title: Wall stretch and thromboxane A₂ activate NO synthase (eNOS) in pulmonary arterial smooth muscle cells via H₂O₂ and Akt-dependent phosphorylation.
abstract: Pulmonary arteries (PAs) have high compliance, buffering the wide ranges of blood flow. Here, we addressed a hypothesis that PA smooth muscle cells (PASMCs) express nitric oxide synthases (NOS) that might be activated by mechanical stress and vasoactive agonists. In the myograph study of endothelium-denuded rat PAs, NOS inhibition (L-NAME) induced strong contraction (96 % of 80 mM KCl-induced contraction (80K)) in the presence of 5 nM U46619 (thromboxane A2 (TXA2) analogue) with relatively high basal stretch (2.94 mN, S(+)). With lower basal stretch (0.98 mN, S(-)), however, L-NAME application following U46619 (TXA2/L-NAME) induced weak contraction (27 % of 80K). Inhibitors of nNOS and iNOS had no such effect in S(+) PAs. In endothelium-denuded S(+) mesenteric and renal arteries, TXA2/L-NAME-induced contraction was only 18 and 21 % of 80K, respectively. Expression of endothelial-type NOS (eNOS) in rat PASMCs was confirmed by RT-PCR and immunohistochemistry. Even in S(-) PAs, pretreatment with H2O2 (0.1-10 μM) effectively increased the sensitivity to TXA2/L-NAME (105 % of 80K). Vice versa, NADPH oxidase inhibitors, reactive oxygen species scavengers, or an Akt inhibitor (SC-66) suppressed TXA2/L-NAME-induced contraction in S(+) PAs. In a human PASMC line, immunoblot analysis showed the following: (1) eNOS expression, (2) Ser(1177) phosphorylation by U46619 and H2O2, and (3) Akt activation (Ser(473) phosphorylation) by U46619. In the cell-attached patch clamp study, H2O2 facilitated membrane stretch-activated cation channels in rat PASMCs. Taken together, the muscular eNOS in PAs can be activated by TXA2 and mechanical stress via H2O2 and Akt-mediated signaling, which may counterbalance the contractile signals from TXA2 and mechanical stimuli.
authors.affiliation: Department of Physiology, Seoul National University College of Medicine, Seoul, 110-799, Korea.|Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799, Korea.|Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul, 110-799, Korea.
authors.forename: Hae Jin
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: Chung-Ang University Red Cross College of Nursing, Seoul, 100-031, Korea.
authors.forename: Hae Young
authors.initials: HY
authors.lastname: Yoo
authors.affiliation: Department of Physiology, Seoul National University College of Medicine, Seoul, 110-799, Korea.|Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799, Korea.
authors.forename: Ji Hyun
authors.initials: JH
authors.lastname: Jang
authors.affiliation: Department of Physiology, Seoul National University College of Medicine, Seoul, 110-799, Korea.|Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799, Korea.
authors.forename: Hai Yue
authors.initials: HY
authors.lastname: Lin
authors.affiliation: Department of Physiology, Seoul National University College of Medicine, Seoul, 110-799, Korea.|Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799, Korea.
authors.forename: Eun Yeong
authors.initials: EY
authors.lastname: Seo
authors.affiliation: Department of Physiology, Seoul National University College of Medicine, Seoul, 110-799, Korea.|Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799, Korea.|Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul, 110-799, Korea.
authors.forename: Yin Hua
authors.initials: YH
authors.lastname: Zhang
authors.affiliation: Department of Physiology, Seoul National University College of Medicine, Seoul, 110-799, Korea. physiolksj@gmail.com.|Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799, Korea. physiolksj@gmail.com.|Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul, 110-799, Korea. physiolksj@gmail.com.
authors.forename: Sung Joon
authors.initials: SJ
authors.lastname: Kim
mesh_terms: D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011651:Pulmonary Artery / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013928:Thromboxane A2 / Q000378:metabolism* / Q000494:pharmacology; D014662:Vasoconstrictor Agents / Q000494:pharmacology*
chemical_list: D014662:Vasoconstrictor Agents; D013928:Thromboxane A2; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D006861:Hydrogen Peroxide; D052250:Nitric Oxide Synthase Type III; D051057:Proto-Oncogene Proteins c-akt
keywords: Endothelial NO synthase; Hydrogen peroxide; Pulmonary artery smooth muscle; Stretch