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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), N(omega)-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 (E(max)) 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 E(max) for ACh were reduced in a dose of 10 mg of L-NAME, and run training restored the reduction of E(max) 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.

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 micromol/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 micromol/L) produced slight relaxations, whereas atenolol (10-100 micromol/L) had no relaxant activity. 5. The NO inhibitor N(G)-nitro-L-arginine methyl ester (100 micromol/L) and the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (1 micromol/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 Ca(2+)-free Krebs' solution, DL-propranolol (10-100 micromol/L) caused marked rightward shift in the concentration-response curves to CaCl(2), with a decrease of maximal responses in tissues with either intact or denuded endothelium. Nifedipine (1 micromol/L) in combination with DL-propranolol virtually abolished the CaCl(2)-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 micromol/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 beta-adrenoceptor blockade.

The aim of this study was to investigate the mechanisms of relaxation to the nitric oxide (NO)-independent soluble guanylyl cyclase (sGC) stimulators 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-4-ylamine (BAY 41-2272) and 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) in the rat mesenteric artery. In endothelium-intact rings, BAY 41-2272 (0.0001-1 microM) and YC-1 (0.001-30 microM) caused concentration-dependent relaxations (pEC(50) values of 8.21 +/- 0.05 and 6.75 +/- 0.06, respectively), which were shifted to the right by 6-fold in denuded rings. The sGC inhibitor H-[1,2,4]oxadiazolo [4,3,-a]quinoxalin-1-one (ODQ) (10 microM) partially attenuated the maximal responses to BAY 41-2272 and YC-1 and displaced their curves to the right by 9- to 10-fold in intact and 3-fold in denuded vessels. The NO synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (100 microM) and the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (100 microM) reduced BAY 41-2272 and YC-1 relaxations, whereas the phosphodiesterase type 5 inhibitor sildenafil (0.1 microM) potentiated these responses. The phosphatase inhibitor calyculin A (50 nM) reduced the relaxant responses, and high concentrations of BAY 41-2272 (1 micorM) and YC-1 (10 microM) inhibited Ca(2+)-induced contractions in K(+)-depolarized rings. BAY 41-2272 (0.1 microM) and YC-1 (1 microM) markedly elevated cGMP levels in an ODQ-sensitive manner. Coincubation of BAY 41-2272 or YC-1 with a NO donor resulted in a synergistic inhibition of phenylephrine-induced contractions paralleled by marked increases in cGMP levels. In conclusion, BAY 41-2272 and YC-1 relax the mesenteric artery through cGMP-dependent and -independent mechanisms, including blockade of Ca(2+) influx. The synergistic responses probably reflect the direct effects of NO and NO-independent sGC stimulators on the enzyme, thus representing a potential therapeutic effect by permitting reductions of nitrovasodilator dose.

Decreases in intrinsic NO cause cerebral vasospasms because of the dysregulation of cGMP formation by NO-mediated pathways. Because 5-cyclopropyl-2-{1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl}pyrimidin-4-ylamine (BAY 41-2272) is a potent soluble guanylyl cyclase (sGC) stimulator in an NO-independent manner, this study aimed to investigate the mechanisms underlying the relaxant effects of BAY 41-2272 in the rat basilar artery. BAY 41-2272 (0.0001 to 1 micromol/L) induced relaxations in a concentration-dependent manner, with pEC50 values of 8.13+/-0.03 and 7.63+/-0.05 in intact and denuded rings, respectively. The sGC inhibitor 1H-[1,2,4] oxadiazolo [4,3,-a]quinoxalin-1-one (ODQ) markedly displaced the curve for BAY 41-2272 to the right in intact or denuded rings (&10-fold). The NO synthesis inhibitor NG-nitro-L-arginine methyl ester caused a rightward shift in the curve for BAY 41-2272 (4-fold), whereas the phosphodiesterase type 5 inhibitor sildenafil enhanced BAY 41-2272-induced relaxations (3- to 4-fold). The Na+-K+-ATPase inhibitor ouabain caused 3-fold rightward shifts in the curves for BAY 41-2272. Ca2+-induced contractions in K+ depolarized rings were significantly attenuated by BAY 41-2272 in an ODQ-insensitive manner. The NO donor glyceryl trinitrate and BAY 41-2272 caused rightward shifts in the contractile responses to serotonin. Their coincubation caused a synergistic inhibition of serotonin-induced contractions. BAY 41-2272 and glyceryl trinitrate increased cGMP levels (but not cAMP) by 10-fold and 4-fold above baseline, respectively, in an ODQ-sensitive manner. cGMP levels increased by 50-fold after coincubation. BAY 41-2272 potently relaxes the rat basilar artery in a synergistic fashion with NO. Targeting the sGC with selective activators, such as BAY 41-2272, may represent a new therapy to treat cerebrovascular disease.

