Mário A. Claudino

Mechanisms underlying relaxation of rabbit aorta by bay 41-2272, a nitric oxide-independent soluble guanylate cyclase activator

1. The compound BAY 41‐2272 (5‐cyclopropyl‐2‐[1‐(2‐fluoro‐benzyl)‐1H‐pyrazolo[3,4‐b]pyridin‐3‐yl]‐pyrimidin‐4‐ylamine) has been described as a potent, nitric oxide (NO)‐independent, stimulator of soluble guanylate cyclase. In the present study, the mechanisms underlying the relaxant effect of BAY 41‐2272 in endothelium‐intact and ‐denuded precontracted rabbit aortic rings were investigated.

Upregulation of gp91phox Subunit of NAD(P)H Oxidase Contributes to Erectile Dysfunction Caused by Long-term Nitric Oxide Inhibition in Rats: Reversion by Regular Physical Training

OBJECTIVES To test the hypothesis that glyco protein 91phox (gp91(phox)) subunit of nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase is a fundamental target for physical activity to ameliorate erectile dysfunction (ED). Vascular risk factors are reported to contribute to ED. Regular physical exercise prevents cardiovascular diseases by increasing nitric oxide (NO) production and/or decreasing NO inactivation. METHODS Male Wistar rats received the NO synthesis inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) for 4 weeks, after which animals were submitted to a run training program for another 4 weeks. Erectile functions were evaluated by in vitro cavernosal relaxations and intracavernous pressure measurements. Expressions of gp91(phox) subunit and neuronal nitric oxidase synthase in erectile tissue, as well as superoxide dismutase activity and nitrite/nitrate (NO(x)) levels were determined. RESULTS The in vitro acetylcholine- and electrical field stimulation-induced cavernosal relaxations, as well as the increases in intracavernous pressure were markedly reduced in sedentary rats treated with l-NAME. Run training significantly restored the impaired cavernosal relaxations. No alterations in the neuronal nitric oxidase synthase protein expression (and its variant penile neuronal nitric oxidase synthase) were detected. A reduction of NO(x) levels and superoxide dismutase activity was observed in l-NAME-treated animals, which was significantly reversed by physical training. Gene expression of subunit gp91(phox) was enhanced by approximately 2-fold in erectile tissue of l-NAME-treated rats, and that was restored to basal levels by run training. CONCLUSIONS Our study shows that ED seen after long-term l-NAME treatment is associated with gp91(phox) subunit upregulation and decreased NO bioavailability. Exercise training reverses the increased oxidative stress in NO-deficient rats, ameliorating the ED.

Exercise training ameliorates the impairment of endothelial and nitrergic corpus cavernosum responses in diabetic rats

AIMS The effect of exercise training (ET) on vascular responsiveness in diabetes mellitus has been largely well studied. However, limited studies have investigated the effects of ET on functional responses of the corpus cavernosum (CC) in diabetic animals. Therefore, the aim of this study was to investigate whether prior ET prevents the impairment of erectile function in streptozotocin-induced diabetic rats. MAIN METHODS Rats were exercised for four weeks prior to the induction of diabetes, and then again for another 4 weeks thereafter. Concentration-response curves to acetylcholine, sodium nitroprusside, Y-27632, BAY 412272 and phenylephrine (PE) were obtained in CC. The excitatory and inhibitory effects of electrical-field stimulation were also evaluated. KEY FINDINGS Plasma SOD levels were markedly decreased in the sedentary diabetic group (D-SD) as compared to control sedentary animals (C-SD), approximately 53% (P<0.05) and this reduction was restored in trained diabetic animals. Physical training restored the impairment of endothelium-dependent and -independent relaxation responses seen in the D-SD group. The potency values for Y-27632 in the CC were significantly reduced in the D-SD group, which was reversed by physical training. The impairment of electrical-field stimulation (EFS)-induced relaxation seen in the D-SD group was restored by physical training. On the other hand, both EFS-induced contractions and concentration-response curves to PE in cavernosal strips were not modified by either diabetes or physical training. SIGNIFICANCE Practice of regular physical exercise may be an important approach in preventing erectile dysfunction associated with diabetes mellitus by re-establishment of the balance between NO production and its inactivation.

Increased Cavernosal Relaxations in Sickle Cell Mice Priapism are Associated with Alterations in the NO‐cGMP Signaling Pathway

