PKC-ζ mediates norepinephrine-induced phospholipase D activation and cell proliferation in VSMC

Jean Hugues Parmentier, Philip Smelcer, Zoran Pavicevic, Edin Basic, Azra Idrizovic, Anne Estes, Kafait Malik

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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-ζ 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-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 [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-ζ mediates PLD activation and cell proliferation elicited by NE in rabbit VSMCs.

Original languageEnglish (US)
Pages (from-to)794-800
Number of pages7
JournalHypertension
Volume41
Issue number3 II
DOIs
StatePublished - Mar 1 2003

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Phospholipase D
Vascular Smooth Muscle
Smooth Muscle Myocytes
Norepinephrine
Cell Proliferation
Oligonucleotides
tert-Butyl Alcohol
1-Butanol
Oleic Acid
Thymidine
Transfection
Plasmids
Ethanol
Phosphotransferases
Phosphorylation
Rabbits

All Science Journal Classification (ASJC) codes

  • Internal Medicine

Cite this

Parmentier, J. H., Smelcer, P., Pavicevic, Z., Basic, E., Idrizovic, A., Estes, A., & Malik, K. (2003). PKC-ζ mediates norepinephrine-induced phospholipase D activation and cell proliferation in VSMC. Hypertension, 41(3 II), 794-800. https://doi.org/10.1161/01.HYP.0000047873.76255.0B

PKC-ζ mediates norepinephrine-induced phospholipase D activation and cell proliferation in VSMC. / Parmentier, Jean Hugues; Smelcer, Philip; Pavicevic, Zoran; Basic, Edin; Idrizovic, Azra; Estes, Anne; Malik, Kafait.

In: Hypertension, Vol. 41, No. 3 II, 01.03.2003, p. 794-800.

Research output: Contribution to journalArticle

Parmentier, JH, Smelcer, P, Pavicevic, Z, Basic, E, Idrizovic, A, Estes, A & Malik, K 2003, 'PKC-ζ mediates norepinephrine-induced phospholipase D activation and cell proliferation in VSMC', Hypertension, vol. 41, no. 3 II, pp. 794-800. https://doi.org/10.1161/01.HYP.0000047873.76255.0B
Parmentier JH, Smelcer P, Pavicevic Z, Basic E, Idrizovic A, Estes A et al. PKC-ζ mediates norepinephrine-induced phospholipase D activation and cell proliferation in VSMC. Hypertension. 2003 Mar 1;41(3 II):794-800. https://doi.org/10.1161/01.HYP.0000047873.76255.0B
Parmentier, Jean Hugues ; Smelcer, Philip ; Pavicevic, Zoran ; Basic, Edin ; Idrizovic, Azra ; Estes, Anne ; Malik, Kafait. / PKC-ζ mediates norepinephrine-induced phospholipase D activation and cell proliferation in VSMC. In: Hypertension. 2003 ; Vol. 41, No. 3 II. pp. 794-800.
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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-ζ 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-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 [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-ζ mediates PLD activation and cell proliferation elicited by NE in rabbit VSMCs.",
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AB - 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 [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-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 [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-ζ mediates PLD activation and cell proliferation elicited by NE in rabbit VSMCs.

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