Urothelium muscarinic activation phosphorylates CBS Ser227 via cGMP/PKG pathway causing human bladder relaxation through H 2 S production

The urothelium modulates detrusor activity through releasing factors whose nature has not been clearly defined. Here we have investigated the involvement of H2S as possible mediator released downstream following muscarinic (M) activation, by using human bladder and urothelial T24 cell line. Carbachol stimulation enhances H2S production and in turn cGMP in human urothelium or in T24 cells. This effect is reversed by cysthationine-β-synthase (CBS) inhibition. The blockade of M1 and M3 receptors reverses the increase in H2S production in human urothelium. In T24 cells, the blockade of M1 receptor significantly reduces carbachol-induced H2S production. In the functional studies, the urothelium removal from human bladder strips leads to an increase in carbachol-induced contraction that is mimicked by CBS inhibition. Instead, the CSE blockade does not significantly affect carbachol-induced contraction. The increase in H2S production and in turn of cGMP is driven by CBS-cGMP/PKG-dependent phosphorylation at Ser(227) following carbachol stimulation. The finding of the presence of this crosstalk between the cGMP/PKG and H2S pathway downstream to the M1/M3 receptor in the human urothelium further implies a key role for H2S in bladder physiopathology. Thus, the modulation of the H2S pathway can represent a feasible therapeutic target to develop drugs for bladder disorders

L-cysteine/hydrogen sulfide pathway: pharmacological approaches in urinary bladder and cardiovascular diseases

