Gender-specific immunological effects of the phosphodiesterase 5 inhibitor sildenafil in healthy mice

Molecular Immunology 56 (2013) 649–659

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Molecular Immunology journal homepage: www.elsevier.com/locate/molimm

Gender-specific immunological effects of the phosphodiesterase 5 inhibitor sildenafil in healthy mice Svetlana Karakhanova a,1 , Yuhui Yang a,b,1 , Julia Link a , Sabine Soltek a , Katharina von Ahn a , Viktor Umansky c,d , Jens Werner a , Alexandr V. Bazhin a,∗ a

Department of General Surgery, University of Heidelberg, Heidelberg, Germany Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China c Skin Cancer Unit, German Cancer Research Center (DKFZ), Heidelberg, Germany d Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, University of Heidelberg, Mannheim, Germany b

a r t i c l e

i n f o

Article history: Received 29 April 2013 Received in revised form 17 June 2013 Accepted 27 June 2013 Available online 2 August 2013 Keywords: cGMP Gender-specific effects Immune cells Immunomodulation Phosphodiesterase 5 Sildenafil

a b s t r a c t Phosphodiesterase 5 (PDE5) is a pharmacological target in erectile dysfunction, pulmonary hypertension and in other indications. In tumor-bearing mice an inhibition of PDE5 with sildenafil prolongs survival of the animals through the augmentation of antitumor immunity, indicating the immunomodulatory properties of this drug. Effects of sildenafil on the immune system in healthy organisms are poorly investigated. In this work we showed that chronic application of sildenafil in healthy mice leads to opposite gender-dependent effects on NK cells, subpopulations of CD4+ and CD8+ T cells, activated conventional T cells, and to a decrease in Gr-1+ CD11b+ immature myeloid cells. Besides, sildenafil treatment decreases the serum concentration of interleukin-6. Ex vivo cultivation of isolated splenocytes with sildenafil results in an increase in CD4+ T cells and a concomitant decrease in B cells and central memory CD8+ T cells. Ex vivo modulatory properties of sildenafil are not gender-specific, indicating the importance of sildenafil’s pharmacokinetics for it immunomodulatory activity in vivo. While the PDE5 expression is equal in the splenocytes from both genders, splenocytes from female mice possess higher basal level of cGMP compared to the male ones. Moreover, cultivation of splenocytes obtained from female but not male mice with sildenafil leads to an increase in cGMP concentration, making sildenafil’s pharmacodynamics also responsible for gender-specific effects of the drug. Thus, this work secures conclusive evidence that the PDE5 inhibitor sildenafil possesses immunomodulatory properties and these effects are gender-specific. Immunological clinical trials are needed to prove the potential immunomodulatory effects of sildenafil in humans. © 2013 Elsevier Ltd. All rights reserved.

1. Introduction Cyclic guanosine monophosphate (cGMP) was one of the first molecules described as the cellular second messenger (Sutherland and Rall, 1958). Its intracellular concentration was shown to be regulated through the cGMP synthesis by guanylyl cyclases (GC, EC 4.6.1.2) and the degradation via dual-specific or cGMPspecific phosphodiesterases (PDE, EC 3.1.4.17). PDE represent a family of eleven enzymes (PDE1-PDE11) that specifically hydrolyze

Abbreviations: cGMP, cyclic guanosine monophosphate; FACS, fluorescence activated cell sorting; GC, guanylyl cyclases; IL, interleukin; mAbs, monoclonal antibodies; NK cells, nature killer cells; NO, nitric oxide; PDE5, phosphodiesterase 5; TCR, T cell receptor. ∗ Corresponding author at: University Hospital Heidelberg, Im Neuenheimer Feld 430, 69120 Heidelberg, Germany. Tel.: +49 6221 5636932; fax: +49 6221 568240. E-mail address: [email protected] (A.V. Bazhin). 1 Equal contribution. 0161-5890/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.molimm.2013.06.021

