Inhibition of endothelial nitric oxide synthase activity by protein kinase C.
Nitric oxide (NO) is an important molecular messenger accounting for endothelium-derived relaxing factor. Recently, NO synthase (NOS) from cultured endothelial cells has been purified and molecularly cloned. To evaluate the effect of phosphorylation by protein kinase C (PKC) and cyclic AMP-dependent protein kinase (PKA) on endothelial constitutive NOS catalytic activity, we incubated purified endothelial NOS with PKC or PKA. Endothelial NOS was stoichiometrically phosphorylated by PKC and PKA. In intact bovine aortic endothelial cells (BAECs), NOS was phosphorylated by stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA). NOS activity measured by the conversion of [3H]arginine to [3H]citrulline in homogenates of BAECs treated with TPA or phorbol 12,13-dibutyrate was reduced by 30%, whereas dibutylyl cyclic AMP did not affect NOS activity. Moreover, we measured NO release from cultured BAECs by a chemiluminescence method to examine the effect of PKC and PKA on endothelial NOS activity. In cultured BAECs, ATP gamma S and A23187 induced NO release in time- and dose-dependent manners. Phorbol esters such as TPA and phorbol 12,13-dibutyrate dose dependently inhibited NO release stimulated by A23187 as well as ATP gamma S. Reduction of NO release by TPA was almost completely prevented by pretreatment with staurosporine, an inhibitor of PKC. NO release by A23187 was increased in PKC-downregulated BAECs. In contrast, dibutylyl cyclic AMP or 8-bromo cyclic GMP had no effect on NO release from BAECs induced by A23187 or ATP gamma S. These results indicate that phosphorylation of NOS by PKC is associated with a reduction of its catalytic activity in vascular endothelial cells.
Hirata
K
K
First Department of Internal Medicine, Kobe University School of Medicine, Japan.
Kuroda
R
R
Sakoda
T
T
Katayama
M
M
Inoue
N
N
Suematsu
M
M
Kawashima
S
S
Yokoyama
M
M
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D019001:Nitric Oxide Synthase; D009573:Nitrites / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism / Q000494:pharmacology*
D009573:Nitrites; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C