INTRODUCTION Priapism is defined as prolonged and persistent penile erection, unassociated with sexual interest or stimulation, and is one of the many serious complications associated with sickle cell disease (SCD). AIM The aim of this study was to evaluate the role of the NO-cGMP signaling pathway in priapism in Berkeley murine model of SCD (SS). METHODS SS mice and C57BL/6 mice (control) penile tissues were removed and the erectile tissue within the corpus cavernosum (CC) was surgically dissected free. The strips were mounted in 10 mL organ baths containing Krebs solution at 37 degrees C (95% O(2), 5% CO(2), pH 7.4), and vertically suspended between two metal hooks. MAIN OUTCOME MEASURES Cumulative concentration-response curves were constructed for acetylcholine (ACh; endothelium-dependent responses), sodium nitroprusside (SNP; endothelium-independent relaxations) and BAY 41-2272 (a potent activator of NO-independent site of soluble guanylate cyclase) in CC precontracted with phenylephrine. Cavernosal responses induced by frequency-dependent electrical field stimulation (EFS) were also carried out to evaluate the nitrergic cavernosal relaxations. RESULTS In SS mice, ACh-induced cavernosal relaxations were leftward shifted by 2.6-fold (P < 0.01) that was accompanied by increases in the maximal responses (78 +/- 5% and 60 +/- 3% in SS and C57B6/6J mice, respectively). Similarly, SNP- and BAY 41-2272-induced CC relaxations were leftward shifted by approximately 3.3- and 2.2-fold (P < 0.01) in SS mice, respectively. A significant increase in maximal responses to SNP and BAY 41-2272 in SS mice was also observed (113 +/- 6% and 124 +/- 5%, respectively) compared with C57B6/6J mice (83 +/- 4% and 99 +/- 2%, respectively). The EFS-induced cavernosal relaxations were also significantly higher SS mice. CONCLUSION These results showed that SS mice exhibit amplified corpus carvenosum relaxation response mediated by NO-cGMP signaling pathway. Intervention in this signaling pathway may be a potential therapeutic target to treat SCD priapism.

Chronic Ethanol Consumption Induces Cavernosal Smooth Muscle Dysfunction in Rats

OBJECTIVES To investigate the effects of chronic ethanol consumption on nitric oxide (NO)-mediated relaxation in rat cavernosal smooth muscle (CSM). METHODS Male wistar rats were divided into 2 groups: control and ethanol. CSM obtained from both groups were mounted in organ chambers for measurement of isometric tension. Contraction of the strips was induced by electrical field stimulation (EFS, 1-32 Hertz) and phenylephrine. We also evaluated the effect of ethanol consumption on the relaxation induced by acetylcholine (0.01-1000 micromol L(-1)), sodium nitroprusside (SNP, 0.01-1000 micromol L(-1)), or EFS (1-32 Hz) in strips precontracted with phenylephrine (10 micromol L(-1)). Blood ethanol, serum testosterone levels, and basal nitrate generation were determined. Immunoexpression of endothelial NO synthase (eNOS) and inducible NO synthase (iNOS) was also accessed. RESULTS Ethanol intake for 4 weeks significantly increased noradrenergic nerve-mediated contractions of CSM in response to EFS. The endothelium-dependent relaxation induced by acetylcholine decreased after the ethanol treatment. Ethanol consumption decreased serum testosterone levels but did not affect the nitrate levels on rat CSM. The mRNA and protein levels for eNOS and iNOS receptors were increased in CSM from ethanol-treated rats. CONCLUSIONS Ethanol consumption reduces endothelium-dependent relaxation induced by acetylcholine, but does not affect SNP or EFS-induced relaxation, suggesting that ethanol disrupts the endothelial function. Despite the overexpression of eNOS and iNOS in ethanol-treated rats, the impaired relaxation induced by acetylcholine may suggest that chronic ethanol consumption induces endothelial dysfunction.

Functional, morphological and molecular characterization of bladder dysfunction in streptozotocin-induced diabetic mice: evidence of a role for L-type voltage-operated Ca2+ channels.

BACKGROUND AND PURPOSE Diabetic cystopathy is one of the most common and incapacitating complications of diabetes mellitus. This study aimed to evaluate the functional, structural and molecular alterations of detrusor smooth muscle (DSM) in streptozotocin‐induced diabetic mice, focusing on the contribution of Ca2+ influx through L‐type voltage‐operated Ca2+ channels (L‐VOCC).

VASORELAXING EFFECTS OF PROPRANOLOL IN RAT AORTA AND MESENTERIC ARTERY: A ROLE FOR NITRIC OXIDE AND CALCIUM ENTRY BLOCKADE