Presumably, the vasorelaxant properties of phosphodiesterase type 5 (PDE5) inhibitors are similar in isolated blood vessels. We aimed to explore the mechanisms underlying the vasorelaxation induced by the selective PDE5 inhibitors sildenafil, vardenafil, and tadalafil in the rat aorta. Aortic rings were mounted in 5-ml organ baths, and concentration-response curves for PDE5 inhibitors (0.0001-10 microM) were constructed in phenylephrine (PE)-precontracted endothelium-intact and -denuded rings. Cyclic nucleotides were measured using enzyme immunoassay kits. Sildenafil, vardenafil, and tadalafil concentration dependently relaxed aortic rings and increased cGMP, but not cAMP, concentrations. Endothelium denudation caused marked rightward shifts in the curves to sildenafil (45-fold), tadalafil (21-fold), and vardenafil (251-fold). Maximal responses to sildenafil and tadalafil were substantially reduced (38 +/- 1% and 53 +/- 2%, respectively), whereas that evoked by vardenafil was not affected. Likewise, inhibition of NO synthase (N(omega)-nitro-L-arginine methyl ester, 100 microM), guanylyl cyclase (1H-[1,2,4]oxadiazolo [4,3,-a]quinoxalin-1-one, 10 microM), or scavenging of NO ([carboxy-PTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide), 100 microM]) caused similar attenuation of the vasorelaxations evoked by PDE5 inhibitors. Sildenafil, tadalafil, and vardenafil significantly potentiated relaxations mediated by glyceryl trinitrate (0.0001-3 microM; 8-13-fold) and atrial natriuretic peptide (0.1-100 nM; 2-3-fold). Contractions evoked by CaCl(2) (0.01-5 mM) in PE-treated rings were significantly reduced (26 +/- 4%) by vardenafil, but not sildenafil or tadalafil, whereas phorbol 12,13-dibutyrate-induced contractions were not affected. Ouabain, cyclopiazonic acid, and calyculin A failed to affect vasorelaxations induced by the PDE5 inhibitors. These results suggest that vardenafil, but not sildenafil or tadalafil, affects Ca(2+) handling in the rat aorta in addition to increasing cGMP levels through inhibition of PDE5 to cause relaxation.

The compound BAY 41-2272 stimulates the soluble guanylyl cyclase in a nitric oxide (NO)-independent manner. We have investigated the potency and efficacy of BAY 41-2272 in the rat anococcygeus muscle, as well as the effects of BAY 41-2272 on NO-mediated anococcygeus relaxations. BAY 41-2272 (0.01-10 microM) potently relaxed precontracted anococcygeus muscle strips, with a pEC(50) value of 6.44 +/- 0.03 and maximum response of 100 +/- 2%. The soluble guanylyl cyclase inhibitor 1H-[1,2,4]-oxidiazolo[4,3-a] quinoxalin-1-one (ODQ, 1 microM) and the NO inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 100 microM) caused significant rightward shifts in the concentration-response curves to BAY 41-2272. The phosphodiesterase type-5 inhibitor tadalafil (0.1 microM) markedly enhanced the relaxations evoked by BAY 41-2272. In addition, BAY 41-2272 increased the duration of nitrergic relaxations by approximately 55%. The relaxations induced by glyceryl trinitrate were also significantly potentiated by BAY 41-2272. In conclusion, BAY 41-2272 interacts with endogenous and exogenous NO causing a potent relaxation of rat anococcygeus muscle.

Herein, we report two cases of pneumothorax and tension pneumopericardium after cardiothoracic surgery. Both patients underwent pericardiotomy during the primary operation and developed pericardial tamponade as a complication. The treatment was tube thoracostomy, and both patients recovered completely.