Background: Recently, several studies have demonstrated that phosphodiesterase type 5 inhibitors (PDE5-Is), drugs widely used as first-line oral treatment of erectile dysfunction (ED), are clinically effective in the treatment of lower urinary tract symptoms (LUTS) secondary to benign prostatic hyperplasia (BPH) and/or ED, although their mechanism of action is still unclear and under investigation. It has been suggested that PDE5-Is beneficial effects on storage symptoms are partially independent from nitric oxide signalling and they cause bladder detrusor relaxation through the involvement of intracellular cyclic nucleotides, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), KATP, BKCa and SKCa channels. Hydrogen sulfide (H2S) is a newly discovered gas-transmitter with myorelaxant properties. It is endogenously generated by cystathionine β- synthase (CBS) and cystathionine γ-lyase (CSE) from L-cysteine. Purpose I: To investigate whether H2S signalling pathway contributes to human bladder function and accounts for sildenafil relaxing effect on human detrusor muscle. Material and methods I: Full thickness bladder samples (detrusor plus urothelium) were obtained by prostatectomy from male patients aged between 61 and 73 years and affected with severe LUTS secondary to BPH. CBS and CSE expression was evaluated by Western blot. H2S production was measured in basal condition and after stimulation with L-cysteine, the endogenous substrate (10 mM), and in response to sildenafil (10 µM; 15, 30, 45 min; or 1, 3, 10, and 30 µM; 30 min), 8-bromo-cGMP (8-Br-cGMP, 100 µM; 30 min) or dibutyryl-cAMP (d-cAMP, 100 µM; 30 min) by using a colorimetric assay. On a stable contraction of bladder strips induced by carbachol, a curve concentration effect of sodium hydrosulfide (NaHS), a stable H2S donor in solution (0.1 µM to 1 mM), or L-cysteine (0.1 µM to 1 mM), or sildenafil (0.1 µM to 10 µM) was performed. To investigate H2S signalling in sildenafil effect, DL-propargylglicine (PPG, 10 mM) and/or aminooxyacetic acid (AOAA, 1 mM) were used as CSE and CBS inhibitors, respectively. Data were expressed as mean ± SEM. Statistical analysis was determined by using ANOVA followed by Bonferroni as a post hoc test. Differences were considered significant when p value was less than 0.05. Results I: Both CBS and CSE were expressed in the human bladder and efficiently converted L-cysteine into H2S. Both NaHS and L-cysteine caused a concentration-dependent relaxation of human bladder strips. Furthermore, sildenafil-induced a relaxation of bladder dome strips and a concentration dependent increase in H2S production. Both effects were significantly reduced by pretreatment of tissue with CBS and CSE inhibitors. Both 8-Br-cGMP and d-cAMP caused a significant increase in H2S production, similar to that induced by sildenafil. Conclusions I: Sildenafil induced relaxation of human bladder involves H2S signalling pathway. The increase in H2S levels triggered by cGMP and cAMP may account for the PDE5-Is beneficial effect in LUTS. Furthermore, it can be hypothesized that the stimulation of L-cysteine/H2S pathway is a common characteristic among all commercially available PDE5-Is. It is also well established that cGMP or cAMP activates protein kinase G (PKG) or protein kinase A (PKA) respectively, which in turn phosphorylates downstream proteins thereby triggering signal transduction. On the basis of the above results, it can be hypothesized that the increase in H2S production triggered by 8-Br-cGMP or d-cAMP involves CBS and/or CSE phosphorylation mediated by PKG or PKA, leading to an increased catalytic activity. Purpose II: To investigate whether cGMP-dependent PKG and/or cAMP-dependent PKA promotes CBS phosphorylation, leading to an increase in H2S production in human urothelium. Material and Methods II: Bladder samples were obtained as previously described in Material and Methods I. Urothelium was carefully dissected and separated from detrusor muscle. Human urothelial T24 cell line wild type (WT), silenced for CBS (CBSΔT24) or site-directed mutated for CBS (HA-CBS S32A, HA-CBS S227A and HA-CBS S525A; the amino acids Ser32, Ser227 and Ser525, the putative phosphorylation sites predicted by GPS 2.1 software, were mutated in Ala) were cultured in DMEM supplemented with FBS 10%. CBS and CSE expression was evaluated in human urothelium and T24 cells, while PKG and PKA expression was evaluated in human urothelium, T24 cell line, detrusor muscle and full thickness bladder by Western blot. H2S production in human urothelium and T24 cells was measured in basal condition and after stimulation with L-cysteine (10 mM), 8-Br-cGMP (100 µM; 30 min for urothelium; 5, 15, 30 min for T24 cells) or d-cAMP (100 µM; 30 min for urothelium; 5, 15, 30 min for T24 cells), in presence or in absence of PKG or PKA inhibitor, such as KT5823 or KT5720, respectively. CBS phosphorylation was evaluated either in a cell free phosphorylation assay and in T24 cells treated with or without 8-Br-cGMP and d-cAMP, in presence or absence of PKG or PKA inhibitor, using antibody against phospho-Ser/Thr (anti-pS/T). CBS phosphorylation and H2S production were also measured in HA-CBS S32A, HA-CBS S227A and HA-CBS S525A mutated T24 cells following the treatment with 8-Br-cGMP (100 µM; 15 min). A specific rabbit polyclonal antibody against phosphorylated CBS at site Ser227 (pCBSSer227) was designed and validated. Data were expressed as mean ± SEM. Statistical analysis was determined by using t Student test or ANOVA followed by Bonferroni as a post hoc test. Differences were considered significant when p value was less than 0.05. Results II: Both CBS and CSE were expressed in human urothelium and T24 cell line, and they efficiently converted L-cysteine into H2S. Treatment of both urothelium and T24 cells with 8-Br-cGMP, significantly increased H2S production in a time dependent manner, reaching the maximum effect at 30 and 15 min, respectively. This effect was markedly reverted by inhibition of PKG with the selective inhibitor KT5823. d-cAMP treatment did not cause any change in H2S production in both urothelium and T24 cells. The increase of H2S in urothelium observed with 8-Br-cGMP treatment was associated to a significant raise in CBS phosphorylation (pCBS) at the same time dosing. Interestingly, d-cAMP did not modify pCBS levels in both urothelium and T24 cells. The lacking of effect on pCBS following d-cAMP treatment was depending on a very weak PKA expression in both urothelium and T24 cell line compared to PKG expression. To identify the PKG phosphorylation site(s), CBS WT or HA-CBS mutants proteins (HA-CBS S32A, HA-CBS S227A, HA-CBS S525A) were subjected to a cell free kinase assay. A significant reduction of phosphorylation of HA-CBS S227A and HA-CBS S525A proteins was observed when compared to CBS WT. The same results were