International Journal of Impotence Research (2006) 18, 432–437 & 2006 Nature Publishing Group All rights reserved 0955-9930/06 $30.00 www.nature.com/ijir ORIGINAL ARTICLE Soluble guanylate cyclase b1-subunit expression is increased in mononuclear cells from patients with erectile dysfunction PJ Mateos-Ca´ceres1, J Garcia-Cardoso2, L Lapuente1, JJ Zamorano-Leo´n1, D Sacrista´n1, TP de Prada1, J Calahorra2, C Macaya1, R Vela-Navarrete2 and AJ Lo´pez-Farre´1 1Cardiovascular Research Unit, Cardiovascular Institute, Hospital Clı´nico San Carlos, Madrid, Spain and 2Urology Department, Fundacio´n Jime´nez Diaz, Madrid, Spain The aim was to determine in circulating mononuclear cells from patients with erectile dysfunction (ED), the level of expression of endothelial nitric oxide synthase (eNOS), soluble guanylate cyclase (sGC) b1-subunit and phosphodiesterase type-V (PDE-V). Peripheral mononuclear cells from nine patients with ED of vascular origin and nine patients with ED of neurological origin were obtained. Fourteen age-matched volunteers with normal erectile function were used as control. Reduction in eNOS protein was observed in the mononuclear cells from patients with ED of vascular origin but not in those from neurological origin. Although sGC b1-subunit expression was increased in mononuclear cells from patients with ED, the sGC activity was reduced. However, only the patients with ED of vascular origin showed an increased expression of PDE-V. This work shows for the first time that, independently of the aetiology of ED, the expression of sGC b1-subunit was increased in circulating mononuclear cells; however, the expression of both eNOS and PDE-V was only modified in the circulating mononuclear cells from patients with ED of vascular origin. International Journal of Impotence Research (2006) 18, 432–437. doi:10.1038/sj.ijir.3901461; published online 9 March 2006 Keywords: molecular biologic study; risk factor Introduction stimulates soluble guanylate cyclase (sGC) and the activation of sGC elevates intracellular levels of Penile erection is regulated by the relaxation of cGMP, resulting in relaxation of the corporal smooth arteriolar and trabecular smooth muscle cells which muscle, dilatation of the penile arteries, arterioles it is mediated by the nitric oxide (NO)–cyclic and sinusoids, and finally, increased arterial inflow guanosine monophosphate (cGMP) pathway. Nitric and passive compression of the penile venous oxide is a nonadrenergic–noncholinergic neuro- outflow.4,5 Soluble guanylate cyclase is a hetero- transmitter that is released from postgangliomic dimer composed of a large (a1) and a small (b1) parasympathetic (cholinergic) nerves in the penis subunits and both subunits are required to produce and NO is also released from the endothelium.1–3 cGMP.6 Cyclic guanosine monophosphate is catabo- Both neuronal NOS (nNOS) isoform and the lized by phosphodiesterase type-V (PDE-V) that it endothelial NOS (eNOS) participate in cavernosal has been identified in human cavernous tissue and relaxation. plays a relevant role in penile erection. However, it Independently of the NO generating systems, a is now known that other tissues expressed PDE-V.7 reduction in the NO-induced vasorelaxation could Leukocytes also produce NO by an eNOS-like also results from impaired downstream signaling NOS and contain sGC.8,9 However, despite of in the vascular smooth muscle cells. Nitric oxide leukocytes are more accessible cells than the vascular cells contained in the corpus cavernosum, the ability of the leukocytes to express eNOS, sGC and PDE-V in patients with erectile dysfunction (ED) Correspondence: Dr AJ Lo´pez-Farre´, Cardiovascular has not been explored. Therefore, the aim of the Research Unit, 7a Norte, Hospital Clı´nico San Carlos, Profesor Martı´n Lagos s/n, Madrid 28040, Spain. present study was to test if mononuclear cells from E-mail: [email protected] patients with ED of vascular and neurological origin Received 26 July 2005; revised 5 January 2006; accepted have modified the level of expression of either 23 January 2006; published online 9 March 2006 eNOS, sGC and PDE-V. Mononuclear cells and erectile dysfunction PJ Mateos-Ca´ceres et al 433 Materials and methods polyacrylamide gels. Equal amounts of proteins (10 mg per lane) estimated by bicinchonic acid Study protocol reagent (Pierce Rockford, IL, USA) were loaded. Male ED patients ranging in age from 33 to 81 years Western blot analysis was performed with a mono- (mean 55.4) were enrolled in the study. All patients clonal antibody against eNOS (1:1500 Transduction were initially evaluated by taking a detailed sexual Laboratories, Lexington, UK), sGC b1-subunit and medical history and diagnosed of ED as (1:1500 Alexis Laboratories, San Diego, CA, USA) evaluated