Neuronal Nitric Oxide Synthase Associated Protein: Nos1ap mediates Sympathoexcitation through Paraventricular Nucleus of the Hypothalamus

Abstract

Exaggerated sympathoexcitation is associated with multiple disorders, including hypertension, heart failure, diabetes leading to various cardiovascular complications. Within the paraventricular nucleus of the hypothalamus (PVN), nitric oxide (NO•) is synthesized by neuronal nitric oxide synthase (NOS1) and acts as a non‐conventional neurotransmitter that inhibits neuronal firing and consequent sympathetic outflow. NOS1 is tethered to N‐methyl D‐aspartate receptor (NMDAR) channels harboring NMDAR subunit 2B by the scaffolding protein postsynaptic density 95 (PSD95). Activation of the NMDAR at the synaptic membrane initiates a Ca2+ influx that stimulates the production of NO•. NOS1 adaptor protein (Nos1ap) competes with PSD95 for NOS1 binding and thus influences the production of NO•. We investigated the effect of over‐expressing Nos1ap in the PVN on sympathetic outflow using Cre recombinase‐conditional Nos1ap‐transgenic (Tg) mice. The Nos1ap Tg mice were microinjected with lentiviral expressing Cre‐GFP or GFP bilaterally into the PVN following our standardized method, referred to as CAPON‐OE and Control, respectively. Two weeks of post‐viral administration, the sympathetic outflow, was evaluated. CAPON‐OE mice displayed a significantly elevated urinary excretion of norepinephrine, an index of overall sympathetic outflow compared to Control (0.64±0.11* vs. 0.17±0.03 ug/day). Basal renal sympathetic nerve activity, measured as a percent of maximal activity was elevated 1.5‐fold in CAPON‐OE (46.09 ± 3.56* vs.31.04 ± 3.24) mice. Furthermore, FosB (a member of the AP‐1 family of transcription regulatory proteins) immunocytochemistry revealed increased expression in the PVN of CAPON‐OE mice when compared to control mice suggesting a chronic activation of PVN neurons after Nos1ap over‐expression. Interestingly, a robust over‐expression of Nos1ap in the PVN of CAPON‐OE (0.49±0.04* vs. 0.21±0.03) compared to control mice does not lead to any significant change in NOS1 expression. However, DAF‐FM staining of PVN slices showed decreased NO• levels in CAPON‐OE mice when compared to control mice suggesting that the decrease in NMDAR‐mediated NOS1 activity due to sequestering of NOS1 by Nos1ap leads to less NO• production. Taken together, our data indicate that specific over‐expression of Nos1ap within the PVN reduces the levels of NO• generated by NOS1, leading to increased sympathetic outflow.Support or Funding InformationSupported by AHA ‐ 14SDG19980007 and NIH grant HL62222.