obtained when the kinase assay was performed on T24 cells WT and T24 mutants HA-CBS S32A, HA-CBS S227A, HA-CBS S525A. Levels of pCBS in HA-CBS S227A and HA-CBS S525A after 8-Br-cGMP treatment were significantly reduced when compared to CBS WT. Furthermore, mutation of CBS Ser227 significantly reduced H2S production in basal condition and following incubation with 8-Br-cGMP compared to CBS WT, while H2S levels were not modified by mutation of CBS Ser525. This result suggested that phosphorylation at Ser227 accounts for H2S production and CBS activity. Finally,the phosphopeptide-specific CBS antibody (anti-pCBSSer227) generated efficiently recognized pCBSSer227 protein in human urothelium and in T24 cells following the treatment with 8-Br-cGMP. Conclusions II: It is known that urothelium influences the contractile state of detrusor smooth muscle and contributes to bladder homeostasis by releasing several agents that modulate muscle contractility. In bladder urothelium PKG/cGMP signalling triggers H2S production through a selective activation of CBS by phosphorylation at Ser227. We suggest that CBS-derived H2S contributes to bladder homeostasis by controlling detrusor muscle contractility. Furthermore, the modulation of intracellular cGMP levels in bladder by using PDE5-Is promotes H2S induction via PKG, contributing to explain the efficacy of PDE5-Is in LUTS therapy. Background: H2S is a physiological signalling molecule with cytoprotective effects in several cardiovascular disease, such as myocardial ischemia (MI) and heart failure. Reactive oxygen species (ROS) represent a major cause of myocardial injury and cellular damage during ischemia/reperfusion. Increasing the activity of cellular antioxidant enzymes, cardiomyocytes can protect myocardium tissue against ischemia/reperfusion injury. H2S is produced in mammalian tissue by three different enzymes: cystathionine γ-lyase (CSE), cystationine β-synthase (CBS) and 3-mercaptopopyruvate sulfurtransferase (3-MST) It has been demonstrated that acute injection of H2S, either prior to ischemia or at reperfusion, markedly ameliorates in vivo myocardial ischemia/reperfusion (MI/R) injury and induces cardioprotection by enhancing eNOS activity, thus increasing myocardial NO bioavailability. Furthermore, H2S exerts antioxidant properties in myocardium through nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signalling. Angiotensin converting enzyme inhibitors (ACEIs) are recommended for the management of myocardial ischemia. Even though ACEIs have been shown to reduce cardiovascular morbidity and mortality in patients affected with myocardial infarction, their cardioprotection is not dependent entirely on inhibition of angiotensin II production. Zofenopril is a sulphydryl-ACEI characterized by high lipophilicity, long-lasting tissue penetration, selective cardiac ACE inhibition and antioxidant and cardioprotective activities. Recently, it has been reported that zofenopril promotes H2S signalling through the upregulation of CSE or by acting as an H2S donor. Thus, it can be hypothesized that the antioxidant and cardioprotective effects of zofenopril may be mediated through H2S and NO dependent signalling. Purpose: To investigate i) the effect of a single administration of zofenopril on myocardial and circulating H2S and NO bioavailability; ii) the effect of zofenopril treatment on the extent of cardiac injury in a murine model of MI/R; 3) the effect of zofenopril therapy on oxidative stress and associated Nrf2 signalling. Material and Methods: Male C57BL/6J mice were treated per oral gavage with vehicle (carboxymethylcellulose 0.02% m/v) or zofenopril calcium {[(1(S), 4(S)]-1(3-mercapto-2 methyl-1-oxopropyl) 4-phenyl-thio-L-proline-S-benzoylester], 10 mg/kg} for 1-8-24 hours. Free H2S levels were measured in heart tissue and plasma by gas chromatography coupled with sulfur chemiluminescence. Nitrite levels in plasma and heart tissue were quantified by using HPLC methods. Gene expression of H2S producing enzymes CBS, CSE and 3-MST was evaluated by RT-qPCR. Myocardial CBS, CSE, 3-MST, eNOS, p-eNOS1177, p-eNOS495, GPX-1, Trx-1, Trx-2, Nrf2, Keap1 protein expression was evaluated by Western blot. Mice were subjected to 45 min of MI by occluding the left coronary artery. Troponin-I levels were measured at 4 hours of reperfusion by using a specific mouse ELISA kit. Infarct size per area at risk (INF/AAR) was determined after 24 hours of reperfusion. Tissue and circulating biomarkers of oxidative stress were determined by measuring malondialdehyde (MDA) and advanced oxidation protein products (AOPP) levels. Data were expressed as mean ± SEM. Statistical analysis was determined by using Student's unpaired, two-tailed t-test or ANOVA followed by Dunnett as a post hoc test. Results: A single zofenopril administration resulted in a significant increase in H2S bioavailability in both heart tissue and plasma at 8 hours of treatment when compared to vehicle. 8 hours were considered the best time dosing to perform the further experiments. A significant increase in tissue and circulating nitrite levels was observed after zofenopril therapy. Myocardial gene expression related to CBS and CSE was not modified by zofenopril treatment, while 3-MST mRNA levels were significantly increased by drug administration. 3-MST as well as CBS and CSE protein levels were similar in both vehicle and zofenopril treated groups. Acute administration of zofenopril promoted eNOS activation through phosphorylation at Ser1177. Furthermore, zofenopril administration 8 hours before MI/R was cardioprotective reducing significantly INF/AAR and circulating troponin-I levels compared to vehicle. Interestingly, zofenopril therapy induced a significant up-regulation of transcription factor Nrf2 without affecting Keap1 levels. Zofenopril therapy induced a significant up-regulation of antioxidant protein, such as GPX-1 and Trx-1 while it did not alter Trx-2 protein expression. Zofenopril treatment markedly reduced the biomarkers of oxidative stress in myocardial tissue (MDA) and plasma (AOPP). Conclusions: Our findings demonstrate that a single administration of zofenopril significantly increases the H2S bioavailability and enhances NO levels in both heart tissue and plasma. The induction in H2S availability occurs mainly in an non enzymatic manner, while the augmented levels of NO are due to the eNOS phosphorylation at the site Ser1177 enhanced by H2S. Furthermore, this study supports the concept that zofenopril-mediated increases in H2S scavenge ROS directly and/or indirectly by activation of Nrf2 increasing antioxidant enzymes expression. In conclusion, our studies demonstrate that zofenopril may prevent cardiac injury during ischemic conditions primarily through the enhancement of cardiovascular antioxidant defense induced by H2S activated Nrf2 signalling