The Rho-kinase pathway mediates Ca2+ sensitization in the penile circulation, which maintains the penis in the flaccid state. We aimed to investigate the functional effect of a novel Rho-kinase inhibitor, H-1152 [(S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine], both in vitro and in vivo as well as to demonstrate the expression of Rho guanine nucleotide exchange factors (RhoGEFs) in the rat corpus cavernosum (CC), by using a semiquantitative reverse transcription-polymerase chain reaction assay to measure their mRNA expression. Cumulative addition of H-1152 (0.001-3 microM) or Y-27632 [0.01-30 microM; (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide] caused sustained relaxations of precontracted CC strips, which were not affected by inhibition of the nitric oxide signaling pathway. Addition of H-1152 (0.1 microM), Y-27632 (1 microM), or sodium nitroprusside (SNP; 0.1 microM) caused rightward shifts in the curves to phenylephrine (PE), but it had little effect on the contractions mediated by electrical field stimulation (EFS). It is noteworthy that when H-1152 or Y-27632 was combined with SNP, a marked synergistic inhibition was noted both on PE- and EFS-induced contractions. Intraperitoneal administration of H-1152 (100 nmol/kg) had a discrete effect on mean arterial pressure and significantly enhanced erectile responses evoked by stimulation of the cavernous nerve. The mRNA expression for PDZ-RhoGEF, p115RhoGEF, and leukemia-associated RhoGEF in cavernosal segments was visualized by electrophoresis on agarose gel. The results indicate that H-1152 is a powerful Rho-kinase inhibitor, giving rise to its therapeutic potential in the treatment of erectile dysfunction. The regulator of G-protein signaling-containing RhoGEFs may represent key components of the molecular mechanisms associated with the abnormal function of the cavernosal smooth muscle.

1. Rho-kinase (ROK) stimulation represents a key step in the maintenance of agonist-induced contraction, an effect counteracted by nitric oxide (NO) released from the endothelium. The aim of the present study was to characterize the involvement of ROK in smooth muscle contraction of the rat coeliac artery using functional and expression studies. 2. Rings of rat coeliac artery were mounted in 5 mL myographs containing warmed and oxygenated Krebs' solution. Rings were connected to isometric transducers and data were recorded in a PowerLab system (ADInstruments, Colorado Springs, CO, USA). After a 60 min equilibration period, preparations were precontracted with phenylephrine (1 micromol/L). Endothelial integrity was assessed by treating the vessels with acetylcholine (1 micromol/L). Expression of ROKalpha, ROKbeta and RhoA was analysed using western blot, whereas Rho guanine nucleotide exchange factors (RhoGEF) were measured at the mRNA level. 3. The addition of Y-27632 (0.01-30 micromol/L) caused sustained relaxation of rings contracted with phenylephrine (PE; 1 micromol/L), with intact or denuded endothelium (pEC50 = 6.38 +/- 0.03 and 5.65 +/- 0.02, respectively). NG-Nitro-L-arginine methyl ester (100 micromol/L) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 micromol/L), but not indomethacin (10 micromol/L), caused marked rightward shifts of the concentration-response curves to Y-27632. The contractile response to KCl (80 mmol/L) was significantly reduced by Y-27632, with a maximal inhibition of 57 +/- 6%. Nifedipine (0.1-100 nmol/L) fully blocked KCl-evoked contractions, but only marginally affected those in response to PE (27 +/- 2% maximal inhibition). At 1 micromol/L, Y-27632 also significantly enhanced relaxations to sodium nitroprusside (SNP; 0.0001-1 micromol/L). 4. At 1 micromol/L, SNP (but not 1 micromol/L Y-27632) significantly elevated the cGMP content above basal levels. Coincubation with SNP and Y-27632 increased cGMP levels, but the results were not significantly different from those in the presence of SNP alone. 5. Western blot analysis revealed the protein expression of RhoA, ROKalpha and ROKbeta. The PDZ-RhoGEF, p115RhoGEF and leukaemia-associated RhoGEF (LARG) mRNA expression in coeliac artery was visualized by electrophoresis on agarose gels. 6. The results clearly demonstrate a role for the RhoA/ROK signalling pathway in the regulation of rat coeliac artery smooth muscle contraction. The findings of the present study suggest that endogenous nitric oxide-induced relaxation is mediated, in part, by inhibition of RhoA/ROK signalling in this tissue.

Beta-Adrenoceptor antagonists may present receptor-independent mechanisms, such as blockade of voltage-gated sodium channels. This study aimed to investigate the effects of non-selective (propranolol), and selective beta1- (atenolol, metoprolol and betaxolol) and beta2-adrenoceptor (ICI 118,551) antagonists in the nitric oxide (NO)-mediated rabbit corpus cavernosum relaxations induced by either electrical field stimulation (EFS) or activators of voltage-gated sodium channels. The sodium channel blockers tetrodotoxin and saxitoxin abolished the relaxations induced by EFS or sodium channel activators of binding site-2 (aconitine and veratridine), site-3 (Ts3 toxin), site-4 (Ts1 toxin) and site-5 (brevetoxin-3). The beta-adrenoceptor antagonists failed to affect the relaxations induced by EFS, aconitine and veratridine. Relaxations induced by Ts3 and Ts1 toxins, as well as brevetoxin-3, were markedly reduced by prior addition of propranolol, betaxolol and ICI 118,551. During the established relaxation induced by Ts3 toxin, propranolol failed to restore the basal tone. In conclusion, beta-adrenoceptor antagonists may cause an allosteric inhibition at the binding site-3, -4 and -5 of voltage-gated sodium channels, leading to blockade of neural NO release.

