Salusin-β is a bioactive peptide with peripheral hypotensive, mitogenic, pro-atherosclerotic effects, and stimulates the arginine vasopressin (AVP) release from perifused rat pituitary (Shichiri et al, 2003). Salusin-β was found in paraventricular nucleus (PVN) and posterior pituitary (Suzuki et al, 2007). We recently found that microinjection of salusin-β into the PVN increased blood pressure and sympathetic outflow in renovascular hypertensive rats but not in sham-operated rats, which was mediated by both circulating AVP and AVP in rostral ventrolateral medulla (Chen et al. 2013). However, the downstream pathway in mediating the effects of salusin-β is unkown. The present study was designed to determine whether superoxide anions in the PVN were involved in the effects of salusin-β, and whether the production of superoxide anions was dependent on protein kinase C (PKC) and NAD(P)H oxidase in 2K1C rats. Renovascular hypertension was induced by two-kidney, one-clip (2K1C) in male Sprague-Dawley rats weighing 160-180 g under anaesthesia (pentobarbital sodium, 60 mg/kg, ip). Acute experiments were carried out 4 weeks after the 2K1C or sham operation under urethane (800 mg/kg) and alpha-chloralose (40 mg/kg) anesthesia intraperitoneally. Renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR) were recorded. Superoxide anion levels and NAD(P)H oxidase activity were measured with chemiluminescence method. Plasma AVP levels were determined with commercial Elisa kits. Values were expressed as mean±SE, compared by T-test or ANOVA (n=6 for each group). PVN microinjection of superoxide anion scavenger PEG-SOD (4 IU) almost abolished the RSNA and MAP responses to salusin-β compared with saline (RSNA change: 1.4±1.9 vs. 10.2±2.7 %, P<0.05; MAP change: 1.1±1.3 vs. 9.7±2.1 mm Hg, P<0.05). Similarly, NAD(P)H oxidase inhibitor apocynin (1 nmol) or PKC inhibitor chelerythrine chloride (CLC, 3 nmol) almostly abolished the effects of salusin-β . Salusin-β in the PVN increased superoxide anion level and NAD(P)H oxidase activity compared with vehicle (26.6±1.6 vs. 13.7±1.5, P<0.01, and 20.8±2.9 vs. 8.6±1.9 mean light units/min/mg protein, P<0.05, respectively), which was abolished by CLC pretreatment. Pretreatment with either PEG-SOD or CLC abolished the increased plasma AVP levels caused by salusin-β (6.7±0.4 and 7.6±0.2 vs. 12.7±0.9 pg/ml, P<0.05 and P<0.01, respectively). These results indicate that superoxide anions in the PVN mediate the effects of salusin-β in the PVN on the RSNA, MAP and AVP release, and that the production of superoxide anions in the PVN was dependent on PKC and NAD(P)H oxidase in 2K1C rats.