(Hypertension. 1995;25:180-185.)
© 1995 American Heart Association, Inc.
Articles |
Inhibition of Endothelial Nitric Oxide Synthase Activity by Protein Kinase C
From the First Department of Internal Medicine, Kobe (Japan) University School of Medicine.
Abstract 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
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 ATPS. 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 ATPS. These results
indicate that phosphorylation of NOS by PKC is associated with a
reduction of its catalytic activity in vascular endothelial cells.
Key Words: nitric oxide • endothelium • protein kinase C
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