Angiotensin II (Ang II) is critical in myocardial pathogenesis, mostly via stimulation of NADPH oxidase(Ref 1, Ref 2). Neuronal nitric oxide synthase (nNOS) has recently been shown to play important roles in modulating myocardial oxidative stress and contractility(Ref 3, Ref 4). Here, we examine whether nNOS is regulated by Ang II and affects NADPH oxidase production of intracellular reactive oxygen species (ROSi) and contractile function in left ventricular (LV) myocytes. Our results showed that Ang II-induced biphasic effects on ROSi and LV myocyte relaxation (TR50) without affecting the amplitude of sarcomere shortening and L-type Ca2+ current density: TR50 was prolonged at 30 min (P<0.001, n=141 with Ang II vs. n=201 in controls) but was shortened after 3hrs (P<0.001, n=106, or after Ang II in vivo treatment, P<0.0001, n=153 compare to that in sham). Correspondingly, ROSi was increased (by 2 fold, P<0.0001, n=14 with Ang II vs. controls, n=16), followed by a reduction to control level (P=0.1, n=33 with Ang II vs. n=35 in controls). Quantitative RT-PCR and immunoblotting experiments showed that Ang II (3hrs) increased the mRNA and protein expression of nNOS (P=0.0002, n=10 for mRNA and P=0.01, n=9 for protein) and increased NO production (nitrite assay, P<0.0001, n=8) in LV myocyte homogenates, suggesting that nNOS activity may be enhanced and involved in mediating the effects of Ang II. Indeed, n(omega)-nitro-l-arginine methyl ester (L-NAME) or a selective nNOS inhibitor, S-methyl-L-thiocitrulline (SMTC) increased NADPH oxidase production of superoxide/ROSi (P<0.0001 between Ang II+SMTC and SMTC only, n=22 and n=27, respectively) and abolished faster myocyte relaxation induced by Ang II (P=0.1 between SMTC and Ang II + SMTC, n=57 and n=90). The positive lusitropic effect of Ang II was not mediated by PKA-, CaMKII-dependent signaling or peroxynitrite. Conversely, inhibition of cGMP/PKG pathway abolished the Ang II-induced faster relaxation (P=0.9 between ODQ and ODQ+Ang II, n=43 & n=32; P=0.8 between KT 5823 & KT 5823+Ang II, n=46 & n=56) by reducing phospholamban (PLN) Ser16 phosphorylation PLN-p/PLN ratio: from P=0.003 between control and Ang II, n=18 to P=0.9 between KT 5823 and KT 5823+Ang II, n=8 or P=0.8 between ODQ and ODQ + Ang II, n=8). Taken together, these results clearly demonstrate that myocardial nNOS is up-regulated by Ang II and functions as an early adaptive mechanism to attenuate NADPH oxidase activity and facilitate myocardial relaxation. Rats (of 8 weeks old) were anesthetized with isoflurane (2.5 %). An osmotic minipump (Alzet model 2004) containing Ang II (200l, 6 mM, infusion rate 125 ng/min/kg) was implanted in the midscapular region under sterile condition. Sham-operated animals underwent the same surgical procedure, except for no pump insertion.