Role of Neural NO Synthase (nNOS) Uncoupling in the Dysfunctional Nitrergic Vasorelaxation of Penile Arteries from Insulin-Resistant Obese Zucker Rats Ana Sa ´ nchez 1 , Cristina Contreras 1 , Marı´a Pilar Martı´nez 2 , Bele ´ n Climent 1 , Sara Benedito 1 , Albino Garcı´a- Sacrista ´n 1 , Medardo Herna ´ ndez 1 , Dolores Prieto 1 * 1 Departamento de Fisiologı ´a, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain, 2 Departamento de Anatomı ´a y Anatomı ´a Patolo ´ gica Comparadas, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain Abstract Objective: Erectile dysfunction (ED) is considered as an early sign of vascular disease due to its high prevalence in patients with cardiovascular risk factors. Endothelial and neural dysfunction involving nitric oxide (NO) are usually implicated in the pathophysiology of the diabetic ED, but the underlying mechanisms are unclear. The present study assessed the role of oxidative stress in the dysfunctional neural vasodilator responses of penile arteries in the obese Zucker rat (OZR), an experimental model of metabolic syndrome/prediabetes. Methods and Results: Electrical field stimulation (EFS) under non-adrenergic non-cholinergic (NANC) conditions evoked relaxations that were significantly reduced in penile arteries of OZR compared with those of lean Zucker rats (LZR). Blockade of NO synthase (NOS) inhibited neural relaxations in both LZR and OZR, while saturating concentrations of the NOS substrate L-arginine reversed the inhibition and restored relaxations in OZR to levels in arteries from LZR. nNOS expression was unchanged in arteries from OZR compared to LZR and nNOS selective inhibition decreased the EFS relaxations in LZR but not in OZR, while endothelium removal did not alter these responses in either strain. Superoxide anion production and nitro-tyrosine immunostaining were elevated in the erectile tissue from OZR. Treatment with the NADPH oxidase inhibitor apocynin or acute incubation with the NOS cofactor tetrahydrobiopterin (BH4) restored neural relaxations in OZR to levels in control arteries, while inhibition of the enzyme of BH4 synthesis GTP-cyclohydrolase (GCH) reduced neural relaxations in arteries from LZR but not OZR. The NO donor SNAP induced decreases in intracellular calcium that were impaired in arteries from OZR compared to controls. Conclusions: The present study demonstrates nitrergic dysfunction and impaired neural NO signalling due to oxidative stress and nNOS uncoupling in penile arteries under conditions of insulin resistance. This dysfunction likely contributes to the metabolic syndrome-associated ED, along with the endothelial dysfunction also involving altered NO signalling. Citation: Sa ´nchez A, Contreras C, Martı ´nez MP, Climent B, Benedito S, et al. (2012) Role of Neural NO Synthase (nNOS) Uncoupling in the Dysfunctional Nitrergic Vasorelaxation of Penile Arteries from Insulin-Resistant Obese Zucker Rats. PLoS ONE 7(4): e36027. doi:10.1371/journal.pone.0036027 Editor: Carlo Gaetano, Istituto Dermopatico dell’Immacolata, Italy Received October 24, 2011; Accepted March 28, 2012; Published April 23, 2012 Copyright: ß 2012 Sa ´nchez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by Grant nu SAF2009-10448 from the Spanish Minister of Science and Innovation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: dprieto@farm.ucm.es Introduction Erectile dysfunction (ED) is currently considered as an early clinical sign of vascular disease due to its high prevalence in patients with cardiovascular risk factors including diabetes, hypertension and hyperlipidemia [1,2]. ED is a common complication and an important cause of decreased quality of life in men with diabetes, male impotence being three times more prevalent in type 1 and type 2 diabetic patients than in the general population [3,4]. Penile erection is a complex and multifactorial hemodynamic process involving organic pathways that require hormonal balance and neuronal and vascular function integrity [5,6]. Erection is initiated by activation of parasympathetic nerves upon sexual stimulation, neural signs from the spinal cord stimulating neuronal nitric oxide synthase (nNOS) activity and NO production from non-adrenergic non-cholinergic (NANC) nerve terminals thereby causing an increase in blood flow to the cavernosal tissue [5,6,7]. Endothelial NOS (eNOS) is then activated by the continued shear stress on the endothelial lining of the sinusoidal spaces and arteries, which continues to produce endothelial-derived NO thus main- taining the sustained phase of penile erection [6,7,8]. Diabetic patients have an increased risk of vascular and nerve dysfunction and both autonomic neuropathy and endothelial dysfunction are considered the main etiological factors in the diabetic ED [9,10]. Hyperglycemia, oxidative stress and altered lipid profiles contribute to vascular complications including peripheral nerve perfusion deficits which play an important role in the etiology of diabetic neuropathy [11]. Impairment of the NO-mediated neural and endothelial relaxations was first PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e36027