cyclic nucleotides and can be classified according to their tissue expression, biochemical properties, regulation, and the sensitivity to various pharmacological agents (Francis et al., 2010). PDE-5 (together with PDE6 and PDE9) belongs to the group of cGMPspecific PDEs, with two ubiquitous isoforms, PDE5A1 and PDE5A2, while PDE-5A3 is specific for smooth muscle cells (Omori and Kotera, 2007). PDE5 is a pharmacological target in erectile dysfunction and pulmonary hypertension (Boswell-Smith et al., 2006; Sanchez Luna et al., 2012). There is a large body of evidence demonstrating that inhibition of PDE5 can also be important for other indications: cognition (Schmidt, 2010) and stroke (Zhang et al., 2005), cardiovascular diseases (Raja, 2006), inhibition of tumor cell growth (Sarfati et al., 2003) and other disorders, in which the benefit of PDE5 inhibition is not easy to explain. Therefore, clinical usage of PDE5 suppression makes the development of PDE5 inhibitors very attractive for clinic and pharmacologic industry. At present, sildenafil is the first-line therapeutic for erectile dysfunction (Corbin

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et al., 2002) and is newly approved for the treatment of pulmonary hypertension. Recently, we and another group have showed that chronic application of sildenafil to tumor-bearing mice prolonged survival of the animals through the augmentation of anti-tumor immunity (Serafini et al., 2006; Meyer et al., 2011). This unexpected effect was due to an inhibition of myeloid-derived suppressor cell (MDSC) function and, as a consequence, to a partial restoration of the T cell receptor (TCR) ␨-chain expression in T cells and of their proliferation (Serafini et al., 2006; Meyer et al., 2011). It was concluded that the PDE5 inhibitor sildenafil possesses certain immunomodulatory properties in tumor-bearing mice. Taking in account the potential immunoregulatory property of sildenafil in tumor-bearing mice, it is important to know whether this drug can induce immunological changes in healthy organisms as well, since sildenafil is a very common medicament, which sales peaked at about 2 million US dollars in 2008. So far, there are only two investigations dealing with effects of PDE inhibitors in healthy mice: Szczypka and Obminska-Mrukowicz (2010a) showed that sildenafil increased the production of interleukin (IL)-1␤ and nitric oxide (NO) by peritoneal macrophages ex vivo, increased the percentage of phagocytosing granulocytes and decreased the percentage of phagocytosing monocytes. In the second work of Szczypka and Obminska-Mrukowicz (2010b), it was demonstrated that sildenafil decreased percentage of B cells while increased percentage of T cells (CD3+ , CD4+ and CD8+ cells) ex vivo. Nevertheless, sildenanfil’s effects on specific lymphocyte subpopulations and other leukocyte subsets were not assessed, especially in vivo. Therefore, the aim of this work was to investigate immunological effects of sildenafil both in vivo and ex vivo. Results of our work present conclusive evidence that this PDE5 inhibitor indeed possesses immunomodulatory properties and these effects are gender-specific. 2. Materials and methods 2.1. Materials The following anti-mouse directly conjugated monoclonal antibodies (mAbs) were used for the fluorescence activated cell sorting (FACS) staining: rat or hamster anti-mouse antibodies CD4-Pacific Blue, CD3e-V500, CD45RB-PE, CD8a-PerCP-Cy5.5, CD62L-APC, CD45R-Pacific Blue, CD80-FITC, I-A[b]-PE, CD11b-PerCP-Cy5.5, CD11c-APC, CD86-PE-Cy7, Gr-1-APC-Cy7, NK1.1-PE, CD69-PerCPCy5.5, and CD25-APC (BD Bioscience, Germany). Fc receptor block (anti-mouse CD16/CD32), and Foxp3-FITC were purchased from eBioscience (Germany). CD19-PE was from Immunotools (Germany). Sildenafil citrate was obtained from Sigma–Aldrich (Germany). Revatio® 20 mg tablets (contained sildenafil) were purchase by Pfizer (Germany). 2.2. Mice C57BL/6 mice (6–8 weeks) were purchased from Charles River (Germany) and kept under specific pathogen-free condition in the animal facility of University Heidelberg (IBF, Heidelberg). Animal experiments were carried out after approval by the authorities (Regierungspraesidium Karlsruhe). 2.3. Sildenafil treatment of mice Drinking water for C57BL/6 mice was supplemented with sildenafil (compounded from pulverized Revatio® tablets) to yield a dose of 20 mg/kg body weight per day. The mice were sacrificed 21

