Involvement of mitogen-activated protein kinase and translocation of cytosolic phospholipase A2 to the nuclear envelope in acetylcholine-induced prostacyclin synthesis in rabbit coronary endothelial cells

H. Kan, Y. Ruan, Kafait Malik

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Abstract

We previously showed that acetylcholine (ACh) stimulates production of prostacyclin, measured as immunoreactive 6-ketoprostaglandin F(1α) (6-keto- PGF(1α)), in coronary endothelial cells (CEC) of rabbit heart by increasing influx of extracellular Ca2+ through a receptor-operated Ca2+ channel and by activating a pertussis toxin-insensitive G protein. The purposes of this study were to elucidate the type of phospholipase A2 (PLA2) involved in 6- keto-PGF(1α) production and the mechanism(s) by which ACh activates PLA2 in cultured CEC. In CEC transiently transfected with cytosolic PLA2 but not secretory PLA2 antisense oligonucleotide, ACh failed to increase 6-keto- PGF(1α); this was prevented by cotransfection with cPLA2 sense oligonucleotide. ACh increased production of prostacyclin and increased protein kinase C (PKC) activity. The PKC inhibitor calphostin C attenuated the ACh-induced increase in PKC activity but not 6-keto-PGF(1α) production. Phorbol-12-myristate-13-acetate and phorbol-12,13-dibutyrate increased PKC activity but failed to alter 6-keto-PGF(1α) production. ACh enhanced the activity of cPLA2 and p42 mitogen-activated protein kinase (MAPK) in cell lysate prepared from CEC. ACh also caused phosphorylation of p42 MAPK and cPLA2, which was inhibited by AG126 {[α-cyano-(3-hydroxy-4- nitro)cinnamonitrile]}, a tyrosine kinase inhibitor known to decrease MAPK activity. In addition, ACh stimulated translocation of cPLA2 from cytosol to nuclear envelope; the translocation of cPLA2 was prevented by removal of extracellular calcium but not by AG126 treatment. Okadaic acid, a protein phosphatase inhibitor, increased cPLA2 activity in cell lysate prepared from CEC but did not alter basal 6-keto-PGF(1α) production in intact CEC; however, ACh-induced 6-keto-PGF(1α) was enhanced by okadaic acid. These data suggest that ACh stimulates prostacyclin synthesis by activation of cPLA2 in a PKC-independent mechanism and that both cPLA2 translocation to nuclear envelope and phosphorylation by MAPK are required for ACh-induced 6-keto- PGF(1α) synthesis in CEC.

Original languageEnglish (US)
Pages (from-to)1139-1147
Number of pages9
JournalMolecular Pharmacology
Volume50
Issue number5
StatePublished - Nov 1 1996

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Cytosolic Phospholipases A2
Nuclear Envelope
Protein Transport
Epoprostenol
Mitogen-Activated Protein Kinases
Acetylcholine
Prostaglandins F
Endothelial Cells
Rabbits
Protein Kinase C
Okadaic Acid
Phospholipases A2
Mitogen-Activated Protein Kinase 1
Phosphorylation
Secretory Phospholipase A2
Phorbol 12,13-Dibutyrate
Protein C Inhibitor
Antisense Oligonucleotides
Phosphoprotein Phosphatases
Pertussis Toxin

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Pharmacology

Cite this

@article{5531041765c7434e8636641818b54fed,
title = "Involvement of mitogen-activated protein kinase and translocation of cytosolic phospholipase A2 to the nuclear envelope in acetylcholine-induced prostacyclin synthesis in rabbit coronary endothelial cells",
abstract = "We previously showed that acetylcholine (ACh) stimulates production of prostacyclin, measured as immunoreactive 6-ketoprostaglandin F(1α) (6-keto- PGF(1α)), in coronary endothelial cells (CEC) of rabbit heart by increasing influx of extracellular Ca2+ through a receptor-operated Ca2+ channel and by activating a pertussis toxin-insensitive G protein. The purposes of this study were to elucidate the type of phospholipase A2 (PLA2) involved in 6- keto-PGF(1α) production and the mechanism(s) by which ACh activates PLA2 in cultured CEC. In CEC transiently transfected with cytosolic PLA2 but not secretory PLA2 antisense oligonucleotide, ACh failed to increase 6-keto- PGF(1α); this was prevented by cotransfection with cPLA2 sense oligonucleotide. ACh increased production of prostacyclin and increased protein kinase C (PKC) activity. The PKC inhibitor calphostin C attenuated the ACh-induced increase in PKC activity but not 6-keto-PGF(1α) production. Phorbol-12-myristate-13-acetate and phorbol-12,13-dibutyrate increased PKC activity but failed to alter 6-keto-PGF(1α) production. ACh enhanced the activity of cPLA2 and p42 mitogen-activated protein kinase (MAPK) in cell lysate prepared from CEC. ACh also caused phosphorylation of p42 MAPK and cPLA2, which was inhibited by AG126 {[α-cyano-(3-hydroxy-4- nitro)cinnamonitrile]}, a tyrosine kinase inhibitor known to decrease MAPK activity. In addition, ACh stimulated translocation of cPLA2 from cytosol to nuclear envelope; the translocation of cPLA2 was prevented by removal of extracellular calcium but not by AG126 treatment. Okadaic acid, a protein phosphatase inhibitor, increased cPLA2 activity in cell lysate prepared from CEC but did not alter basal 6-keto-PGF(1α) production in intact CEC; however, ACh-induced 6-keto-PGF(1α) was enhanced by okadaic acid. These data suggest that ACh stimulates prostacyclin synthesis by activation of cPLA2 in a PKC-independent mechanism and that both cPLA2 translocation to nuclear envelope and phosphorylation by MAPK are required for ACh-induced 6-keto- PGF(1α) synthesis in CEC.",
author = "H. Kan and Y. Ruan and Kafait Malik",
year = "1996",
month = "11",
day = "1",
language = "English (US)",
volume = "50",
pages = "1139--1147",
journal = "Molecular Pharmacology",
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TY - JOUR

T1 - Involvement of mitogen-activated protein kinase and translocation of cytosolic phospholipase A2 to the nuclear envelope in acetylcholine-induced prostacyclin synthesis in rabbit coronary endothelial cells

AU - Kan, H.

