Jean-Hugues Parmentier

20-Hydroxyeicosatetraenoic Acid Mediates Angiotensin Ii-Induced Phospholipase D Activation in Vascular Smooth Muscle Cells

Angiotensin II (Ang II) activates cytosolic phospholipase A2 (cPLA2) and phospholipase D (PLD) in rabbit vascular smooth muscle cells (VSMCs). Ang II also activates ras/mitogen-activated protein (MAP) kinase in VSMCs; this activation is mediated by 20-hydroxyeicosatetraenoic acid (HETE) and 12(S)-HETE, which are metabolites of arachidonic acid generated by cytochrome P450 4A and lipoxygenase, respectively, produced on activation of cPLA2. The purpose of this study was to determine if Ang II–induced PLD activation in VSMCs is mediated through the ras/extracellular signal-regulating kinase (ERK) pathway by arachidonic acid metabolites that are generated consequent to cPLA2 stimulation. Inhibitors of PLD (C2 ceramide), phosphatidate phosphohydrolase (propranolol), and diacylglycerol lipase (RHC 80267) attenuated Ang II–induced arachidonic acid release. Ang II–induced PLD activation, as measured by [3H]phosphatidylethanol production, was inhibited by C2 ceramide but not by propranolol or RHC 80267. Ang II–induced PLD activation was decreased by the inhibitor methyl arachidonylfluorophosphate (MAFP) and the antisense oligonucleotide of cPLA2. Inhibitors of lipoxygenases (baicalein) and cytochrome P450 4A (ODYA) attenuated Ang II–induced PLD activation. 20-HETE and 12(S)-HETE increased PLD activity. Inhibitors of ras farnesyltransferase (FPT III and BMS-191563) and MAP kinase kinase (UO126) attenuated the increase in PLD activity elicited by 20-HETE and Ang II. PLD2 was the main isoform activated by Ang II in VSMCs. These data suggest that the CYP4A metabolite 20-HETE, which is generated from arachidonic acid after cPLA2 activation by Ang II, stimulates the ras/MAP kinase pathway, which in turn activates PLD2 and releases further arachidonic acid for prostaglandin synthesis through the phosphatidate phosphohydrolase/diacylglycerol lipase pathway.

Small GTP binding protein Ras contributes to norepinephrine-induced mitogenesis of vascular smooth muscle cells

Norepinephrine stimulates release of arachidonic acid from tissue lipids. Arachidonic acid metabolites generated through the lipoxygenase and cytochrome P-450 pathways but not cyclooxygenase stimulate mitogen activated protein (MAP) kinase activity and proliferation of vascular smooth muscle cells (VSMC). Moreover, norepinephrine has been shown to activate the Ras/MAP kinase pathway through generation of cytochrome P450 metabolite of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE). The purpose of this study was to investigate the contribution of Ras in norepinephrine-induced mitogenesis in aortic VSMC. Farnesylation of Ras by farnesyl transferase is required for its full activation. Norepinephrine-induced DNA synthesis, as measured by [3H]-thymidine incorporation, was attenuated by inhibitors of Ras farnesyl transferase FPT III and BMS-191563. These agents also inhibited 20-HETE-stimulated [3H]-thymidine incorporation. In cells transiently transfected with dominant negative Ras (RasN17), norepinephrine, and 20-HETE-induced proliferation of VSMC was attenuated. Both norepinephrine and 20-HETE increased localization of Ras to plasma membrane and MAP kinase activity; FPT III attenuated these effects. These data suggest that VSMC proliferation induced by norepinephrine and 20-HETE is mediated by Ras/MAP kinase pathway.

PKC-ζ 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-&zgr; 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 &mgr;mol/L, 30 minutes) and myristoylated PKC-&zgr; pseudosubstrate peptide inhibitor (25 &mgr;mol/L, 1 hour). Transfection of VSMCs with PKC-&zgr; antisense, but not sense, oligonucleotides, which reduced PKC-&zgr; 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-&zgr; plasmid but not in those transfected with wild-type PKC-&zgr;. NE increased immunoprecipitable PKC-&zgr; activity and phosphorylation, reaching a maximum at 2 and 5 minutes, respectively. NE-induced increase in PKC-&zgr; activity was inhibited by Ro 31-8220 and by the pseudosubstrate inhibitor. Treatment of VSMCs for 48 hours with PKC-&zgr; 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-&zgr; mediates PLD activation and cell proliferation elicited by NE in rabbit VSMCs.

