Donnarumma, Erminia (2015) L-cysteine/hydrogen sulfide pathway: pharmacological approaches in urinary bladder and cardiovascular diseases. [Tesi di dottorato]

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Abstract

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.

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