AU - Ruan, Y.

AU - Malik, Kafait

PY - 1996/11/1

Y1 - 1996/11/1

N2 - We previously showed that acetylcholine (ACh) stimulates production of prostacyclin, measured as immunoreactive 6-ketoprostaglandin F(1α) (6-keto- PGF(1α)), in coronary endothelial cells (CEC) of rabbit heart by increasing influx of extracellular Ca2+ through a receptor-operated Ca2+ channel and by activating a pertussis toxin-insensitive G protein. The purposes of this study were to elucidate the type of phospholipase A2 (PLA2) involved in 6- keto-PGF(1α) production and the mechanism(s) by which ACh activates PLA2 in cultured CEC. In CEC transiently transfected with cytosolic PLA2 but not secretory PLA2 antisense oligonucleotide, ACh failed to increase 6-keto- PGF(1α); this was prevented by cotransfection with cPLA2 sense oligonucleotide. ACh increased production of prostacyclin and increased protein kinase C (PKC) activity. The PKC inhibitor calphostin C attenuated the ACh-induced increase in PKC activity but not 6-keto-PGF(1α) production. Phorbol-12-myristate-13-acetate and phorbol-12,13-dibutyrate increased PKC activity but failed to alter 6-keto-PGF(1α) production. ACh enhanced the activity of cPLA2 and p42 mitogen-activated protein kinase (MAPK) in cell lysate prepared from CEC. ACh also caused phosphorylation of p42 MAPK and cPLA2, which was inhibited by AG126 {[α-cyano-(3-hydroxy-4- nitro)cinnamonitrile]}, a tyrosine kinase inhibitor known to decrease MAPK activity. In addition, ACh stimulated translocation of cPLA2 from cytosol to nuclear envelope; the translocation of cPLA2 was prevented by removal of extracellular calcium but not by AG126 treatment. Okadaic acid, a protein phosphatase inhibitor, increased cPLA2 activity in cell lysate prepared from CEC but did not alter basal 6-keto-PGF(1α) production in intact CEC; however, ACh-induced 6-keto-PGF(1α) was enhanced by okadaic acid. These data suggest that ACh stimulates prostacyclin synthesis by activation of cPLA2 in a PKC-independent mechanism and that both cPLA2 translocation to nuclear envelope and phosphorylation by MAPK are required for ACh-induced 6-keto- PGF(1α) synthesis in CEC.

AB - We previously showed that acetylcholine (ACh) stimulates production of prostacyclin, measured as immunoreactive 6-ketoprostaglandin F(1α) (6-keto- PGF(1α)), in coronary endothelial cells (CEC) of rabbit heart by increasing influx of extracellular Ca2+ through a receptor-operated Ca2+ channel and by activating a pertussis toxin-insensitive G protein. The purposes of this study were to elucidate the type of phospholipase A2 (PLA2) involved in 6- keto-PGF(1α) production and the mechanism(s) by which ACh activates PLA2 in cultured CEC. In CEC transiently transfected with cytosolic PLA2 but not secretory PLA2 antisense oligonucleotide, ACh failed to increase 6-keto- PGF(1α); this was prevented by cotransfection with cPLA2 sense oligonucleotide. ACh increased production of prostacyclin and increased protein kinase C (PKC) activity. The PKC inhibitor calphostin C attenuated the ACh-induced increase in PKC activity but not 6-keto-PGF(1α) production. Phorbol-12-myristate-13-acetate and phorbol-12,13-dibutyrate increased PKC activity but failed to alter 6-keto-PGF(1α) production. ACh enhanced the activity of cPLA2 and p42 mitogen-activated protein kinase (MAPK) in cell lysate prepared from CEC. ACh also caused phosphorylation of p42 MAPK and cPLA2, which was inhibited by AG126 {[α-cyano-(3-hydroxy-4- nitro)cinnamonitrile]}, a tyrosine kinase inhibitor known to decrease MAPK activity. In addition, ACh stimulated translocation of cPLA2 from cytosol to nuclear envelope; the translocation of cPLA2 was prevented by removal of extracellular calcium but not by AG126 treatment. Okadaic acid, a protein phosphatase inhibitor, increased cPLA2 activity in cell lysate prepared from CEC but did not alter basal 6-keto-PGF(1α) production in intact CEC; however, ACh-induced 6-keto-PGF(1α) was enhanced by okadaic acid. These data suggest that ACh stimulates prostacyclin synthesis by activation of cPLA2 in a PKC-independent mechanism and that both cPLA2 translocation to nuclear envelope and phosphorylation by MAPK are required for ACh-induced 6-keto- PGF(1α) synthesis in CEC.

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VL - 50

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EP - 1147

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