Arachidonic acid-derived oxidation products initiate apoptosis in vascular smooth muscle cells

The mechanism of arachidonic acid (AA)-induced apoptosis in vascular smooth muscle cells (VSMCs) was studied in the A-10 rat aortic smooth muscle cell line. Treatment of serum-deprived VSMCs with 50 microM AA for 24 h resulted in a loss of cell viability. The apoptotic effect of AA was characterized by annexin V binding, sub-G1 population of cells, cell shrinkage and chromatin condensation. AA-induced VSMC death was attenuated by antioxidants alpha-tocopherol and glutathione, the hydrogen peroxide (H2O2) scavenger catalase and by serum proteins, albumin and gamma globulins. Moreover, the AA peroxidation products, 12(S)-hydroperoxyeicosatetraenoic acid (HPETE), 15(S)-HPETE, 4-hydroxy-2-nonenal (HNE) and malondialdehyde (MDA) caused VSMC apoptosis. These data suggest an oxidative mechanism of AA-induced VSMC death. The apoptotic effect of AA was pH-dependent, being inhibited by extracellular alkalinization to pH 8.0. AA inhibited serum-stimulated cell cycle progression in quiescent cells, but not in proliferating cells. In conclusion, AA, through its oxidation products causes VSMC apoptosis. Antioxidants, by inhibiting VSMC apoptosis, may prevent consequent pathological events such as atherosclerotic plaque rupture.

Calcium and Protein Kinase C (PKC)-Related Kinase Mediate α1A-Adrenergic Receptor-Stimulated Activation of Phospholipase D in Rat-1 Cells, Independent of PKC

A previous study conducted in rat-1 cells expressing α1A-adrenergic receptors showed that phenylephrine (PHE) stimulates phospholipase D (PLD) activity. This study was conducted to determine the contribution of protein kinase C (PKC) to PHE-induced PLD activation in these cells. PKC inhibitors bisindolylmaleimide (BIM) I and Ro 31-8220, but not Gö 6976 or a pseudosubstrate peptide inhibitor of PKCα, decreased PLD activity and arachidonic acid release elicited by PHE. However, antisense oligonucleotides directed against PKC α, δ, ε, and η reduced PKC isoform levels by about 80% but failed to alter PHE-induced PLD activation, indicating that these PKC isoforms are not involved in PLD activation elicited by α1A-adrenergic receptor stimulation. Ectopic expression of a kinase-deficient mutant of the PKC-related kinase PKN significantly attenuated PHE-induced PLD activation. On the other hand, BIM I and Ro 31-8220 blocked PHE-mediated increase in intracellular Ca2+ but Gö 6976 and the peptide inhibitor did not. In the absence of extracellular Ca2+, PHE failed to increase PLD activity. These results indicate that α1A-adrenergic receptor-stimulated PLD activation is mediated by a mechanism independent of PKCα, δ, ε, and η, but dependent on a PKC-related kinase, PKN. Moreover, PKC inhibitors BIM I and Ro 31-8220 block PHE-induced PLD activity by inhibiting calcium signal. Caution should be used in interpreting the data obtained with PKC inhibitors in vivo.

Protein kinase Cζ regulates phospholipase D activity in rat-1 fibroblasts expressing the α1A adrenergic receptor

BackgroundPhenylephrine (PHE), an α1 adrenergic receptor agonist, increases phospholipase D (PLD) activity, independent of classical and novel protein kinase C (PKC) isoforms, in rat-1 fibroblasts expressing α1A adrenergic receptors. The aim of this study was to determine the contribution of atypical PKCζ to PLD activation in response to PHE in these cells.ResultsPHE stimulated a PLD activity as demonstrated by phosphatidylethanol production. PHE increased PKCζ translocation to the particulate cell fraction in parallel with a time-dependent decrease in its activity. PKCζ activity was reduced at 2 and 5 min and returned to a sub-basal level within 10–15 min. Ectopic expression of kinase-dead PKCζ, but not constitutively active PKCζ, potentiated PLD activation elicited by PHE. A cell-permeable pseudosubstrate inhibitor of PKCζ reduced basal PKCζ activity and abolished PHE-induced PLD activation.Conclusionα1A adrenergic receptor stimulation promotes the activation of a PLD activity by a mechanism dependent on PKCζ; Our data also suggest that catalytic activation of PKCζ is not required for PLD stimulation.