by the IIEF standardized questionnaire.10 or PDE-V (1:2500 Calbiochem, La Jolla, CA, USA). Six patients did not achieve a full rigid erection, Specific eNOS, sGC b-subunit and PDE-V proteins three patients showed a short-length full erection were detected by enhanced chemoluminiscense and nine patients showed lack of erection. Patients (ECL, Amersham Corp) and evaluated by densito- were divided into two categories according to the metry (Molecular Dynamics). Prestained protein echo-Doppler analysis after intravenous prostaglan- markers (Sigma) were used for molecular mass din E1 infusion. The vascular ED group included determinations. patients (n ¼ 9; mean age 55.6 years) that showed alterations to prostaglandin E1, a vasodilator, during the echo-Doppler examination based on the peak Determination of soluble guanylate cyclase activity systolic velocity and resistance index. In this group, 5 Â 106 mononuclear cells per tube were incubated the patients also showed previous history of vascu- at 371C in RPMI medium containing 0.025% bovine lar risk factors. In this regard, one patient had serum albumin for 15 min. To investigate the history of hypertension, two had hyperlipemia, two functional activity of sGC, mononuclear cells were had ischemic heart disease associated with hyper- incubated in the presence and in the absence of a lipemia and four patients had not vascular risk NO-donor, sodium nitroprusside (10À5 mol/l; SNP) factors. and a PDE inhibitor, 3-isobutil-1-methylxanthine The second group was the neurological ED group (10À4 mol/l; IBMX) that prevent cGMP breakdown. that included patients (n ¼ 9; mean age 56.6 years) Afterwards, mononuclear cells were lysed in 1 mol/l with ED of neurological origin each of whom ClH in ethanol. After having been mixed, the achieved a full rigid erection after prostaglandin E1 homogenates were centrifuged (12 000 g, 15 min at injection. This group included two patients with 41C) and the supernatants were recovered and dried radical prostatectomy, one patient with cortical by speed-vac. Cyclic guanosine monophosphate was atrophy, two patients with diabetes and four measured in acetylated samples with a kit from patients with peripheral neuropathy. Patients taking Amersham International as reported.14 PDE-V inhibitors in the 6 months previous to the study were excluded. Fourteen age-matched (mean age 54.7 years) with normal erectile function were Statistical analysis used as reference for control values. These age- Results are expressed as mean7s.e.m. Comparisons matched patients were going to be operated of were performed by ANOVA. Bonferroni’s correction Cryptorchidism, Varicocele and Hydrocele. All sub- for multiple comparisons was used to determine the jects gave fully informed consent and the study was level of significance of the P-value. Po0.05 was approved by the local Ethic Committee. The study considered significant. was blind for the investigators that determined the molecular parameters and analyzed the results. Blood samples were drawn from a peripheral vein. Mononuclear cells were isolated by Results Ficoll-Hypaque centrifugation and the layer con- taining leukocytes and lymphocytes was removed Endothelial nitric oxide synthase expression and immediately frozen at À801C for molecular in mononuclear cells determinations. A significant reduction in the level of eNOS protein expressed in mononuclear cells obtained from patients with ED was observed compared with those from age-matched controls (Figure 1). Determination of endothelial nitric oxide synthase, When ED patients were divided as ED of vascular soluble guanylate cyclase b1 subunit and and neurological origin, only mononuclear cells phosphodiesterase type-V from the vascular ED patients showed a significant The expression of eNOS, sGC b1-subunit and reduction of the eNOS protein level compared PDE-V proteins was analyzed by Western blot with those from age-matched controls (Figure 2). as described.11,12 In brief, mononuclear cells were Endothelial nitric oxide synthase expression resuspended and solubilized in Laemmli buffer also tended to be reduced in the mononuclear cells containing 2-mercaptoethanol.13 The proteins from patients with ED of neurological origin obtained were separated in denaturing SDS/10% although it did not reach statistical significance International Journal of Impotence Research Mononuclear cells and erectile dysfunction PJ Mateos-Ca´ceres et al 434 100 300 Control Control E.D. E.D. 250 80 * * 200 60 150 40 100 sGC arbitrary units (a.u.) eNOS arbitrary units (a.u.) 20 50 0 0 Figure 1 Bar graph showing the densitometric analysis of the Figure 3 Bar graph showing the densitometric analysis of the Western blots that detected the expression of the endothelial Western blots