: P
authors.lastname: Smelcer
authors.forename: Zoran
authors.initials: Z
authors.lastname: Pavicevic
authors.forename: Edin
authors.initials: E
authors.lastname: Basic
authors.forename: Azra
authors.initials: A
authors.lastname: Idrizovic
authors.forename: Anne
authors.initials: A
authors.lastname: Estes
authors.forename: Kafait U
authors.initials: KU
authors.lastname: Malik
mesh_terms: D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D004789:Enzyme Activation; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D009638:Norepinephrine / Q000494:pharmacology*; D010739:Phospholipase D / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000502:physiology*; D011817:Rabbits; D000099221:Protein Kinase C zeta
chemical_list: D009638:Norepinephrine; D010739:Phospholipase D; D011493:Protein Kinase C; D000099221:Protein Kinase C zeta