Regulation of Nitric Oxide Synthase‐3 (NOS‐3) by cGMP‐Dependent Protein Kinase (PKG) in Ovine Lung Microvascular Endothelial Cells (MVECs)

• Published in: The FASEB journal 2006-03, Vol.20 (4), p.A720-A720
• Main Authors: John, Theresa A., Ibe, Basil O., Raj, J. Usha
• Format: Article
• Language: English
• ISSN: 0892-6638; 1530-6860
• DOI: 10.1096/fasebj.20.4.A720

After shear stress, NOS‐3 is phosphorylated at serine 116 (pSer116) (J Biol Chem, 1999). Whether phosphorylation at this site is related to activation of NOS‐3 and the endogenous kinase(s) involved are not known. We investigated the role of PKG, an endogenous kinase, as a candidate in newborn lamb lung explant MVECs expressing CD‐31 and factor VIII. To determine PKG expression, MVECs were immunolabeled with rabbit anti‐PKG antibody (Ab) and counter‐labeled with goat anti‐rabbit‐alexa 568 Ab for microscopy. Fetal lamb vein smooth muscle cells (SMCs) were positive controls. To determine the role of PKG in nitric oxide (NO) production, we measured NO using DAF‐FM (0.8 μM) in MVECs, with and without PKG inhibition by Rp‐8‐Bromo‐PET‐cGMPS (BPC) (0.3–30 μM). To confirm NO production was from constitutive NOS, we used 1 mM L‐NAME or 3‐imino‐4‐methyl‐piperidine (IMP) to inhibit it. We also determined the effect of stimulation of PKG on NOS‐3 and pSer116‐NOS immunofluorescence using cy‐3 in 30 μM 8‐bromo‐cGMP (BC) and 170 μM detaNONOate (DNN) treated MVECs. NO production was augmented at 1h by 3 μM BPC; 39.39 ± 4 % increase over controls (p 70% by both L‐NAME and IMP, indicating that NO measured in our experiments is due to NOS‐3 activity. With immunofluorescence, we found that PKG is expressed in MVECs and was mainly nuclear. PKG is both nuclear and cytoplasmic in SMCs. NOS expression was decreased by BC and DNN but pSer116‐NOS expression was increased by both drugs, mainly in the nuclear region. We conclude that endothelial PKG possibly phosphorylates NOS‐3 at serine 116 and has an inhibitory effect on NOS‐3 activity and NO production. Supported by NHLBI RO1 HL059435, HL077819 and MIRS HL059435 to T. John.