Hydrogen Sulfide and Urogenital Tract.

In this chapter the role played by H2S in the physiopathology of urogenital tract revising animal and human data available in the current relevant literature is discussed. H2S pathway has been demonstrated to be involved in the mechanism underlying penile erection in human and experimental animal. Both cystathionine-ß synthase (CBS) and cystathionine-γ lyase (CSE) are expressed in the human corpus cavernosum and exogenous H2S relaxes isolated human corpus cavernosum strips in an endothelium-independent manner. Hydrogen sulfide pathway also accounts for the direct vasodilatory effect operated by testosterone on isolated vessels. Convincing evidence suggests that H2S can influence the cGMP pathway by inhibiting the phosphodiesterase 5 (PDE-5) activity. All these findings taken together suggest an important role for the H2S pathway in human corpus cavernosum homeostasis. However, H2S effect is not confined to human corpus cavernosum but also plays an important role in human bladder. Human bladder expresses mainly CBS and generates in vitro detectable amount of H2S. In addition the bladder relaxant effect of the PDE-5 inhibitor sildenafil involves H2S as mediator. In conclusion the H2S pathway is not only involved in penile erection but also plays a role in bladder homeostasis. In addition the finding that it involved in the mechanism of action of PDE-5 inhibitors strongly suggests that modulation of this pathway can represent a therapeutic target for the treatment of erectile dysfunction and bladder diseases.

A new therapeutic approach to erectile dysfunction: urotensin-II receptor high affinity agonist ligands.

Urotensin-II (U-II) is a cyclic peptide that acts through a G protein-coupled receptor (urotensin-II receptor [UTR]) mainly involved in cardiovascular function in humans. The urotensinergic system is also implicated in the urogenital tract. Indeed, U-II relaxes human corpus cavernosum strips and causes an increase in intracavernous pressure (ICP) in rats. In light of this, the U-II/UTR pathway can be considered a new target for the treatment of erectile dysfunction. On this hypothesis, herein we report on two new UTR high affinity-agonists, P5U (H-Asp-c[Pen-Phe-Trp-Lys-Tyr-Cys]-Val-OH) and UPG84(H-Asp-c[Pen-Phe-DTrp-Orn-(pNH 2 ) Phe-Cys]-Val-OH). The effects of P5U and UPG84 were each compared separately with U-II by monitoring the ICP in anesthetized rats. Intracavernous injection of U-II (0.03-1 nmol), P5U (0.03-1 nmol) or UPG84 (0.03-1 nmol) caused an increase in ICP. P5U, in particular, elicited a significant increase in ICP as compared to U-II. The observed effect by using P5U at a dose of 0.1 nmol per rat was comparable to the effect elicited by U-II at a dose of 0.3 nmol. Moreover, UPG84 at the lowest dose (0.03 nmol) showed an effect similar to the highest dose of U-II (1 nmol). Furthermore, UPG84 was found to be more effective than P5U. Indeed, while the lowest dose of P5U (0.03 nmol) did not affect the ICP, UPG84, at the same dose, induced a prominent penile erection in rat. These compounds did not modify the blood pressure, which indicates a good safety profile. In conclusion, UPG84 and P5U may open new perspectives for the management of erectile dysfunction.

New insight into the binding mode of peptides at urotensin-II receptor by Trp-constrained analogues of P5U and urantide.

Urotensin II (U-II) is a disulfide bridged peptide hormone identified as the ligand of a G-protein-coupled receptor. Human U-II (H-Glu-Thr-Pro-Asp-c[Cys-Phe-Trp-Lys-Tyr-Cys]-Val-OH) has been described as the most potent vasoconstrictor compound identified to date. We have recently identified both a superagonist of human U-II termed P5U (H-Asp-c[Pen-Phe-Trp-Lys-Tyr-Cys]-Val-OH) and the compound termed urantide (H-Asp-c[Pen-Phe-D-Trp-Orn-Tyr-Cys]-Val-OH), which is the most potent UT receptor peptide antagonist described to date. In the present study, we have synthesized four analogues of P5U and urantide in which the Trp(7) residue was replaced by the highly constrained L-Tpi and D-Tpi residues. The replacement of the Trp(7) by Tpi led to active analogues. Solution NMR analysis allowed improving the knowledge on conformation-activity relationships previously reported on UT receptor ligands.