days later and their spleens were removed to prepare a splenocyte single cell suspension as described elsewhere (Yang et al., 2012). 2.4. FACS analysis of murine splenocytes Freshly isolated spleen cell suspension was resuspended in the stain buffer (PBS and 1 mM EDTA). The cells were counted and the density was adjusted to 4 × 107 /mL. The unspecific binding caused by the Fc receptors was blocked by incubating the cells with antimouse CD16/CD32 antibody (1 ␮L for 2 × 106 cells) at 4 ◦ C in the dark for 10 min. Then 50 ␮L cell suspension (2 × 106 cells) was incubated with 50 ␮L of the stain buffer containing various mAbs at 4 ◦ C in the dark for 15 min. The amount of each antibody had been titrated before the experiment. After two-time washing with the stain buffer, the cells were resuspended in 100 ␮L of the stain buffer and transferred to a 5 ml polystyrene tube containing 300 ␮L of the stain buffer for FACS analysis. Foxp3 staining buffer set was used for intracellular staining, according to the manufacturer’s instruction. The following panels with the different combinations of antibodies were used in our experiments: (1) panel for CD4+ T and CD8+ T cells. Different subsets of CD4+ T cells were characterized as naïve T cells (CD62L+ CD45RBhi ), effector T cells (CD62L− CD45RBhi ), central memory T cells (Tcm , CD62L+ CD45RBlo ) and effector memory T cells (Tem , CD62L− CD45RBlo ); the corresponding subsets of CD8+ T cells were naïve T cells (CD62L+ CD44− ), effector T cells (CD62L− CD44− ), central memory T cells (Tcm , CD62L+ CD44+ ), effector memory T cells (Tem , CD62L− CD44+ ); (2) panel for Treg/NK/NKT cells. Treg cells were characterized as Foxp3+ CD25+ within the CD3+ CD4+ T cell population. NK cells (CD3− NK1.1+ ) and NKT cells (CD3+ NK1.1+ ) were identified, and their activation state was measured by the expression of CD69; (3) panel for dendritic cells. Mature conventional dendritic cells (cDC, CD11chi CD11b+ ) and mature plasmacytoid dendritic cells (pDC, CD11cint CD45R+ ) were further characterized by the high expression of MHC-II molecules (I-Ab ). The activation state of both DC subsets were measured by the expression of CD80 (B7-1) and CD86 (B7-2); (4) Panel for granulocyte/macrophage/monocyte. Here CD11b+ Gr-1+ cells were analyzed. All the gates were set according to the corresponding fluorescence minus one (FMO) control. 2.5. In vitro treatment of murine splenocytes with sildenafil citrate After the preparation of splenocyte single-cell suspension, cells were seeded in the 6-well plate at the density of 106 cells/mL. Then the cells were treated with different concentrations of sildenafil citrate (75 nM, 750 nM and 7.5 ␮M) or vehicle control in 5% CO2 at 37 ◦ C for 24 h. Afterwards all the cells, including the adherent cells, were recovered for FACS analysis, or for RNA isolation, or cGMP measurement. 2.6. RNA isolation and real-time RT-PCR analysis Total RNA from splenocytes was isolated using RNeasy mini kit (Qiagen, Germany) according to the manufacturer’s instructions. RNA concentrations were determined using a NanoPhotometerTM Pearl (SERVA Electrophoresis, Germany). Real-time RT-PCR analysis was done as described elsewhere (Bazhin et al., 2008), using the SYBR-Green system and primers for PDE5a, ˇ-actin and GAPDH provided by Qiagen (Germany), and measured using a Light-Cycler (Roche, Germany). For each experiment, a melting curve analysis and a gel electrophoresis of PCR products were performed to exclude primer dimers. Data were analyzed using the comparative Ct method (Schmittgen and Livak, 2008). Measurements were performed in triplicate.

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2.7. cGMP measurement cGMP was measured with the DetectX® Cyclic GMP Colorimetric EIA Kit from ArborAssays (USA) in a regular format as described by the manufacturer. Briefly, harvested cells (about 107 cells) were lysed with the provided diluent. 50 ␮L of the lysates or standards (provided with the kit) were added into the IgG pre-coated wells. After incubation with different kit reagents at room temperature, the plates were read on a plate reader. Standard concentrations of cGMP (from 0.5 pmol/mL to 32 pmol/mL) were simultaneously proceeded to estimate cGMP concentration in the probes of interest. Each sample was measured in duplicate, and the final concentration of cGMP normalized on cell numbers was determined. 2.8. Analysis of cytokines with a luminex assay Analysis of IL-2, IL-1␤, IL-6, IL-10 and VEGF was performed as described elsewhere (Shevchenko et al., 2012) using MILLIPLEX® MAP Kit (Millipore GmbH, Schwalbach/TS, Germany). Briefly, 25 ␮L serum were incubated with color-coded beads coated with the capture antibodies for the respective cytokines overnight at 4 ◦ C. After washing, the beads were incubated with biotinylated secondary detection antibodies for each cytokine for 1 h at room temperature, followed by the incubation with streptavidin-phycoerythrin for 30 min at room temperature. Finally, measurement using Luminex® 100/200 System was performed. According to the standard curves, the concentration of the respective cytokines was calculated and presented in pg/mL. 2.9. Statistical analysis All statistical analyses were performed using GraphPad Prism Version 5.01. Distributions of continuous variables were described

