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Enos-Uncoupling in Age-Related Erectile Dysfunction

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Enos-Uncoupling in Age-Related Erectile Dysfunction International Journal of Impotence Research (2011) 23, 43–48 & 2011 Macmillan Publishers Limited All rights reserved 0955-9930/11 www.nature.com/ijir ORIGINAL ARTICLE eNOS-uncoupling in age-related erectile dysfunction JM Johnson, TJ Bivalacqua, GA Lagoda, AL Burnett and B Musicki Department of Urology, The Johns Hopkins University, Baltimore, MD, USA Aging is associated with ED. Although age-related ED is attributed largely to increased oxidative stress and endothelial dysfunction in the penis, the molecular mechanisms underlying this effect are not fully defined. We evaluated whether endothelial nitric oxide synthase (eNOS) uncoupling in the aged rat penis is a contributing mechanism. Correlatively, we evaluated the effect of replacement with eNOS cofactor tetrahydrobiopterin (BH4) on erectile function in the aged rats. Male Fischer 344 ‘young’ (4-month-old) and ‘aged’ (19-month-old) rats were treated with a BH4 precursor sepiapterin (10 mg/kg intraperitoneally) or vehicle for 4 days. After 1-day washout, erectile function was assessed in response to electrical stimulation of the cavernous nerve. Endothelial dysfunction (eNOS uncoupling) and oxidative stress (thiobarbituric acid reactive substances, TBARS) were measured by conducting western blot in penes samples. Erectile response was significantly reduced in aged rats, whereas eNOS uncoupling and TBARS production were significantly increased in the aged rat penis compared with young rats. Sepiapterin significantly improved erectile response in aged rats and prevented increase in TBARS production, but did not affect eNOS uncoupling in the penis of aged rats. These findings suggest that aging induces eNOS uncoupling in the penis, resulting in increased oxidative stress and ED. International Journal of Impotence Research (2011) 23, 43–48; doi:10.1038/ijir.2011.2; published online 3 February 2011 Keywords: erection; oxidative stress; penis; tetrahydrobiopterin Introduction excessive cGMP degradation by upregulated PDE5,19 reduced activation of protein kinase G-I by cGMP20 ED is highly associated with aging.1–3 According to and increased oxidative stress.21 However, the source the Massachusetts Male Aging Study, 52% of men of reactive oxygen species in the aged penis is not beyond 40 years of age have some degree of ED known and the mechanisms of decreased endothelial and it is projected to affect 322 million men nitric oxide signaling are only partially elucidated. worldwide by 2025.4 Age-related ED is character- Oxidative stress is an important factor contribut- ized by decreased nerve and endothelium-mediated ing to endothelial dysfunction associated with corpus cavernosum relaxation,5 decreased androgen aging. Nicotinamide adenine dinucleotide phos- levels,6 pathological remodeling of the pudendal phate oxidases, xanthine oxidase, cyclooxygenases, artery,7 reduction in smooth muscle content,8–11 mitochondrial electron transport and eNOS uncou- impaired growth factor and cytokine signaling12,13 pling are potential vascular sources of reactive and upregulation of the RhoA/Rho-kinase contrac- oxygen species.22 eNOS uncoupling is a switch in tile pathway14 in the penis. Decreased endothelial the enzymes’ activity from a nitric oxide- to a signaling is attributed to impaired endothelial nitric predominantly superoxide-producing enzyme when 2,15–17 oxide synthase (eNOS) expression, decreased tetrahydrobiopterin (BH4), an essential eNOS cofac- eNOS phosphorylation,17 decreased content of the tor, is at suboptimal levels.23,24 Decreased nitric 18 nitric oxide synthase (NOS) substrate L-arginine, oxide production and increased superoxide produc- tion by uncoupled eNOS further enforce oxidative stress by perpetuating eNOS uncoupling through peroxynitrite formation, thereby resulting in chronic Correspondence: Dr B Musicki, Department of Urology, endothelial dysfunction. Although eNOS uncou- Johns Hopkins Hospital, Marburg 405, 600 North Wolfe Street, Baltimore, MD 21287, USA. pling has been largely observed in vitro and in the general diseased vasculature,23,24 including that of E-mail: [email protected] 25,26 Received 22 August 2010; revised 14 December 2010; the penis, its role in the regulation of eNOS accepted 31 December 2010; published online 3 February function and dysfunction in the penis, and the 2011 functional relevance of eNOS uncoupling in ED eNOS uncoupling in the aged rat penis JM Johnson et al 44 associated with aging, are unknown. The aim of this on 4–20% Tris gels at low temperature (below 15 1C) study was to investigate whether eNOS uncoupling and transferred to polyvinylidene difluoride mem- serves as a potential basis for increased oxidative brane. Membranes were probed