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Wall stretch and thromboxane A2 activate NO synthase (eNOS) in pulmonary arterial smooth muscle cells via H2O2 and Akt-dependent phosphorylation

  • Muscle physiology
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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) Ser1177 phosphorylation by U46619 and H2O2, and (3) Akt activation (Ser473 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.

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Authors and Affiliations

  1. Department of Physiology, Seoul National University College of Medicine, Seoul, 110-799, Korea

    Hae Jin Kim, Ji Hyun Jang, Hai Yue Lin, Eun Yeong Seo, Yin Hua Zhang & Sung Joon Kim

  2. Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul, 110-799, Korea

    Hae Jin Kim, Ji Hyun Jang, Hai Yue Lin, Eun Yeong Seo, Yin Hua Zhang & Sung Joon Kim

  3. Ischemic/Hypoxic Disease Institute, Seoul National University College of Medicine, Seoul, 110-799, Korea

    Hae Jin Kim, Yin Hua Zhang & Sung Joon Kim

  4. Chung-Ang University Red Cross College of Nursing, Seoul, 100-031, Korea

    Hae Young Yoo

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This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (Nos. 2007-0056092 and 2011-0017370) and also by the Brain Korea 21 PLUS Program.

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ESM 1

Supplementary Figure S1. Angiotensin II treatment induced a significant transient contraction of S(+) PA. However, no contractile response to the addition of L-NAME was observed (.). Data from six repeated experiments are summarized (B). Supplementary Figure S2. Intermediate level of basal stretch (1.96 mN) was applied, S(−/+) PA. In total eight cases of experiments, six S(−/+) PA showed minute contractile response to L-NAME (A), and only two cases showed strong contraction (B). (PPT 282 kb)

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Kim, H.J., Yoo, H.Y., Jang, J.H. et al. Wall stretch and thromboxane A2 activate NO synthase (eNOS) in pulmonary arterial smooth muscle cells via H2O2 and Akt-dependent phosphorylation. Pflugers Arch - Eur J Physiol 468, 705–716 (2016). https://doi.org/10.1007/s00424-015-1778-1

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