by means, SE, median, 25% and 75% percentiles, and were presented as box-and-whiskers plots or as column bar graphs. D’Agostino and Pearson omnibus normality tests were conducted to estimate the distribution of data. The null hypothesis (mean values were equal) versus the alternative hypothesis (mean values were not equal) was tested for more than two groups by one-way ANOVA with the Dannett’s post hoc test, and for two groups by unpaired two-tailed t-test for normal distributed variants or by Mann–Whitney test for nonparametric distributed data.

3. Results 3.1. Different in vivo effects of sildenafil on immune cell populations in healthy male and female mice To investigate the immunological effects of sildenafil, we performed an immunophenotyping of splenocytes from healthy mice by FACS analysis. In these experiments, healthy wild-type mice were treated with 20 mg/kg of sildenafil per day for 3 weeks. Upon the treatment, mice were visibly neither physiologically nor behaviourally affected. In addition, mice did not show any splenomegaly or microsplenia. The percentage of T cells, DC, Gr1+ CD11b+ leukocytes, NK and NKT cells were analyzed. The gating strategy for leukocytes is shown in Fig. S1. First, the percentage of T cells and their memory/naïve state were accessed. In the whole population of our experimental mice including equal proportion of male and female animals, we did not find any difference in the percentage of T cells or in their memory/naïve state between treated and control mice (Fig. S2). Surprisingly, analyzing our data based on the mouse gender, we observed some different effects of sildenafil in male and female mice (Figs. 1 and 2). Whereat, sildenafil treatment has a tendency

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Fig. 6. MNC and leukocyte subpopulations after in vitro cultivation with sildenafil of splenocytes obtained from all (A), male and female (B) mice, and (C) CD8+ Tcm -cells in all animals and in both genders. Data from two independent experiments are presented as column bar graphs (n = 8 for (A, C), and n = 4 for (B, C)), *p < 0.05, **p < 0.01 and ***p < 0.001; control group vs. treatment group.

to decrease the percentage of CD4+ T cells and to increase the percentage of CD8+ T cells in male but not in female mice (Figs. 1 and 2). Concerning the memory/naïve state of T cells, we found opposite effects of sildenafil on CD4+ T cell subpopulations in males and females, i.e. a decrease in percentage of Tem and Tcm in males while an increase in percentage of these cells in females (Fig. 1). Besides, we observed an increase in percentage of naïve CD4+ T cells after the treatment in male mice. In the subpopulations of CD8+ T cells merely a decrease in percentage of the CD8+ Tcm cells was found in males (Fig. 2). With regard to Treg, conventional T cells (Tcon), activated Tcon, NK and NKT cells, sildenafil treatment showed no effect on these cell populations in the whole gender-mixed population (Fig. S3). Gender-specific analysis of the data revealed different effects of sildenafil on activated Tcon and NK cells: there was a decrease in the male population while an increase in female mice (Fig. 3). However, there was still no effect of sildenafil either on Tcon, Treg or NKT cells (Fig. 3). Treatment with sildenafil did not affect the percentage of DC in the whole mouse population or in gender-specific one (Fig. S4). Moreover, the treatment did not change also the maturation and activation state of these cells irrespective of the mouse gender (Figs. S5 and S6). Since sildenafil can abolish immunosuppressive effects of Gr1+ CD11b+ immature myeloid cells in tumor-bearing mice (Meyer et al., 2011) we wanted to investigate this subpopulation in healthy mice after sildenafil treatment. In the whole population of animals sildenafil did not affect the percentage of these cells (Fig. 4A). However, such treatment led to a decrease in percentage of Gr1+ CD11b+ cells in the spleen of female but not of male mice (Fig. 4B). Next we performed an analysis of different factors (IL-2, IL-6, IL10, IL-1␤ and VEGF) in serum of the experimental animals (Fig. 5). Sildenafil diminished the IL-6 level both in the female group and in

