Hanbo Hu, Meiguo Xin, Leonid L. Belayev, Jianliang Zhang, Edward R. Block and Jawaharlal M. Patel. Autoinhibitory domain fragment of endothelial NOS enhances pulmonary artery vasorelaxation by the NO/cGMP pathway. Am. J. Physiol. Lung Cell Mol. Physiol. 00: L000-L000, 2004.--- The catalytic activity of endothelial cell nitric oxide synthase (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. Since AID and AAF had similar effects on activation of eNOS, AAF and AAF-SR were used for further evaluation. Although AAFstimulation increased the catalytic activity of PKC- in PAEC, AAF-mediated activation of eNOS was independent of phosphorylation of Ser-1177 or Thr-495 and/or expression of eNOS protein. AAF-stimulation of PAEC increased NO and cGMP production which were attenuated by pretreatment with an 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 pre-contracted 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.