MTFP1 loss sensitizes to mtDNA-driven sterile inflammation and heart failure in mice

International audienceIntroductionMitochondrial integrity is paramount to heart homeostasis. Under certain stress conditions, mitochondrial DNA (mtDNA) can leak into the cytosol and trigger type I innate immunity via the DNA sensing cGAS-STING pathway, the downstream interferon (IFN) regulatory factor 3 (IRF3) and cell surface receptor IFNAR1. Our previous work shows that loss of the inner membrane (IM) protein Mitochondrial Fission Process 1 (MTFP1) in cardiomyocytes of mice (cMKO) disturbs mitochondrial IM integrity leading to the activation of immune responses and a progressive dilated cardiomyopathy (DCM), which culminates into heart failure (HF) and middle-aged death.ObjectiveInvestigate the impact of MTFP1 loss on mtDNA driven type I immune response and inflammation.MethodIn vitro, Mtfp1-/- mouse embryonic fibroblasts (MEFs) depleted or not of mtDNA, Sting or Irf3 were subjected to mitochondrial outer membrane permeabilization to assess the Interferons Stimulated Genes (ISGs) expression and IRF3 phosphorylation. In vivo, cMKO mice were additionally deleted of Sting or Ifnar1 and subjected to echocardiography to assess DCM progression.ResultsUpon treatment, Mtfp1-/- MEFs display increased ISGs expression and IRF3 phosphorylation compared to WT cells, which is abrogated by the depletion of mtDNA, STING or IRF3 in these cells. In vivo, whole body deletion of STING in cMKO mice mitigates HF, whereas IFNAR1 ablation fully rescues cardiac dysfunction and remodeling and extends lifespan of mice.ConclusionCollectively, our data suggest a role of MTFP1 in restraining mtDNA-dependent inflammation, which in vivo fosters HF in the cMKO mice

