Regulation of endothelial nitric oxide synthase: involvement of protein kinase G 1 beta, serine 116 phosphorylation and lipid structures.
1. 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 micromol/L) with and without prior stimulation with the PKG activator 8-bromo-cGMP (8-Br-cGMP; 0.3 and 3 micromol/L) or prior PKG inhibition with beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothionate (BPC; 0.3 and 3 micromol/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 1 beta 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 1 beta gave a punctate appearance, indicating its presence in intracellular vesicles. Nitric oxide production decreased by approximately 20% with 300 nmol/L and 3 micromol/L 8-Br cGMP (P < 0.05) and increased by 20.8 +/- 3.7% with 3 micromol/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 micromol/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 1 beta, 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 1 beta is colocalized with pSer116-NOS, indicating that PKG action may involve serine 116 phosphorylation on NOS.
John
Theresa A
TA
Division of Neonatology, Harbor UCLA Medical Center, Los Angeles Biomedical Research Institute, Torrance, California 90502, USA.
Ibe
Basil O
BO
Raj
J Usha
JU
D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D003505:Cyclodextrins / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000201:enzymology; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008168:Lung / Q000098:blood supply* / Q000196:embryology; D008563:Membrane Lipids / Q000378:metabolism*; D008833:Microcirculation / Q000201:enzymology; D008856:Microscopy, Fluorescence / Q000379:methods; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009603:Nitroso Compounds / Q000494:pharmacology; D015388:Organelles / Q000187:drug effects / Q000201:enzymology*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021122:Protein Subunits / Q000378:metabolism; D021381:Protein Transport; D012694:Serine / Q000378:metabolism; D012756:Sheep; D013997:Time Factors
C503301:4-amino-5-methylamino-2',7'-difluorofluorescein diacetate; C104224:8-bromo-beta-phenylethenoguanosine 3',5'-cyclic monophosphate; D003505:Cyclodextrins; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008563:Membrane Lipids; D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; D047428:Protein Kinase Inhibitors; D021122:Protein Subunits; C094210:2,2'-(hydroxynitrosohydrazono)bis-ethanamine; C016276:8-bromocyclic GMP; D009569:Nitric Oxide; D012694:Serine; D052250:Nitric Oxide Synthase Type III; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP