Autoinhibitory domain fragment of endothelial NOS enhances pulmonary artery vasorelaxation by the NO-cGMP pathway.
Catalytic activity of eNOS is regulated by multiple posttranscriptional mechanisms, including a 40-amino acid (604-643) autoinhibitory domain (AID) located in the reductase domain of the eNOS protein. We examined whether an exogenous synthetic AID, an 11-amino acid (626-636) fragment of AID (AAF), or scrambled AAF (AAF-SR), enhanced eNOS activity and NO-cGMP-mediated vasorelaxation using pulmonary artery (PA) endothelial/smooth muscle cell (PAEC/PASM) coculture, isolated PA segment, and isolated lung perfusion models. Incubation of isolated total membrane fraction of PAEC with AID or AAF resulted in concentration-dependent loss of eNOS activity. In contrast, incubation of intact PAEC with AID or AAF but not AAF-SR caused concentration- and time-dependent activation of eNOS. Because AID and AAF had similar effects on activation of eNOS, AAF and AAF-SR were used for further evaluation. Although AAF stimulation increased catalytic activity of PKC-alpha in PAEC, AAF-mediated activation of eNOS was independent of phosphorylation of Ser1177 or Thr495 and/or expression of eNOS protein. AAF stimulation of PAEC increased NO and cGMP production, which were attenuated by pretreatment with the eNOS inhibitor l-NAME. AAF caused time-dependent vasodilation of U-46619-precontracted endothelium-intact but not endothelium-denuded PA segments, and this response was attenuated by l-NAME. AAF, but not AAF-SR, also caused vasorelaxation in an ex vivo isolated mouse lung perfusion model precontracted with U-46619. Incubation with fluorescence-labeled AAF demonstrated translocation of AAF in PAEC in culture, isolated PA, and isolated intact lungs. These results demonstrate that AAF-stimulated vasodilation is mediated via activation of eNOS and enhanced NO-cGMP production in PA and intact lung.
Hu
Hanbo
H
Research Service (151 VA Medical Center, 1601 SW Archer Road, Gainesville, FL 32608-1197, USA.
Xin
Meiguo
M
Belayev
Leonid L
LL
Zhang
Jianliang
J
Block
Edward R
ER
Patel
Jawaharlal M
JM
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D006152:Cyclic GMP / Q000502:physiology*; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology; D023421:Models, Animal; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000737:chemistry / Q000494:pharmacology*; D052250:Nitric Oxide Synthase Type III; D010446:Peptide Fragments / Q000737:chemistry; D011651:Pulmonary Artery / Q000187:drug effects / Q000502:physiology*; D013552:Swine; D014664:Vasodilation / Q000187:drug effects*
D010446:Peptide Fragments; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D006152:Cyclic GMP