Involvement of nitric oxide and hydrogen sulfide in glucorticoid-induced hypertension in rat

Excess of glucocorticoid, either endogenous, as in Cushing syndrome, or exogenous, via pharmacological administration of glucorticoids, induces hypertension. This form of hypertension is commonly related to activation of the mineralcortocoids receptor whereas it certainly has a role (Baid S et al., 2004). However, evidences indicate that glucocorticoid elevated blood pressure independently by mineralcorticoid receptor, in both humans and animal model (Kalimi M et al., 1989; Bertagna C et al., 1986). Furthermore, glucorticoid receptor is present in both vascular smooth muscle (Provencher PH et al., 1995) and endothelium (Wallerath T et al., 1999). To date, the mechanism(s) underlying glucocorticoid induced increase in blood pressure is still unclear. The gaseous transmitters nitric oxide (NO) and hydrogen sulphide (H2S) together with endothelial-derived hyperpolarizing factor (EDHF) play a key role in the regulation of vascular homeostasis. In cardiovascular system, NO derived from endothelial NO synthase (eNOS) while H2S is predominantly produced by cisthationine-β synthase (CBS) and/or cystathionine-γ lyase (CSE). Published data suggest that H2S is EDHF (d'Emmanuele di Villa Bianca et al., 2011; Tang G et al., 2013). Therefore, we investigated the involvement of NO and H2S/EDHF signalling in glucocorticoid-induced hypertension. Male Wistar rats were treated with dexamethasone (DEX, 1.5mg/kg subcutaneously) or vehicle (saline) for 8 days. During treatment, blood pressure was recorded by using a tail cuff apparatus, in conscious rats. Perfused arterial mesenteric plexus was used to evaluate the NO and the EDHF contribute. Thereafter, western blot study was performed to appreciate phoshorylated-eNOS/eNOS ratio or CBS and CSE expression in mesenteric tissue. The production of NO and H2S was also measured. DEX treatment caused a significant increase in blood pressure. The contribute of NO mediated vasodilation was higher in the mesenteric bed of DEX-treated rats. Conversely, EDHF-mediated vasodilatation resulted significantly reduced in DEX group. In line with these findings, the peNOS/ eNOS ratio as well as NO production were significantly increased in DEX group compared with vehicle. On the other hand, CBS and CSE expression was markedly reduced in DEX group and well correlated with H2S production. Consistently with the fact that H2S is EDHF, this latter result strongly support the reduction in EDHF-vasodilation occurring in DEX-group. In conclusion, our data indicate that exists a cross-talk between NO and H2S in glucorticoidinduced hypertension. In this scenario H2S pathway could represent a novel target opening new pharmacological strategies in the control of blood pressure. Baid S and Nieman LK. Curr Hypertens Rep 2004; 6:493-9. d'Emmanuele et al., JPET 2011; 337:59-64. Kalimi M et al., Am J Physiol 1989; 256:E682–E685. Bertagna X et al., J Clin Endocrinol Metab 1986 63:639-43. Wallerath T. et al., Proc Natl Acad Sci USA 1999; 13357-13362. Provencher PH et al., J Steroid Biochem Mol Biol 1995; 2:219-25. Tang G et al., Antioxid Redox Signal. 2013 [Epub ahead of print]

Anti-virulence therapy against Pseudomonas aeruginosa: identification of antibiofilm drugs and development of inhalable Niclosamide and Flucytosine Formulations

