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REGULATION OF ENDOTHELIAL NITRIC OXIDE SYNTHASE: INVOLVEMENT OF PROTEIN KINASE G 1β, SERINE 116 PHOSPHORYLATION AND LIPID STRUCTURES
Author(s) -
John Theresa A,
Ibe Basil O,
Raj J Usha
Publication year - 2008
Publication title -
clinical and experimental pharmacology and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.752
H-Index - 103
eISSN - 1440-1681
pISSN - 0305-1870
DOI - 10.1111/j.1440-1681.2007.04801.x
Subject(s) - endothelial nitric oxide synthase , phosphorylation , serine , chemistry , nitric oxide , nitric oxide synthase , cgmp dependent protein kinase , microbiology and biotechnology , protein kinase a , biochemistry , kinase , enzyme , biology , mitogen activated protein kinase kinase , enos , organic chemistry
SUMMARY1 Endothelial nitric oxide synthase (NOS3) is important for vascular homeostasis. The role of protein kinase G (PKG) in regulation of NOS3 activity was studied in primary cultures of newborn lamb lung microvascular endothelial cells (LMVEC). 2 We determined the presence of PKG in fetal and neonatal LMVEC as well as subcellular localization of PKG isoforms in the neonatal cells by fluorescence immunohistochemistry. We used diaminofluorescein (DAF) fluorophore to measure nitric oxide (NO) production from neonatal LMVEC. We confirmed that NO measured was from constitutive NOS3 by inhibiting it with NOS inhibitors. 3 To identify a role for PKG in basal NO production, we measured NO release from LMVEC cells using 4‐amino‐5‐methylamino‐2′,7′‐difluorofluorescein (DAF‐FM; 0.5–0.8 µmol/L) with and without prior stimulation with the PKG activator 8‐bromo‐cGMP (8‐Br‐cGMP; 0.3 and 3 µmol/L) or prior PKG inhibition with β‐phenyl‐1, N 2‐etheno‐8‐bromoguanosine‐3′,5′‐cyclic monophosphorothionate (BPC; 0.3 and 3 µmol/L). With the same drugs, we determined the role of PKG on cellular expression of NOS3 and serine 116 phosphorylated NOS (pSer116‐NOS) by qualitative and quantitative immunofluorescence assays, as well as western blotting. 4 Because PKG 1β was distributed throughout the cytosol in a punctate expression, we used 2 mmol/L cyclodextrin, a cholesterol extractor, to determine a role for lipid vesicles in PKG regulation of NO production. 5 Protein kinase G 1β gave a punctate appearance, indicating its presence in intracellular vesicles. Nitric oxide production decreased by approximately 20% with 300 nmol/L and 3 µmol/L 8‐Br cGMP ( P < 0.05) and increased by 20.8 ± 3.7% with 3 µmol/L BPC ( P < 0.001), indicating that both stimulated and basal PKG activity has inhibitory effects on basal NOS3 function. Nitric oxide synthase immunofluorescence and immunoblot expression were decreased and pSer116‐NOS immunofluorescence was increased by 800 nmol/L 8‐Br‐cGMP and 170 µmol/L ( Z )‐1‐[2‐(2‐aminoethyl)‐ N ‐(2‐ammonio‐ethyl)amino]diazen‐1‐ium‐1, 2‐diolate (DETANONOate). The effect of cyclodextrin indicated that cholesterol extraction interfered with PKG inhibition of NOS. Further examination of pSer116‐NOS by immunohistochemistry showed it abundant in the endoplasmic reticulum and colocalized with PKG 1β, especially in nuclear vesicles. 6 We conclude that endothelial PKG is involved in endogenous regulation of basal NOS3 activity with the involvement of lipid structures, the endoplasmic reticulum and the nucleus. Protein kinase G 1β is colocalized with pSer116‐NOS, indicating that PKG action may involve serine 116 phosphorylation on NOS.