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

Ji Hyun Jang, Jung Nyeo Chun, Shigeo Godo, Guangyu Wu, Hiroaki Shimokawa, Chun Zi Jin, Ju Hong Jeon, Sung Joon Kim, Zhe Hu Jin, Yin Hua Zhang

Research output: Contribution to journalArticle

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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.

Original languageEnglish (US)
JournalBasic Research in Cardiology
Volume110
Issue number3
DOIs
StatePublished - May 1 2015

Fingerprint

Angiotensin Type 2 Receptor
Nitric Oxide Synthase Type III
Cardiac Myocytes
Angiotensin II
Up-Regulation
Nitrosation
Cell Membrane
Membranes
Myocardium
1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt
Biotinylation
Nitric Oxide Synthase Type I
Angiotensin Type 1 Receptor
Losartan
NG-Nitroarginine Methyl Ester
Gene Deletion
Nitroprusside
Site-Directed Mutagenesis
Muscle Cells
Hypertrophy

Keywords

  • Angiotensin II
  • Angiotensin type 2 receptor
  • Cardiac myocyte
  • eNOS
  • nNOS

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)

Cite this

ROS and endothelial nitric oxide synthase (eNOS)-dependent trafficking of angiotensin II type 2 receptor begets neuronal NOS in cardiac myocytes. / Jang, Ji Hyun; Chun, Jung Nyeo; Godo, Shigeo; Wu, Guangyu; Shimokawa, Hiroaki; Jin, Chun Zi; Jeon, Ju Hong; Kim, Sung Joon; Jin, Zhe Hu; Zhang, Yin Hua.

In: Basic Research in Cardiology, Vol. 110, No. 3, 01.05.2015.

Research output: Contribution to journalArticle

Jang, Ji Hyun ; Chun, Jung Nyeo ; Godo, Shigeo ; Wu, Guangyu ; Shimokawa, Hiroaki ; Jin, Chun Zi ; Jeon, Ju Hong ; Kim, Sung Joon ; Jin, Zhe Hu ; Zhang, Yin Hua. / ROS and endothelial nitric oxide synthase (eNOS)-dependent trafficking of angiotensin II type 2 receptor begets neuronal NOS in cardiac myocytes. In: Basic Research in Cardiology. 2015 ; Vol. 110, No. 3.
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AU - Chun, Jung Nyeo

AU - Godo, Shigeo

AU - Wu, Guangyu

AU - Shimokawa, Hiroaki

AU - Jin, Chun Zi

AU - Jeon, Ju Hong

AU - Kim, Sung Joon

AU - Jin, Zhe Hu

AU - Zhang, Yin Hua

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N2 - 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.

AB - 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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