Early aggressive and maintenance antibiotic therapies prolong cystic fibrosis (CF) patient life, but are not able to eradicate Pseudomonas aeruginosa lung infection. Anti-virulence drugs represent a promising therapeutic option in CF. These new drugs could alleviate the severity of the infection, reduce lung inflammation, and help antibiotics in eradicating the chronic P.aeruginosa lung infection. The long times and high costs required for the evelopment of ???brand new??? anti-virulence drugs can be saved by repurposing ???old??? drugs already used in humans for different purposes. Indeed, we have recently shown that the antimycotic drug flucytosine and the anthelmintic drug niclosamide can be repurposed to suppress P. aeruginosa virulence in vitro and in animal models of infection. However, the old drugs need to be re-formulated for the new clinical application, taking into consideration the peculiarities of CF patients. Objectives 1) To finalize pre-clinical studies concerning flucytosine and niclosamide, by developing and validating inhalable formulations for use in CF therapy. 2) To discovery other drugs with anti-virulence activity against P. aeruginosa. Materials and methods 1) Novel inhalable liquid and dry powders formulations of flucytosine and niclosamide will be developed. In vitro cytotoxicity and activity studies will be performed in lung epithelium cell cultures and on a large number of P. aeruginosa strains isolated from CF patients. Then, toxicity/pharmacokinetic of the developed drug formulations upon inhalation will be evaluated in rats. Finally, the best drug formulations will be validated in a mouse model of lung infection. 2) Purpose-built biosensors able to identify inhibitors of biofilm formation will be used for the screening of 1600 ???old??? drugs. The inhibitory activity of the best anti-biofilm drugs will be validated on P. aeruginosa CF strains

H₂S Prodrug, SG-1002, Protects against Myocardial Oxidative Damage and Hypertrophy In Vitro via Induction of Cystathionine β-Synthase and Antioxidant Proteins

Endogenously produced hydrogen sulfide (H2S) is critical for cardiovascular homeostasis. Therapeutic strategies aimed at increasing H2S levels have proven cardioprotective in models of acute myocardial infarction (MI) and heart failure (HF). The present study was undertaken to investigate the effects of a novel H2S prodrug, SG-1002, on stress induced hypertrophic signaling in murine HL-1 cardiac muscle cells. Treatment of HL-1 cells with SG-1002 under serum starvation without or with H2O2 increased the levels of H2S, H2S producing enzyme, and cystathionine β-synthase (CBS), as well as antioxidant protein levels, such as super oxide dismutase1 (SOD1) and catalase, and additionally decreased oxidative stress. SG-1002 also decreased the expression of hypertrophic/HF protein markers such as atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), galectin-3, TIMP1, collagen type III, and TGF-β1 in stressed HL-1 cells. Treatment with SG-1002 caused a significant induction of cell viability and a marked reduction of cellular cytotoxicity in HL-1 cells under serum starvation incubated without or with H2O2. Experimental results of this study suggest that SG-1002 attenuates myocardial cellular oxidative damage and/or hypertrophic signaling via increasing H2S levels or H2S producing enzymes, CBS, and antioxidant proteins

L-Cys/CSE/H2S pathway modulates mouse uterus motility and sildenafil effect

Sildenafil, a selective phosphodiesterase type 5 (PDE5) inhibitor, commonly used in the oral treatment for erectile dysfunction, relaxes smooth muscle of human bladder through the activation of hydrogen sulfide (H2S) signaling. H2S is an endogenous gaseous transmitter with myorelaxant properties predominantly formed from L-cysteine (L-Cys) by cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). Sildenafil also relaxes rat and human myometrium during preterm labor but the underlying mechanism is still unclear. In the present study we investigated the possible involvement of H2S as a mediator of sildenafil-induced effect in uterine mouse contractility. We firstly demonstrated that both enzymes, CBS and CSE were expressed, and able to convert L-Cys into H2S in mouse uterus. Thereafter, sildenafil significantly increased H2S production in mouse uterus and this effect was abrogated by CBS or CSE inhibition. In parallel, L-Cys, sodium hydrogen sulfide or sildenafil but not D-Cys reduced spontaneous uterus contractility in a functional study. The blockage of CBS and CSE reduced this latter effect even if a major role for CSE than CBS was observed. This data was strongly confirmed by using CSE−/− mice. Indeed, the increase in H2S production mediated by L-Cys or by sildenafil was not found in CSE−/− mice. Besides, the effect of H2S or sildenafil on spontaneous contractility was reduced in CSE−/− mice. A decisive proof for the involvement of H2S signaling in sildenafil effect in mice uterus was given by the measurement of cGMP. Sildenafil increased cGMP level that was significantly reduced by CSE inhibition. In conclusion, L-Cys/CSE/H2S signaling modulates the mouse uterus motility and the sildenafil effect. Therefore the study may open different therapeutical approaches for the management of the uterus abnormal contractility disorders