1 Propranolol has been prescribed successfully to patients with cardiovascular diseases, but the exact mechanisms by which it reduces peripheral vascular resistance have been poorly investigated. 2 The present study was designed to investigate the relaxing effects of propranolol in the rat isolated aorta and mesenteric artery, focusing on the contribution of the nitric oxide (NO)–cGMP pathway and calcium entry blockade. Relaxation responses to propranolol were obtained in precontracted rat aortic and mesenteric artery rings. 3 dl‐Propranolol (10–100 mmol/L) produced concentration‐dependent relaxations in the aorta and mesenteric artery rings with intact endothelium. The isomers d‐ and l‐propranolol produced relaxation responses that were equipotent to the racemic mixture. 4 Metoprolol (10–100 mmol/L) produced slight relaxations, whereas atenolol (10–100 mmol/L) had no relaxant activity. 5 The NO inhibitor NG‐nitro‐l‐arginine methyl ester (100 mmol/L) and the soluble guanylate cyclase inhibitor 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (1 mmol/L), as well as removal of the endothelium, significantly reduced the relaxation responses induced by the lower concentrations of propranolol without affecting maximal responses. In addition, dl‐propranolol markedly increased cGMP levels in endothelium‐intact preparations. 6 In Ca2+‐free Krebs’ solution, dl‐propranolol (10–100 mmol/L) caused marked rightward shift in the concentration–response curves to CaCl2, with a decrease of maximal responses in tissues with either intact or denuded endothelium. Nifedipine (1 mmol/L) in combination with dl‐propranolol virtually abolished the CaCl2‐induced contractile responses. 7 The relaxation responses induced by dl‐propranolol were significantly reduced in aortic and mesenteric rings precontracted with phorbol‐12,13‐dibutyrate (1 mmol/L). 8 In conclusion, dl‐propranolol relaxes arterial smooth muscle by mechanisms involving activation of the NO–cGMP pathway and calcium influx blockade, independent of b‐adrenoceptor blockade.

Oxidative stress associated with middle aging leads to sympathetic hyperactivity and downregulation of soluble guanylyl cyclase in corpus cavernosum

Impairment of nitric oxide (NO)-mediated cavernosal relaxations in middle age contributes to erectile dysfunction. However, little information is available about the alterations of sympathetic neurotransmission and contraction in erectile tissue at middle age. This study aimed to evaluate the alterations of the contractile machinery associated with tyrosine hydroxylase (TH) in rat corpus cavernosum (RCC) at middle age, focusing on the role of superoxide anion. Male Wistar young (3.5-mo) and middle-aged (10-mo) rats were used. Electrical-field stimulation (EFS)- and phenylephrine-induced contractions were obtained in RCC strips. Levels of reactive-oxygen species (ROS) and TH mRNA expression, as well as protein expressions for α₁/β₁-subunits of soluble guanylyl cyclase (sGC), in RCC were evaluated. The neurogenic contractile responses elicited by EFS (4-32 Hz) were greater in RCC from the middle-aged group that was accompanied by elevated TH mRNA expression (P < 0.01). Phenylephrine-induced contractions were also greater in the middle-aged group. A 62% increase in ROS generation in RCC from middle-aged rats was observed. The mRNA expression for the α₁A-adrenoceptor remained unchanged among groups. Protein levels of α₁/β₁-sGC subunits were decreased in RCC from the middle-aged compared with young group. The NADPH oxidase inhibitor apocynin (85 mg·rat(-1)·day(-1), 4 wk) fully restored the enhanced ROS production, TH mRNA expressions, and α₁/β₁-subunit sGC expression, indicating that excess of superoxide anion plays a major role in the sympathetic hyperactivity and hypercontractility in erectile tissue at middle age. Reduction of oxidative stress by dietary antioxidants may be an interesting approach to treat erectile dysfunction in aging population.

Long-term oral treatment with BAY 41-2272 ameliorates impaired corpus cavernosum relaxations in a nitric oxide-deficient rat model.

Study Type – Therapy (case control) 
Level of Evidence 3b

Protective effect of prior physical conditioning on relaxing response of corpus cavernosum from rats made hypertensive by nitric oxide inhibition.

The aim of this work was to evaluate the influence of run training on the responsiveness of corpus cavernosum (CC) from rats made hypertensive by treatment with nitric oxide (NO) synthesis inhibitor. Wistar rats were divided into sedentary control (C-SD), exercise training (C-TR), Nω-nitro-L-arginine methyl ester (L-NAME) sedentary (LN-SD) and L-NAME trained (LN-TR) groups. The run training program consisted in 8 weeks in a treadmill, 5 days/week, each session lasted 60 min. L-NAME treatment (2 and 10 mg/rat/day) started after 4 weeks of prior physical conditioning and lasted 4 weeks. Concentration–response curves were obtained for acetylcholine (ACh), sodium nitroprusside (SNP), sildenafil and BAY 41-2272. The effect of electrical field stimulation (EFS) on the relaxations responses of CC was evaluated. Run training prevented the arterial hypertension induced by L-NAME treatment (LN-SD: 135±2 and 141±2 mm Hg for both doses of L-NAME) compared to LN-SD groups (154±1 and 175±2 mm Hg, for 2 and 10 mg of L-NAME, respectively). Run training produced an increase in the maximal responses (Emax) of CC for ACh (C-SD: 47±3; C-TR: 52±1; and LN-TR: 53±3%) and SNP (C-SD: 89±1; C-TR: 98±1; and LN-TR: 95±1%). Both potency and Emax for ACh were reduced in a dose of 10 mg of L-NAME, and run training restored the reduction of Emax for ACh. No changes were found for BAY 41-2271 and sildenafil. Relaxing responses to EFS was reduced by L-NAME treatment that was restored by prior physical conditioning. In conclusion, our study shows a beneficial effect of prior physical conditioning on the impaired CC relaxing responses in rats made hypertensive by chronic NO blockade.