Abstract 15161: Angiotensin Ii Receptors and Nadph Oxidase Mediate Angiotensin Ii Stimulation Of Neuronal Nitric Oxide Synthase Protein Expression and Activity in Murine Left Ventricular Myocardium
Nitric oxide (NO) deficiency is responsible for increased oxidative stress and myocardial pathogenesis. Convincing evidence has shown that the protein expression and activity of neuronal nitric oxide synthase (nNOS) are increased in hypertrophic and failing heart. So far, mechanism mediating nNOS up-regulation in the myocardium under stress remains unidentified. Here, we examine the hypothesis that angiotensin II (Ang-II), a potent trigger of oxidative stress, stimulates the protein expression and activity of nNOS, which in turn, improves diastolic function in rat left ventricular (LV) myocytes. Our results showed that Ang-II (1 μM, 3 hrs) increased mRNA and protein expressions of nNOS (P=0.0002, n=10 for mRNA & P=0.03, n=9 for protein) in rat LV myocyte homogenates. NO production (nitrite assay) was also greater by Ang II (P<0.0001, n=8), which was abolished by a selective nNOS inhibitor, S-methyl-L-thiocitrulline (SMTC, 100 nM, P<0.001, n=12). Similar to Ang-II, a specific agonist of type 2 Ang-II receptor (AT2R), CGP-42112A (1 μM) increased nNOS protein expression (P=0.04, n=6). Conversely, AT2R antagonist, PD123319 (1 μM) abolished the effect of Ang-II on nNOS protein expression (P<0.001, n=5) and activity (P=0.001, n=12), suggesting that AT2R mediates the effect of Ang-II. Surprisingly, a type 1 Ang-II receptor (AT1R) antagonist, losartan (1 μM) exerted similar effects to that of PD123319. In line, both NADPH oxidase inhibitor, apocynin (100 μM) and superoxide scavenger, tiron (1 mM), blocked Ang-II-stimulation of nNOS protein expression (P=0.02, n=5) and activity (P<0.0001, n=6). Furthermore, Ang-II stimulation of nNOS, in turn, facilitated LV myocyte relaxation and reduced NADPH oxidase production of superoxide. Pretreatment of LV myocytes with losartan, PD123319, apocynin, tiron, respectively, abolished Ang-II-induced and nNOS-mediated faster LV myocyte relaxation. These results suggest that Ang-II stimulates nNOS protein expression and activity in cardiac myocytes via both AT1R and AT2R receptors. NADPH oxidase and intracellular ROS function as upstream transcriptional regulators of nNOS. Our results, for the first time, demonstrate the mechanism that links pathogenic Ang-II to nNOS transcription in mammalian myocardium.
- © 2012 by American Heart Association, Inc.