Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC154

Poster Communications

Functional roles of neuronal nitric oxide synthase in NADPH oxidase activity and contraction in angiotensin II-treated rat ventricular myocytes

J. Chung1, C. Jin1, S. Kim1, Y. Zhang1

1. Department of Physiology, Seoul National University, College of Medicine, Seoul, Korea, Republic of.


Angiotensin II (Ang II) activation of NADPH oxidase is crucial to myocardial pathogenesis. It has been demonstrated that the expression and activities of cardiac nitric oxide synthases (NOS) are up-regulated downstream of Ang II stimulation. However, the functional role of nNOS in left ventricular (LV) myocyte of Ang II remains undetermined. Our results show that Ang II (1 μM) significantly prolonged LV myocyte relaxation at 30 min (TR50, field stimulation at 2 Hz, 36oC, P<0.001, between Ang II vs. controls, n=141 and n=201, respectively) whereas such an effect was absent at 1hr or at 2hrs (P=0.6 or P=0.6 comparing to that in controls, n=38 & 41). Surprisingly, TR50 became facilitated after 3hrs incubation with Ang II (P<0.001, n=88) and maintained at this level up to 6hrs (P=0.001 between Ang II 6hrs vs. control, n=49 for Ang II). Similar faster LV myocyte relaxation was also observed with lower doses of Ang II comparing to those of control (3hrs, Ang II 10 nM: P=0.03, n=52; Ang II 100 nM: P<0.001, n=40) or between LV myocytes isolated from rats treated with Ang II via mini-osmotic pump (4 weeks) and those from sham-operated groups (P<0.0001, n=97 vs. 99). The effect was mediated by ATR1, NADPH oxidase and ATR2. Sarcomere shortening was unaffected. Concomitant to time-dependent biphasic changes in TR50 by Ang II, intracellular reactive oxygen species (ROS, 2',7'-dichlorodihydrofluorescein diacetate, H2DCFDA, 10 μM) was increased at 30 min (P=0.02, Ang II vs. control, n=7 and n=10) but decreased to control level after 3hrs (P=0.09, Ang II vs. control, n=33 and n=35). N(omega)-nitro-l-arginine methyl ester (L-NAME, 1 mM) or nNOS inhibitor, S-methyl-L-thiocitrulline (SMTC, 100 nM) elevated intracellular ROS above the control level in the presence of Ang II (P<0.0001 between LV myocytes in Ang II+L-NAME vs. that with L-NAME only, n=29 and n=22; P<0.0001 between LV myocytes in Ang II+SMTC vs. that with SMTC only, n=22 and n=27) and abolished Ang II-facilitation of LV myocyte relaxation (P=0.08, between SMTC only and Ang II + SMTC, n=57 and n=90), suggesting a role for nNOS. Indeed, real time RT-PCR showed nearly two-fold increase of nNOS mRNA. A reducing agent, DTT (1mM, but not peroxynitrite decomposition catalyst, FePPTS, 10 mM or inhibitors of PKA and CaMKII) prevented Ang II-induced faster relaxation (P=0.5 between DTT and DTT+Ang II, n=47 and n=52). The results clearly demonstrate that myocardial nNOS-derived NO plays an important role in attenuating NADPH oxidase-derived ROS to facilitate myocardial relaxation downstream of Ang II through a mechanism involves s-nitrosylation.

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