Soluble Guanylate Cyclase B1-Subunit Expression Is Increased in Mononuclear Cells from Patients with Erectile Dysfunction

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ORIGINAL ARTICLE Soluble guanylate cyclase b1-subunit expression is increased in mononuclear cells from patients with erectile dysfunction

PJ Mateos-Caćeres1, J Garcia-Cardoso2, L Lapuente1, JJ Zamorano-Leoń1, D Sacristań1, TP de Prada1, J Calahorra2, C Macaya1, R Vela-Navarrete2 and AJ Lo´pez-Farre´1

1Cardiovascular Research Unit, Cardiovascular Institute, Hospital Clıńico San Carlos, Madrid, Spain and 2Urology Department, Fundacioń Jimeńez 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 Clıńico San Carlos, Profesor Martıń 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-Caćeres 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 containing 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 that detected the expression of the sGC b1-subunit in mononuclear cells obtained from age-matched controls and nitric oxide synthase protein in circulating mononuclear cells 7 obtained from age-matched controls and patients with erectile erectile dysfunction patients. Results are mean s.e.m. *Po0.05 with respect to control. dysfunction. Results are represented as mean7s.e.m. *Po0.05 with respect to control.

E.D. Control Vasc Neu E.D. 70 kDa sGC Control Vasc Neu

140 kDa eNOS 42 kDa β-Actin

250 * 42 kDa β-Actin *

100 200

80 150 Control Vascular Neurological 60 * 100 Control Vascular 40 Neurological 50 sGC arbitrary units (a.u.)

20 0 eNOS arbitrary units (a.u.) Figure 4 Representative Western blot demonstrating sGC 0 b1-subunit expression in mononuclear cells obtained from age- matched controls and from patients with erectile dysfunction of Figure 2 Representative Western blot showing the expression of vascular and neurological origin. At the bottom is shown the endothelial nitric oxide synthase protein in mononuclear cells densitometric analysis of the Western blots. Results are shown as from age-matched control and from patients with erectile mean7s.e.m. *Po0.05 with respect to control. dysfunction of vascular and neurological origin. The expression of b-actin is also showed. At the bottom is shown the densitometric analysis of the Western blots. Results are shown as mean7s.e.m. *Po0.05 with respect to control. Soluble guanylate cyclase b1-subunit and phosphodiesterase type-V in mononuclear cells Western blot analysis demonstrated a significant (Figure 2). No changes in b-actin expression, a increase in the expression of sGC b1-subunit protein constitutive protein used as control, were observed in mononuclear cells from patients with ED (Fig- in the mononuclear cells from control, vascular and ure 3). The level of sGC-b1 subunit expressed in neurological ED patients supporting the specificity mononuclear cells obtained from patients with ED of the modifications observed in the eNOS expres- of neurological and vascular origin appeared to be sion (Figure 2). very similar (Figure 4).

International Journal of Impotence Research Mononuclear cells and erectile dysfunction PJ Mateos-Ca´ceres et al 435 Control Table 1 cGMP production by mononuclear cells from patients with erectile dysfunction of vascular and neurological origin 250 E.D. 6 * cGMP (fmol/5 Â 10 cells)

200 Control Vascular Neurological

Basal 19.573 16.473.1 10.173.2*,** , 150 SNP 169.1721.1 97.776* 67.8710.4* ** SNP þ IBMX 227.4724.1 157.7714.2* 105.1716.5*,**

7 100 Results are represented as mean s.e.m. *Po0.05 with respect to age-matched control. **Po0.05 with respect to mononuclear cells from erectile dysfunction patients of vascular origin. Abbreviations: cGMP, cyclic guanosine monophosphate; SNP,

PDE-V arbitrary units (a.u.) 50 sodium nitroprusside; IBMX, 3-isobutil-1-methylxanthine.

0 origin was very similar to that observed in the Figure 5 Bar graph showing the densitometric analysis of the mononuclear cells from age-matched controls (Fig- Western blots that detected the expression of the phosphodiester- ure 6). ase type-V protein in circulating mononuclear cells obtained from age-matched controls and patients with erectile dysfunction Results are represented as mean7s.e.m. *Po0.05 with respect to control. Cyclic guanosine monophosphate levels in mononuclear cells Basal cGMP levels in mononuclear cells from ED E.D. patients were found reduced compared with those Control Vasc Neu from age-matched controls (control: 19.573; ED: 7 6 100 kDa PDE-V 13.5 2 fmol/5 Â 10 cells; Po0.05). The basal cGMP levels were reduced in mononuclear cells from patients with ED of neurological origin (Table 1). 400 Control Mononuclear cells from ED patients of vascular * origin also showed a reduced basal cGMP levels Vascular compared with control although it did not reach Neurological statistical signification (Table 1). Stimulation of 300 mononuclear cells with the NO-donor, SNP (10À5 mol/l) increased cGMP levels in mononuclear cells from both controls and ED patients of vascular and neurological origin (Table 1). However, the 200 cGMP levels observed in SNP (10À5 mol/l)-stimulated mononuclear cells from patients of neurological origin were lower than that observed in 10À5 mol/l SNP-stimulated mononuclear cells from PDE-V arbitrary units (a.u.) 100 ED patients of vascular origin (Table 1). We further analyzed the activity of sGC by stimulating sGC with SNP and inhibiting PDE with IBMX (10À4 mol/l). As shown in Table 1, the 0 blockade of PDE by IBMX enhanced the activity of Figure 6 Representative Western blot demonstrating the expres- sGC induced by SNP in the mononuclear cells from sion of phosphodiesterase type-V in mononuclear cells from all the experimental groups although sGC activity age-matched control and patients with erectile dysfunction of remained higher in controls than in ED patients vascular and neurological origin. At the bottom is shown the densitometric analysis of the Western blots. Results are repre- (Table 1). In addition, cGMP levels in SNP þ IBMX- sented as mean7s.e.m. *Po0.05 with respect to control. incubated mononuclear cells was also higher in ED patients of vascular origin than those from neurological origin (Table 1). The expression of PDE-V was increased in the mononuclear cells from patients with ED (Figure 5). However, the expression of PDE-V was only in- Discussion creased in the patients with ED of vascular origin (Figure 6). The expression of PDE-V in the mono- Nitric oxide produced by the neuronal NOS isoform nuclear cells from patients with ED of neurological contained in the nerve fibers has been identified as

