Protein kinase G may exert pro-degradation inhibition on nitric oxide synthase.
Nitric oxide synthase (NOS) is regulated by protein-protein interactions. We had earlier shown that PKG inhibits activated NOS in endothelial cells and speculated that PKG phosphorylation of NOS terminates its activity. The present work examines if PKG activation increases breakdown of NOS. Diamino-fluorescein fluorescence spectrometry of real time NO production was used to establish that isolated ovine lung microvascular endothelial cells responded to PKG modulation as previously reported. Fluorescence activated cell sorter (FACS) analysis was used to establish that 8-Br-cGMP, a PKG activator, caused carboxy terminal deletion on NOS, a sign of degradation. Western blot analysis was used to investigate NOS fragments in control and 5 min 8-Br-cGMP treated cells. PKG activator 8-Br-cGMP, at 20 nM, 200 nM, and 2 μM, decreased nitric oxide production in a dose dependent manner (p<0.05 in all cases). PKG inhibitors: 100 μM Rp-8-Br-PET-cGMPS, 50 nM Rp-8-pCPT-cGMPS, or 4 μM Rp-8-Br-cGMPS Na significantly increased NO production (p<0.05) showing that PKG normally inhibits basal NO production. 8-Br-cGMP (100 nM) abrogated the elevation in NO production produced by the PKG inhibitors. FACS analysis revealed that PKG decreased NOS carboxy terminal labeling. Western blot analysis revealed that 8-Br-cGMP increased N-terminal serine-116 phosphorylated NOS fragments of molecular weights of about 60, 50 and 35 kDa. PKG may be a post-activation inhibitor of NOS, possibly important for the degradation of the spent enzyme.
Department of Pharmacology, Lagos State University College of Medicine, Lagos, Nigeria. theresaadebola@yahoo.com
Theresa Adebola
TA
John
D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002478:Cells, Cultured; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D008168:Lung / Q000098:blood supply*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D012694:Serine; D012756:Sheep; D013050:Spectrometry, Fluorescence; D013997:Time Factors
D020536:Enzyme Activators; D010446:Peptide Fragments; D047428:Protein Kinase Inhibitors; D009569:Nitric Oxide; D012694:Serine; D019001:Nitric Oxide Synthase; D017869:Cyclic GMP-Dependent Protein Kinases