with polyclonal stress in the penis, contributing to ED, using the rabbit anti-eNOS antibody (BD Transduction Labora- 25 aged-rat model. Correlatively, we evaluated BH4 tories, San Diego, CA, USA) at 1:700 dilution. eNOS replacement as an intervention for restoring erectile uncoupling is inversely related to the ratio of active function. eNOS dimers to inactive eNOS monomers. The ratio was determined with arbitrary units and expressed relative to the ratio for vehicle-treated young rats. Materials and methods Animal model Thiobarbituric acid reactive substances (TBARS) Male Fischer 344 ‘aged’ (19-month-old, 14 rats) and production ‘young’ (4-month-old, 14 rats) rats were used Thiobarbituric acid reactive substance (TBARS) (National Institute of Aging, Bethesda, MD, USA). assay, which evaluates lipid peroxidation, serves Rats were injected intraperitoneally with sepiapter- as an indicator of oxidative stress in tissues. The rat in, a BH4 precursor (10 mg/kg, Sigma Aldrich, St penis was pulverized and resuspended at a concen- Louis, MO, USA) or vehicle (3.3% DMSO/saline) for tration of 100 mg/ml in phosphate buffered saline. 4 consecutive days. This time period was chosen on TBARS production was measured according to the the basis of previous studies in non-penile vascu- manufacturer’s instructions using a TBARS assay lature, which demonstrated improvement in eNOS- kit (ZeptoMetrix Corp., Buffalo, NY, USA), as 25 mediated responses by sepiapterin.27,28 Erectile described. Samples were analyzed spectrofluoro- function and penes collection for molecular studies metrically in a microplate reader (BMG Labtech, were performed after a 1-day washout period. All Durham, NC, USA). Results were expressed relative animal procedures were conducted in accordance to vehicle-treated young rats. with the ethical standards of the Johns Hopkins University School of Medicine Guidelines for the Care and Use of Animals. Statistical analysis Statistical analysis was performed by using one-way analysis of variance, followed by Newman–Keuls Physiologic erection studies multiple comparison test or by t-test when appro- ± Animals were anesthetized with intraperitoneal priate. The data were expressed as the mean s.e.m. injection of 50 mg/kg ketamine/5 mg/kg xylazine. A A value of Po0.05 was considered to be statistically bipolar electrode attached to a Grass Instruments significant. S48 stimulator (Grass Instruments, Quincy, MA, USA) was placed around the cavernous nerve, Results as described.17 The right carotid artery was cannu- lated with polyethylene tubing (PE-50), anticoagu- Effect of aging and sepiapterin treatment on erectile lated with 20 U/ml heparin solution for continuous monitoring of systemic blood pressure. To measure function intracavernosal pressure (ICP), the shaft of the penis Erectile response, expressed as ICP/MAP, was was denuded of skin and fascia and the left corpus significantly (Po0.05) decreased in aged rats com- cavernosum was perforated with a 27-gauge needle pared with young rats at all voltages of electrical connected through PE-50 to a pressure transducer stimulation of the cavernous nerve (Figure 1). (DI-190; Dataq Instruments, Akron, OH, USA), as Sepiapterin treatment significantly (Po0.05) described.17 ICP was measured at increasing vol- increased the ICP/MAP ratio in aged rats at 2 tages. ICP responses were calculated as stimulated and 4 V, compared with aged vehicle-treated rats. ICP above baseline pressure, expressed per mean Sepiapterin treatment did not affect the ICP/MAP arterial pressure (MAP) using MATLAB software ratio in young rats (Figure 1). (Mathworks, Natick, MA, USA). Effect of aging and sepiapterin treatment on eNOS Low-temperature sodium dodecyl sulfate- uncoupling polyacrylamide gel electrophoresis (SDS-PAGE) Aged rat penes exhibited significantly decreased Minced penile tissue was homogenized and par- (Po0.05) ratio of eNOS dimers to monomers, tially purified for NOS by affinity binding to 20, compared with young rat penes (Figure 2), indicat- 50-ADP Sepharose, an NADP structural analog ing increased eNOS uncoupling in the aged rat immobilized on Sepharose 4B, which binds and penis. Sepiapterin treatment did not affect eNOS immobilizes enzymes requiring this cofactor.29 uncoupling in the penis of young or aged rats Partially purified NOS samples were resolved compared with vehicle-treated rats (Figure 2). International Journal of Impotence Research eNOS uncoupling in the aged rat penis JM Johnson et al 45 Figure 1 Effect of aging and sepiapterin treatment on penile erection. Erectile response is indicated by intracavernosal pressure (ICP) indexed to systemic blood pressure (mean arterial pressure, MAP). Each bar represents the mean±s.e.m. of 4–6 rats; *Po0.05 vs young vehicle; þ Po0.05 vs aged vehicle. Veh, vehicle; Sep, sepiapterin. Effect of aging and sepiapterin
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