the whole population of treated mice but not in male mice separately analyzed (Fig. 5). Furthermore, we also found a tendency of an increase in IL-2 in serum of female but not of male mice after sildenafil treatment (Fig. 5). No influence of sildenafil on serum levels of IL-10, IL-1␤ or VEGF was observed (Fig. 5). Thus, sildenafil has some immunomodulatory properties in healthy mice, which are gender-specific. This sex-related effects of sildenafil could be manifested due to the gender-dependent differences in pharmacokinetics (resorption, distribution, metabolism and excretion of sildenafil) or in pharmacodynamics (biochemical effects of sildenafil).

3.2. Direct effects of sildenafil on splenocytes ex vivo Next, we aimed to understand whether pharmacokinetics, pharmacodynamics or both are responsible for such gender-dependent effects of sildenafil. For this purpose, we isolated splenocytes from healthy mice (male and female), cultivated them ex vivo for 24 h with different concentrations of sildenafil (75 nM, 750 nM and 7.5 ␮M) and finally performed a FACS analysis. We found that sildenafil induced no cytotoxicity of mononuclear cells (MNC) (Fig. 6A). This drug did not affect CD8+ T cells, but at the highest concentration (7.5 ␮M), increased the percentage of CD4+ T cells and diminished the amount of B cells (Fig. 6A). These effects were not gender-specific, since the splenocytes from both genders - male and female manifested the same features after sildenafil treatment (Fig. 6B). In respect to the naïve/memory phenotype of T lymphocytes, we found that sildenafil at 7.5 ␮M concentration reduced the amount of CD8+ Tcm cells both in the whole population, and separately in male or female mice (Fig. 6C). Sildenafil did not affect other subpopulations of CD8+ T-cells and subpopulations of CD4+ T-cells (Fig.

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Fig. 7. Relative expression of PDE5a (A) and basal cGMP level (B) in splenocytes obtained from male and female mice, and effects of sildenafil on basal cGMP expression in different genders (C). Data from two independent experiments are presented as box-and-whiskers plots or as column bar graphs (n = 5 for A and n = 8 for B and C), *p < 0.05 and ***p < 0.001; control group vs. treatment group.

S7). We also found no ex vivo effects of sildenafil on Tcon, activated Tcon, Treg, NK and NKT cells (Fig. S7). Since the ex vivo effects of sildenafil are not gender-dependent we assumed that the in vivo differences of sildenafil could be due to different pharmacokinetics properties of sildenafil in male and female animals. 3.3. Gender-specific effects of sildenafil on the basal cGMP level in splenocytes of healthy mice In the last part of the present work we dealt with pharmacodynamics effects of sildenafil, i.e. with the biochemical features of this drug. Since sildenafil is a specific inhibitor of PDE5, which hydrolyzes cGMP, this should lead to a reduced level of this cellular secondary messenger. One possibility could be that murine immune cells have different PDE5 expression levels in male and female animals. To prove this hypothesis, we performed a real-time PCR analysis of the PDE5a gene expression in isolated splenocytes from male and female mice. The level of PDE5a expression in splenocytes was equal in both genders (Fig. 7A). Thus, we could exclude the difference in the PDE5a expression level in the murine splenocytes as a reason for the observed gender-specific effects. Another possibility could be that different genders have various basal level of cGMP that would affect biochemical effects of sildenafil. We measured the basal cGMP level in murine splenocytes and found that male mice have lower level of cGMP as compared to female animals (Fig. 7B), suggesting that

sildenafil might have different effects in different genders because of the various basal level of cGMP. To prove this assumption, we isolated splenocytes from male and female mice, cultivated them with different concentrations of sildenafil (75 nM, 750 nM and 7.5 ␮M) and finally measured the cellular cGMP level. Interestingly, we found an increase in cGMP level (because of PDE5 inhibition) only in splenocytes isolated from female mice as compared to untreated group (Fig. 7C). Thus, pharmacodynamics of sildenafil could also be responsible for different effect of the drug in different genders.