International Journal of Impotence Research Mononuclear cells and erectile dysfunction PJ Mateos-Ca´ceres et al 436 an important mediator of penile erection.15 Recent cGMP signaling in mouse thoracic aorta. Therefore, evidences have suggested that the eNOS isoform has the reduced expression of eNOS protein found in also a relevant role in penile erection.16 the mononuclear cells from ED patients could be Erectile dysfunction may causes endothelial dys- involved in the upregulation of the sGC b1-subunit. function. An important concept related to endo- Further studies are needed to explore it. thelial dysfunction is that it is a systemic disease. Soluble guanylate cyclase is an heterodimer For example, it has been demonstrated correlation composed of a large (a1) and a small b1 subunits between endothelial dysfunction in the coronary and both sGC subunits are required for the enzy- arteries and in the forearm circulation.17 Moreover, matic activity of sGC.6 Therefore, upregulation of 18 Foresta et al. have recently demonstrated that the the sGC b1-subunit may increase the ability of this number of circulating endothelial progenitor cells enzyme to produce cGMP. However, a reduced was reduced in ED patients as a sign of systemic cGMP production was found when mononuclear endothelial dysfunction. The activity of leukocytes cells from the patients with ED were stimulated with is increased by the endothelial dysfunction. Since the exogenous NO-donor, SNP. Initially, it seems to leukocytes contain the entire machinery of the NO/ be a paradoxical result since the upregulation of sGC cGMP system, we analyzed whether ED may be was accompanied with cGMP reduction. Therefore, reflected in modifications of the NO/cGMP system we determined the level of expression of PDE-V in in circulating leukocytes. the mononuclear cells. Western blot experiments Circulating mononuclear cells from patients with showed that mononuclear cells expressed PDE-V. ED of vascular origin showed a reduced of eNOS Previously, Tenor et al.25 and Sarfeti et al.26 have protein expression. However, the level of eNOS showed, by functional experiments, that lympho- protein expressed in mononuclear cells from cytes contain PDE-V activity. We showed here for the patients with ED of neurological origin was first time the expression of PDE-V by Western blot in not statistically modified, although tended to be human mononuclear cells. diminished with respect to that of age-matched Phosphodiesterase type-V expression was only control. found upregulated in mononuclear cells from The mechanisms underlying the downregulation patients with ED of vascular origin. Therefore, we of eNOS protein in mononuclear cells from ED determined the activity of sGC in the mononuclear patients of vascular origin were not established cells by stimulating sGC with SNP and blocking the in the present study. In vitro studies have shown cGMP catabolism with IBMX, an inhibitor of PDE that inflammation, and more particularly cytokines activities. These experiments showed that sGC are intimately involved in the downregulation activity was reduced in the mononuclear cells from of eNOS protein in both the endothelium and ED patients compared with control. Moreover, sGC leukocytes.19,20 In this regard, a recent review from activity was significatively lower in mononuclear Jeremy et al.21 and Giugliano et al.22 have suggested cells from ED patients of neurological origin than in a relationship between ED and inflammation. The those from vascular origin. Taken together, these expression of eNOS protein seem to be preserved findings suggest that, independently of eNOS in the mononuclear cells from the patients with protein expression, the reduction of cGMP produc- ED of neurological origin, which could be related tion in mononuclear cells from the ED patients of to a lower production of inflammatory mediators vascular origin may be related to both upregulation than in the patients with ED of vascular origin. of PDE-V and reduction in the sGC activity, whereas Another possibility may be that the level of in the patients of neurological origin may be other eNOS regulatory factors, such as vascular attributed to a decreased sGC activity. endothelial growth factor which it has been demon- As limitations of the study, the present experi- strated to restore ED,23 could be different between mental design does not allow us to determine the patients with ED of vascular and neurological origin. implication of the changes observed in the expres- Further studies are warranted to clarify these sion of enzymes involved in the NO-cGMP generat- hypotheses. ing system on the functionality of mononuclear cells Independently of the level of expression of the of ED patients. eNOS protein, the sGC, the main receptor for NO, In summary, the present work shows for the first was upregulated in mononuclear cells obtained time that, independently of the aetiological origin of from ED patients. The upregulation of the sGC b1- the ED, sGC b1-subunit is upregulated in circulating subunit in mononuclear cells was independent of mononuclear cells from patients with ED whereas the aetiological origin of the ED. PDE-V expression was only modified in the patients We have not determined the precise mechanisms with ED of vascular origin. A more reliable biomar- by which sGC may be upregulated in mononuclear ker associated with ED was the evaluation of the sGC cells from patients with ED. Indeed, it is not well b1-subunit than both eNOS and PDE-V expression. known the mediators involved in the regulation of Therefore, the better understanding of the regulatory the expression of sGC. In this regard, Hussain et al.24 mechanisms of sGC may lead to new strategies in the have showed that NO reduction upregulated sGC- prevention and treatment of ED.

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