Sildenafil effect on the human bladder involves the L-cysteine/hydrogen sulfide pathway: a novel mechanism of action of phosphodiesterase type 5 inhibitors

Several studies have documented a relationship between male sexual dysfunction and lower urinary tract symptom (LUTS) (Speakman MJ, 2009). LUTS are common in men with ED and there is a strong correlation between the severity of LUTS and the degree of erectile dysfunction in all age groups, which suggests a causal relationship or, more possibly, the presence of common pathogenetic pathways (Speakman MJ, 2009). Phosphodiesterase type 5 inhibitors (PDE5-Is), commonly used in ED therapy, were recently found to be effective in the treatment of LUTS (?ckert S et al., 2011) although their mechanism of action is still unclear. PDE5-Is cause bladder smooth muscle relaxation through a mechanism partially independent from nitric oxide (Oger S et al., 2010). Hydrogen sulfide (H2S) is a newly discovered gas-transmitter with myorelaxant properties. It is synthesized predominantly from L-cysteine (L-cys) by the enzymes cystathionine-β- synthase (CBS) and cystathionine-γ-lyase (CSE) (Wang R, 2012). The aim of our study was to evaluate whether L-cys/H2S pathway is involved in the effect of sildenafil on human detrusor dome. In this study have been used samples obtained from patients undergoing open prostatectomy for benign prostatic hyperplasia and bladder outlet obstruction. The expression of CBS, CSE was evaluated by western blot analysis. CBS and CSE activity was detected measuring H2S production by a colorimetric assay in basal and stimulated conditions with L-cys (Stipanuk MH et al., 1982). The effect of sildenafil (1, 3, 10, and 30μM), 8-bromo-cyclic guanosine monophosphate (8-bromo-cGMP; 100 μM) and dibutyryl-ciclic adenosine monophosphate (dibutyryl-cAMP; 100 μM) on H2S production was also evaluated. The relaxation-response to Lcys (0.1μM to 10mM), sodium hydrogen sulfide (NaHS, 0.1μM to 10mM) and sildenafil (0.1 μM to 10 μM) was assessed on carbachol pre-contracted detrusor strips. CBS and CSE inhibitors were used to demonstrate the involvement of H2S signaling in sildenafil effect. We found that human bladder expresses CBS and CSE and that tissue homogenates significantly convert L-cys into H2S. NaHS or L-cys relaxed in a concentration-dependent manner the human bladder strips. Sildenafil caused a relaxation of pre-contracted bladder dome strips in a concentration-dependent manner and this effect was significantly reduced by CSE and CBS inhibition. In addition, sildenafil caused a concentration-dependent increase in H2S production in human tissue. Similarly, both 8-bromo-cGMP and d-cAMP caused an increase in H2S production. In conclusion we have shown that L-cysteine/H2S pathway has a functional role in human bladder representing a new therapeutical target in LUTS. At last but not least, sildenafil effect involves H2S signaling trough cGMP and cAMP. This evidence may clarify the beneficial effect of PDE5-Is either in LUTS or ED treatment. Speakman MJ (2009) Curr Pharm Des 15:3502-3505. ?ckert S et al. (2011). Br J Clin Pharmacol 72:197-204. Oger S et al. (2010). Br J Pharmacol 160:1135-43. Wang R. (2012). Physiol Rev 92:791-896. Stipanuk MH et al. (1982). Biochem J 206:267-277

Involvement of Hydrogen Sulfide (H2S) in Dexamethasone (DEX) Induced Hypertension in Rat