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 (H(2)S) is a newly discovered transmitter with myorelaxant properties. It is predominantly formed from L-cysteine by cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE). OBJECTIVE: To evaluate whether the L-cysteine/H(2)S 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. H(2)S 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 µM), 8-bromo-cyclic guanosine monophosphate (8-bromo-cGMP; 100 µM) or dibutyryl-cyclic adenosine monophosphate (dibutyryl-cAMP; 100 µM). A curve concentration effect of sodium hydrosulfide (NaHS), H(2)S donor (0.1 µM to 10mM), L-cysteine (0.1 µM to 10mM), and sildenafil (0.1-10 µM) was performed on precontracted detrusor dome strips. To investigate H(2)S 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 H(2)S. Both NaHS and L-cysteine relaxed human strips. Sildenafil caused (1) a relaxation of bladder dome strips and (2) a concentration-dependent increase in H(2)S 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 H(2)S production. CONCLUSIONS: The sildenafil relaxant effect on the human bladder involves the H(2)S 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 H(2)S pathway in the human bladder may open new therapeutic approaches.

An ex vivo standardized assay to measure human platelet cGMP.

INTRODUCTION: Nitric oxide (NO) acts a pleiotropic biomodulator in several systems, including the cardiovascular, nervous and immune systems. The intracellular levels of cyclic guanylate monophosphate (cGMP) can be increased by NO or by inhibiting the breakdown to 5'cGMP operated by the cyclic nucleotide phosphodiesterases (PDEs). Platelets are anuclear circulating cells that are rich in both soluble guanylyl cyclase and PDEs. The purpose of this study was to standardize cGMP determination in human platelets. METHODS: Fresh blood samples were obtained from a group of healthy volunteers in order to obtain washed platelets. Platelet (3×10(4)/µl or 5×10(5)/µl) cGMP levels were measured in basal and stimulated conditions. To stimulate platelets two different NO-donors were used: sodium nitroprusside (SNP; 10, 100, 1000 µM) or diethylamine NONOate (DEA-NONOate; 1, 10, 100 µM). Different times of incubation were also studied (5, 15 and 30 min). As positive control has been used ODQ a well known inhibitor of guanylyl cyclase. Platelet cGMP accumulation was measured by using a standard ELISA kit using different sample dilutions. RESULTS: The optimal stimulus was DEA-NONOate, the optimal washed platelet concentration was 5×10(5)/µl, incubation time was 30 min and dilution to be used was 1:2. DISCUSSION: Platelets represent a valuable marker to investigate the effect of drugs interfering with the cGMP cascade. This standardized assay allows to measure ex vivo the inhibitory (PDE inhibitors) or stimulatory effect (NO donors) of drugs given in vivo to humans.

Hydrogen sulphide induces mouse paw oedema through activation of phospholipase A2.

BACKGROUND AND PURPOSE: Hydrogen sulphide (H(2)S), considered as a novel gas transmitter, is produced endogenously in mammalian tissue from L-cysteine by two enzymes, cystathionine ß-synthase and cystathionine γ-lyase. Recently, it has been reported that H(2)S contributes to the local and systemic inflammation in several experimental animal models. We conducted this study to investigate on the signalling involved in H(2)S-induced inflammation. EXPERIMENTAL APPROACH: L-cysteine or sodium hydrogen sulphide (NaHS) was injected into the mouse hind paw and oedema formation was evaluated for 60 min. In order to investigate H(2)S-induced oedema formation, we used 5-HT and histamine receptor antagonists, and inhibitors of K(ATP) channels or arachidonic acid cascade. Prostaglandin levels were determined in hind paw exudates by radioimmunoassay. Paws injected with L-cysteine or NaHS were examined by histological methods. KEY RESULTS: Both NaHS and L-cysteine caused oedema characterized by a fast onset which peaked at 30 min. This oedematogenic action was not associated with histamine or 5-HT release or K(ATP) channel activation. However, oedema formation was significantly inhibited by the inhibition of cyclooxygenases and selective inhibition of phospholipase A(2). Prostaglandin levels were significantly increased in exudates of hind paw injected with NaHS or L-cysteine. The histological examination clearly showed an inflammatory state with a loss of tissue organization following NaHS or L-cysteine injection. CONCLUSIONS AND IMPLICATIONS: Phospholipase A(2) and prostaglandin production are involved in pro-inflammatory effects of H(2)S in mouse hind paws. The present study contributes to the understanding of the role of L-cysteine/H(2)S pathway in inflammatory disease.

