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ROS and endothelial nitric oxide synthase (eNOS)-dependent trafficking of angiotensin II type 2 receptor begets neuronal NOS in cardiac myocytes

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Abstract

Angiotensin II (Ang II), a potent precursor of hypertrophy and heart failure, upregulates neuronal nitric oxide synthase (nNOS or NOS1) in the myocardium. Here, we investigate the involvement of type 1 and 2 angiotensin receptors (AT1R and AT2R) and molecular mechanisms mediating Ang II-upregulation of nNOS. Our results showed that pre-treatment of left ventricular (LV) myocytes with antagonists of AT1R or AT2R (losartan, PD123319) and ROS scavengers (apocynin, tiron or PEG-catalase) blocked Ang II-upregulation of nNOS. Surface biotinylation or immunocytochemistry experiments demonstrated that AT1R expression in plasma membrane was progressively decreased (internalization), whereas AT2R was increased (membrane trafficking) by Ang II. Inhibition of AT1R or ROS scavengers prevented Ang II-induced translocation of AT2R to plasma membrane, suggesting an alignment of AT1R-ROS-AT2R. Furthermore, Ang II increased eNOS-Ser1177 but decreased eNOS-Thr495, indicating concomitant activation of eNOS. Intriguingly, ROS scavengers but not AT2R antagonist prevented Ang II-activation of eNOS. NOS inhibitor (L-NG-Nitroarginine Methyl Ester, L-NAME) or eNOS gene deletion (eNOS−/−) abolished Ang II-induced membrane trafficking of AT2R, nNOS protein expression and activity. Mechanistically, S-nitrosation of AT2R was increased by sodium nitroprusside (SNP), a NO donor. Site-specific mutagenesis analysis reveals that C-terminal cysteine 349 in AT2R is essential in AT2R translocation to plasma membrane. Taken together, we demonstrate, for the first time, that Ang II upregulates nNOS protein expression and activity via AT1R/ROS/eNOS-dependent S-nitrosation and membrane translocation of AT2R. Our results suggest a novel crosstalk between AT1R and AT2R in regulating nNOS via eNOS in the myocardium under pathogenic stimuli.

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Acknowledgments

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2013068067); by the Brain Korea 21 Graduate Programme of the Korean Ministry of Education, Science and Technology, Seoul National University Hospital, the Korean Society of Hypertension (2013), SK Telecom Research Fund (no. 3420130290) and from the National Natural Science Foundation of China (NSFC, 31460265). National Institutes of Health grant R01GM076167.

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Authors and Affiliations

  1. Department of Physiology & Biomedical Sciences, Hypoxia Research Institute, Institute of Human-Environment Interface Biology, College of Medicine, Seoul National University, 103 Dae Hak Ro, Chong no Gu, 110-799, Seoul, Korea

    Ji Hyun Jang, Jung Nyeo Chun, Ju Hong Jeon, Sung Joon Kim & Yin Hua Zhang

  2. Yanbian University Hospital, Yanji, China

    Chun Zi Jin, Zhe Hu Jin & Yin Hua Zhang

  3. Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan

    Shigeo Godo & Hiroaki Shimokawa

  4. Department of Pharmacology and Toxicology, Medical College of Georgia, Georgia Regents University, Augusta, Georgia, USA

    Guangyu Wu

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  1. Ji Hyun Jang
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Jang, J.H., Chun, J.N., Godo, S. et al. ROS and endothelial nitric oxide synthase (eNOS)-dependent trafficking of angiotensin II type 2 receptor begets neuronal NOS in cardiac myocytes. Basic Res Cardiol 110, 21 (2015). https://doi.org/10.1007/s00395-015-0477-6

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