Abstract 088: Overexpression of Neuronal Nitric Oxide Synthase Associated Protein in the Paraventricular Nucleus Contributes to Increased Sympathoexcitation in Male Mice by Modulating Subcellular Localization of Neuronal Nitric Oxide Synthase

Abstract

Nitric oxide (NO•) bioavailability within the paraventricular nucleus (PVN) plays a pivotal role in the pathogenesis of many cardiovascular diseases including hypertension. Aligning neuronal nitric oxide synthase (NOS1) with N -methyl-D-aspartate (NMDA) receptor by tethering to PSD95 (postsynaptic density 95 protein) allows the production of NO• initiated by an influx of Ca 2+ in neurons within the PVN. NOS1 associated protein (Nos1ap) competes with PSD95 for NOS1 binding and thus influences the production of NO•. We investigated the effect of specifically overexpressing Nos1ap in the PVN on sympathetic outflow using cre recombinase-conditional Nos1ap over-expression transgenic(Tg) mice injected with lentivirus expressing Cre-GFP or GFP into the PVN. Two weeks of post-viral administration, the sympathetic outflow was evaluated in both male and female mice. Male-Cre-Nos1ap mice displayed a significantly elevated urinary excretion of norepinephrine, an index of overall sympathetic outflow compared to GFP-Nos1ap (0.64±0.11* vs. 0.17±0.03 ug/day) while female littermates showed no difference (0.44±0.06 vs. 0.36±0.03 ug/day). Basal renal sympathetic nerve activity assessed as a percent of maximal activity was elevated 1.5-fold in Cre-Nos1ap male mice (46.1 ± 3.6* vs.31.0 ± 3.2) while there was no significant difference in Cre-Nos1ap female mice(22.6± 1.2 vs. 24.8 ± 2.6) compared to respective GFP controls. Interestingly, female mice had higher levels of basal NOS1 in the PVN compared to male littermates (0.79 ±0.01* vs. 0.37±0.05). Further, a robust overexpression of Nos1ap in the PVN of Cre-Nos1ap (0.49±0.04* vs. 0.21±0.03 male; 0.53±0.08* vs. 0.28±0.04 female) compared to GFP-Nos1ap mice does not lead to any change in NOS1, NMDA receptor(NR)2A, NR2B, while NR1 expression tended to increase in both sexes. Overexpression of Nos1ap in NG108 neurons, in vitro, led to relocalization of 46% of membrane-associated NOS1 to the cytosolic compartment, suggesting that the decrease in NMDA receptor-mediated NOS1 activity leads to reduced NO• production. Taken together, our data indicate that overexpression of Nos1ap within the PVN reduces the levels of NO• generated by NOS1 leading to increased sympathetic outflow in male mice but not in female mice.