Recombinant human erythropoietin prevents lipopolysaccharide-induced vascular hyporeactivity in the rat.

Erythropoietin (EPO) is a hypoxia-inducible hormone that is essential for normal erythropoiesis in the bone marrow. Administration of recombinant human-EPO is currently being used for the therapy of anemia associated with chronic renal failure and cancer. Moreover, EPO reduces organ injury in experimental hemorrhagic as well as in splanchnic artery occlusion shock and preserves cardiac function after experimental cardiac I/R. Erythropoietin receptors are widely distributed in the cardiovascular system, including endothelial, smooth muscle, cardiac, and other cell types, and nonhematopoietic effects of EPO are increasingly recognized. Thus, the vasculature may be a biological target of EPO. Therefore, the aim of our study was to investigate whether EPO exerts a protective effect in septic shock by modulating vascular dysfunction and hyporeactivity. Rats received EPO (300 U/kg, i.v.) or vehicle 30 min before and 1 and 3 h after LPS (8 x 10 U/kg, i.v.). In vivo and ex vivo (aortic rings) experiments were performed to evaluate the vascular response to contracting and vasodilating agents. The expression of iNOS, intercellular adhesion molecule 1, poly(ADP)ribose polymerase, Bcl-xl, and Bcl-2 was evaluated by Western blot analysis in the rat aorta. We demonstrate that EPO significantly prevents LPS-induced vascular hyporeactivity and endothelial dysfunction. Interestingly, EPO inhibits the increase in iNOS, poly(ADP)ribose polymerase, and intercellular adhesion molecule 1 expression in the aorta of endotoxemic rats and attenuated the decline in the expression of both Bcl-xl and Bcl-2 caused by LPS. In conclusion, our data support the view that EPO has important nonerythropoietic effects protecting organ and tissue against injury and indicate that EPO may be useful in the therapy of patients with septic shock.

Sphingosine 1-phosphate induces endothelial nitric-oxide synthase activation through phosphorylation in human corpus cavernosum.

Sphingosine 1-phosphate (S1P) is the natural ligand for a specific G protein-coupled receptors. In endothelial cells, S1P has been shown to modulate the activity of the endothelial nitric-oxide synthase (eNOS) through phosphorylation operated by Akt. Nitric oxide (NO) produced by neuronal nitric-oxide synthase and eNOS plays a central role in triggering and maintaining penile erection. This study has assessed the possibility of a similar cross-talk between eNOS and S1P in human corpus cavernosum and whether this interaction is connected to penile vascular response. Quantitative reverse transcription-polymerase chain reaction demonstrated the presence of S1P(1), S1P(2), and S1P(3) receptors in both the human corpus cavernosum (HCC) and the penile artery. S1P on its own did not relax or contract HCC strips, but on the other hand, incubation with S1P (0.1 microM) caused a 6-fold increase in relaxation induced by a subliminal dose of acetylcholine. This effect is dependent upon eNOS activation through an Akt-dependent phosphorylation, as demonstrated by pharmacological modulation with l-nitroarginine methyl ester and wortmannin and by Western blot studies. In human tissue, S1P seems to be the possible candidate for the activation of the eNOS calcium-independent pathway. This pathway may represent a new therapeutic area of intervention in erectile dysfunction (ED) to develop a way to selectively promote NO production at the endothelial level. This approach could also be used to enhance phosphodiesterase 5 therapy in patients with ED that are poor responders, such as in the case of diabetes.

Synthesis by microwave irradiation of a substituted benzoxazine parallel library with preferential relaxant activity for guinea pig trachealis.

An efficient, facile, and practical parallel combinatorial synthesis of substituted-benzoxazines under microwave irradiation was described. The procedure involved the use of a microwave oven especially designed for organic synthesis suitable for parallel synthesis of solution libraries. A demonstration 19-membered library of substituted N,N-dimethyl- and N-methyl-benzoxazine amide derivatives, structurally related to the potassium channel opener cromakalim, was generated by both conventional and microwave procedures, achieving a reduction from 7 h to 30-36 min in library generation time for the microwave approach. All the synthesized compounds were tested using the in vitro models of rat aorta and guinea pig trachea rings pre-contracted with phenylephrine and carbachol, respectively. All N,N-dimethyl amide derivatives showed a relaxant activity higher on guinea pig trachea rings than on rat aorta rings.

