At1r/Ros/Enos/At2r Axis in Angiotensin Ii Upregulation of Neuronal Nitric Oxide Synthase in Cardiomyocytes

Objective: Angiotensin II (Ang II) is a potent precursor of hypertrophy and heart failure. However, neuronal nitric oxide synthase (nNOS or NOS1) is upregulated by Ang II, suggesting pro-protective mechanisms of Ang II signaling in the heart. Until recently, the mechanisms mediating Ang II-inducing nNOS remained unclear. The main purpose was to identify cellular mechanisms of Ang II type I and type II receptors and the subsequent signaling cascades mediating nNOS upregulation in cardiomyocytes. Design and method: nNOS expression was detected in left ventricular (LV) myocytes from SD rats (8-10 weeks, male) pre-treated with Ang II with and without antagonists of AT1R or AT2R (losartan, PD123319) and ROS scavengers (apocynin, tiron or PEG-catalase). Surface biotinylation and immunocytochemistry experiments were conducted to examine AT1R expression in plasma membrane. eNOS inhibitors and LV myocytes from eNOS gene deletion mice were used to test the effects of eNOS in the signaling pathway. Site-specific mutagenesis analysis was used to specify s-nitrosylation cysteine residue of AT2R for its membrane translocation. Results: Our results showed that Ang II increased nNOS protein expression but losartan, PD123319 and apocynin, tiron or PEG-catalase blocked Ang II-upregulation of nNOS. AT1R expression was progressively internalization with Ang II treatment accompanied by membrane trafficking of AT2R. Inhibition of AT1R or ROS scavengers prevented Ang II-induced translocation of AT2R to plasma membrane. Furthermore, eNOS-Ser1177 was increased by Ang II but eNOS-Thr495 was decreased, and ROS scavengers prevented Ang II-activation of eNOS. L-NG-Nitroarginine Methyl Ester (L-NAME) or eNOS gene deletion abolished Ang II-induced membrane trafficking of AT2R, nNOS protein expression and activity. Mechanistically, NO induces S-nitrosation of AT2R and site-specific mutagenesis analysis reveals that C-terminal cysteine 349 in AT2R is essential in AT2R translocation to plasma membrane. Conclusions: Here, we demonstrate that Ang II upregulates nNOS protein expression and activity via AT1R/ROS/eNOS-dependent S-nitrosation and membrane translocation of AT2R. Crosstalk between AT1R and AT2R in regulating nNOS via eNOS is important in cardiac protection under pathogenic stimuli.