Introduction: Glucocorticoid (GC) excess is related to hypertension. The deletion of endothelial GC-receptors abrogates the blood pressure increase, suggesting GC-induced hypertension is endothelium-dependent. In response to shear stress endothelium releases nitric oxide, endothelial derived hyperpolarizing factor (EDHF) and prostacyclin. Recently H2S has been proposed as a candidate for EDHF. H2S is mainly produced by the enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) from L-cysteine. The aim of this study was to investigate the EDHF/H2S signaling in GC-hypertension. Methods: Male Wistar rats were treated with DEX (1.5 mg/kg/sc) or vehicle (VEH) for 8 days. Systolic blood pressure (SBP) was monitored every 2 days. EDHF was evaluated in mesenteric plexus and carotid artery performing a concentration-effect curve of acetylcholine in presence of indomethacin (INDO) and nitro-L-arginine methyl ester (L-NAME). Apamin (APA) plus charibdotoxin (CTX), SKCa and BKCa inhibitors, or propargylglycine (PAG), CSE inhibitor, were used. CBS and CSE levels were analyzed by immunoblot. H2S levels were measured by a colorimetric assay. Results: DEX treatment significantly increased SBP compared to VEH (*p<0.05, **p<0.01, ***p<0.001 at days 2-4, 6, 8 respectively). EDHF-mediated relaxation of mesenteric bed or carotid artery was markedly reduced in DEX group compared to VEH (***p<0.001). APA and CTX as well as PAG abolished EDHF-mediated relaxation in DEX or VEH group (***,°°°p<0.001 respectively). CBS and CSE levels were significantly reduced in mesenteric plexus and carotid artery in DEX group (*p<0.05). The H2S production was markedly reduced in mesenteric plexus and carotid artery (*p<0.05, **p<0.01 respectively) as well as plasmatic H2S levels (*p<0.05) in DEX rats compared to VEH. Conclusions: Our data demonstrate that GC-excess induces an impairment of H2S/EDHF signaling indicating an additional cause of GC-mediated hypertension

Sildenafil Effect on the Human Bladder Involves the L-cysteine/Hydrogen Sulfide Pathway: A Novel Mechanism of Action of Phosphodiesterase Type 5 Inhibitors

Background: Phosphodiesterase type 5 inhibitors (PDE5-Is) are effective in the treatment of lower urinary tract symptom (LUTS), although their mechanism of action is still unclear. PDE5-Is cause bladder detrusor relaxation, and this effect is partially independent of nitric oxide. Hydrogen sulfide (H2S) is a newly discovered transmitter with myorelaxant properties. It is predominantly formed from L-cysteine by cystathionineb- synthase (CBS) and cystathionine-g-lyase (CSE). Objective: To evaluate whether the L-cysteine/H2S pathway contributes to the relaxing effect of sildenafil on the human detrusor dome. Design, setting, and participants: Samples of bladders obtained from men undergoing open prostatectomy for benign prostatic hyperplasia (BPH) were used. The presence of CBS and CSE enzymes was assessed by western blot. H2S production was measured by a colorimetric assay in basal and stimulated conditions with L-cysteine and in response to sildenafil (1, 3, 10, and 30 mM), 8-bromo–cyclic guanosine monophosphate (8-bromo– cGMP; 100 mM) or dibutyryl–cyclic adenosine monophosphate (dibutyryl-cAMP; 100 mM). A curve concentration effect of sodium hydrosulfide (NaHS), H2S donor (0.1 mM to 10 mM), L-cysteine (0.1 mM to 10 mM), and sildenafil (0.1–10 mM) was performed on precontracted detrusor dome strips. To investigate H2S signaling in a sildenafil effect, CBS and CSE inhibitors were used. Outcome measurements and statistical analysis: Analysis of variance was used, followed by the Bonferroni post hoc test. Results and limitations: CBS and CSE are present in the human bladder dome and efficiently convert L-cysteine into H2S. Both NaHS and L-cysteine relaxed human strips. Sildenafil caused (1) a relaxation of bladder dome strips and (2) a concentrationdependent increase in H2S production. Both effects were significantly reduced by CBS and CSE inhibitors. Similar to sildenafil, both 8-bromo-cGMP and dibutyryl-cAMP caused an increase in H2S production. Conclusions: The sildenafil relaxant effect on the human bladder involves the H2S signaling pathway. This effect may account in part for the efficacy of PDE5-Is in LUTS. A better definition of the pathophysiologic role of the H2S pathway in the human bladder may open new therapeutic approaches