New lignans from the roots of Valeriana prionophylla with antioxidative and vasorelaxant activities.

Two new 7,9':7',9-diepoxylignan glycosides have been isolated from the roots of Valeriana prionophylla. Their structures have been established on the basis of 1D and 2D NMR experiments as prinsepiol-4-O-beta-D-glucopyranoside (1) and fraxiresinol-4'-O-beta-D-glucopyranoside (2). In addition, 8-hydroxypinoresinol-4'-O-beta-d-glucopyranoside (3), 8-hydroxypinoresinol (4), prinsepiol (5), and chlorogenic acid were isolated. Compounds 1, 3, 4, and 5 were evaluated for their antioxidative properties in Trolox equivalent antioxidant activity (TEAC) and chemiluminescence (CL) assays. The same compounds were tested for their vascular activity in rat aorta rings. The aglycones 4 and 5 displayed powerful antioxidant activity; in addition, aglycone 4 showed a higher vasorelaxant activity than compounds 1, 3, and 5.

Pyrrolidine dithiocarbamate reduces renal dysfunction and injury caused by ischemia/reperfusion of the rat kidney.

Dithiocarbamates can modulate the expression of genes associated with inflammation or development of ischemia/reperfusion injury. Here, we investigate the effects of pyrrolidine dithiocarbamate, an inhibitor of nuclear factor (NF)-kappaB activation, on the renal dysfunction and injury caused by ischemia/reperfusion of the rat kidney. Bilateral clamping of renal pedicles (45 min) followed by reperfusion (6 h) caused significant renal dysfunction and marked renal injury. Pyrrolidine dithiocarbamate (100 mg/kg, administered i.v.) significantly reduced biochemical and histological evidence of renal dysfunction and injury caused by ischemia/reperfusion of the rat kidney. Furthermore, pyrrolidine dithiocarbamate markedly reduced the expression of inducible nitric oxide synthase (iNOS) protein and significantly reduced serum levels of nitric oxide. Finally, pyrrolidine dithiocarbamate inhibited the activation of NF-kappaB by preventing its translocation from the cytoplasm into the nuclei of renal cells. These results demonstrate that pyrrolidine dithiocarbamate reduces renal ischemia/reperfusion injury and that dithiocarbamates may provide beneficial actions against ischemic acute renal failure.

Involvement of beta 3-adrenergic receptor activation via cyclic GMP- but not NO-dependent mechanisms in human corpus cavernosum function.

The beta(3)-adrenoreceptor plays a major role in lipolysis but the role and distribution of beta(3)-receptors in other specific sites have not been extensively studied. beta(3)-adrenergic receptors are present not only in adipose tissue but also in human gall bladder, colon, prostate, and skeletal muscle. Recently, beta(3)-adrenergic receptor stimulation was shown to elicit vasorelaxation of rat aorta through the NO-cGMP signal transduction pathway. Here we show that beta(3)-receptors are present in human corpus cavernosum and are localized mainly in smooth muscle cells. After activation by a selective beta(3)-adrenergic receptor agonist, BRL 37344, there was a cGMP-dependent but NO-independent vasorelaxation that was selectively blocked by a specific beta(3)-receptor antagonist. In addition, we report that the human corpus cavernosum exhibits basal beta(3)-receptor-mediated vasorelaxant tone and that beta(3)-receptor activity is linked to inhibition of the RhoA/Rho-kinase pathway. These observations indicate that beta(3)-receptors may play a physiological role in mediating penile erection and, therefore, could represent a therapeutic target for treatment of erectile dysfunction.

Synthesis and vasorelaxant activity of new 1,4-benzoxazine derivatives potassium channel openers.

As part of a search for new potassium channel openers, the synthesis and vasorelaxant activity of new 1,4-benzoxazine derivatives derived from transformation of the benzopyran skeleton of cromakalim were described. Several new 1,4-benzoxazine derivatives were provided with significant vasorelaxant activity with an overall pharmacological behavior similar to CRK (1f, 1i, 2d, 2e, 2f and 2i).