4. Discussion Taking in the account the immunomodulatory properties of sildenafil in tumor-bearing mice and the high frequency of sildenafil prescription by medicines, the main aim of this work was to investigate immunological effects of sildenafil in healthy mice. Our date demonstrated that sildenafil has immunomodulatory effects in vivo. These effects are (i) different in male and female mice, and (ii) the gender-dependent differences are due to the pharmacokinetics and pharmacodynamics of this drug. We showed in vivo that sildenafil possessed a modest immunosuppressive effect in male mice reflected by a decrease in frequencies of activated Tcon and NK cells. On the contrary, in female mice we observed an immunostimulatory effect of sildenafil represented by an increase in frequencies of activated

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Tcon, NK cells and an elevated IL-2 levels in serum, while the frequency of Gr-1+ CD11b+ immature myeloid cells (as a counterpart of myeloid-derived suppressor cells in tumor-bearing hosts (Gabrilovich and Nagaraj, 2009)) and the IL-6 concentration in serum were decreased. Moreover, the balance between naïve and memory subpopulations of CD4+ T cells was differently affected by sildenafil in male and female mice. While we observed a decrease in percentage of CD4+ Tem and CD4+ Tcm cells in males, these sildenafil effects were opposite in females. Since we did not observe such effects of sildenafil in ex vivo experiments, we could exclude its direct effect on the expression of subpopulation markers used in our panels (except CD62L and CD44 as markers for CD8+ Tcm ). Thus, we conclude that sildenafil indeed shifts the balance between immune stimulatory and suppressive cell, and influences the distribution of subpopulations in the memory T cell compartment in healthy mice. In general, differences in immunological parameters between the genders are already known (Fish, 2008) and could be explained by endocrine and genetic differences between males and females (for review see (Klein, 2012)). The endocrine influence on immune system can be illustrated by the fact that testosterone reduces the NK cell activity (Hou and Zheng, 1988) and production of proinflammatory cytokines (Rettew et al., 2008), increasing the synthesis of anti-inflammatory cytokines (D’Agostino et al., 1999). On the other hand, estrogens regulate differentiation of DC and other antigenpresenting cells (Paharkova-Vatchkova et al., 2004; Escribese et al., 2008). In addition, it has also been proposed that estrogens could affect the immunosuppressive function of Treg under partial control of the immune regulatory molecule B7-H1 (Lin et al., 2010). From the genetic point of view, some genes on the X chromosome play a role in functions of the immune system (for review see (Libert et al., 2010)). Therefore, we as well can say that “immune cells have a sex” (Klein, 2012) that could be manifested in different gender-mediated effects of some drugs on immune cells. It has been shown that melatonin possesses sex-dependent effects on the development of systemic lupus erythematosus (JimenezCaliani et al., 2006). Thus, our gender-specific effects of sildenafil are in line with the above-mentioned publications. Based on our data, we concluded that the mechanisms underling the different effects of sildenafil are represented by the pharmacokinetics and pharmacodynamics of the drug. From the clinical pharmacology it is known that anti-hypertensive drugs have sexrelated effects due to their gender specific pharmacokinetics and pharmacodynamics (Ueno and Sato, 2012). In general, pharmacokinetics depends on the gender-differences in expression of gene coding for hepatic drug metabolizing enzymes (i.e. cytochrome P450, glutathione transferase etc.) (Scandlyn et al., 2008; Waxman and Holloway, 2009). Sildenafil is cleared predominantly in hepatic microsomes by CYP3A4 (Hyland et al., 2001). At the same time, human CYP3A4 displayed higher expression at the mRNA and protein levels in women than in men (Hunt et al., 1992). Therefore, men and women should have also different bioavailability of the drug, and consequently, they can have a different reaction to sildenafil. In line with this suggestion, ex vivo experiments showed no differences in effects of sildenafil in male and female splenocytes, underlying the importance of the pharmacokinetics in the sex-dependent variability of these effects in healthy mice. However, we could not exclude that sildenafil may interfere with all steps of cGMP metabolism, manifesting in the difference of cGMP levels from male and female splenocytes. Indeed, we showed that splenocytes from female mice possess higher basal cGMP level than the male ones. It is tempting to speculate that not only PDE5, inhibited by sildenafil, is important for cGMP level in splenocytes, but also the anabolic pathway of cGMP metabolism, namely GC could also have an influence on the cGMP level what in turn would affect the reaction to sildenafil.

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