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. 2. Male New Zealand white rabbits were anaesthetized with pentobarbital sodium. Aortic rings were transferred to 10 mL organ baths containing oxygenated and warmed Krebs' solution. Tissues were connected to force-displacement transducers and changes in isometric force were recorded. Aortic rings were precontracted submaximally with phenylephrine (1 micromol/L). 3. The addition of BAY 41-2272 (0.01-10 micromol/L) to the organ bath produced concentration-dependent relaxations of the aortic rings with a higher potency in endothelium-intact (pEC50 6.59 +/- 0.05) compared with endothelium-denuded (pEC50 6.19 +/- 0.04; P < 0.05) preparations. No differences in maximal responses were observed in either preparation. The NO synthesis inhibitor NG-nitro-L-arginine methyl ester (100 micromol/L) produced a 2.1-fold rightward shift in endothelium-intact (P < 0.01) rings, but had no effect in endothelium-denuded rings. The soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 micromol/L) caused significant rightward shifts of the concentration-response curves to BAY 41-2272 of 4.9- and 2.6-fold in endothelium-intact and -denuded rings, respectively. The phosphodiesterase-5 inhibitor sildenafil (0.1 micromol/L) significantly potentiated the relaxant effects of BAY 41-2272 in both endothelium-intact and -denuded rings. 4. At 1 micromol/L, BAY 41-2272 significantly elevated the aortic cGMP content above basal levels in both endothelium-intact and -denuded rings. Furthermore, ODQ reduced BAY 41-2272-elicited increases in cGMP content by 17 and 90% in endothelium-intact and -denuded rings, respectively (P < 0.01). 5. In conclusion, BAY 41-2272 potently relaxes endothelium-intact and -denuded rabbit aortic rings. The basal release of endothelium-derived NO enhances BAY 41-2272-induced relaxations, suggesting a synergistic effect of BAY 41-2272 and NO on soluble guanylate cyclase. In addition, the endothelium-independent relaxation involves both GMP-dependent and -independent mechanisms.

Stimulation of the RhoA/Rho-kinase (ROK) signaling represents a key step in the maintenance of agonist-induced contraction of smooth muscle. We aimed to demonstrate Ca(2+) sensitization in rat anococcygeus and retractor penis muscles and to identify the molecular expression of major components of this pathway. Both anococcygeus and retractor penis showed a similar expression of RhoA, ROKalpha, and ROKbeta at the protein level as well as the mRNA for RhoGEFs. Cumulative addition of the ROK inhibitors H-1152 (0.001-3 microM), Y-27632 (0.01-30 microM) or HA-1077 (0.01-30 microM) caused sustained relaxations of precontracted smooth muscle strips. Ca(2+) sensitization induced by phenylephrine, norepinephrine and carbachol was markedly antagonized by all three ROK inhibitors. In addition, the contractile response to KCl-induced depolarization was highly sensitive to these ROK inhibitors. H-1152 was approximately 8-20 more potent than Y-27632 and HA-1077 to inhibit contraction. Electrical field stimulation (EFS, 1-32 Hz) caused transient contractions in both anococcygeus and retractor penis muscle, which were blocked by tetrodotoxin (1 microM), phentolamine (1 microM) or bretylium tosylate (30 microM). Similarly, H-1152 (0.1-1 microM), Y-27632 (1-10 microM) or HA-1077 (1-10 microM) significantly reduced EFS-evoked contractions in a concentration-dependent manner. The results indicate that the RhoA/ROK-mediated Ca(2+) sensitization pathway is expressed in anococcygeus and retractor penis muscles and enhances contractions produced by receptor-dependent and independent mechanisms.

Erectile dysfunction (ED) is a highly prevalent and often under-treated condition. Erection is basically a spinal reflex that can be initiated by recruitment of penile afferents but also by visual, olfactory and imaginary stimuli. The generated nervous signals will influence the balance between contractile and relaxant factors, which control the degree of contraction of penile corporal cavernosal smooth muscles and, thus, determine the erectile state of the penis. The different steps involved in neurotransmission, impulse propagation and intracellular transduction of neural signals may be changed in different types of ED. Recent studies have revealed important roles for the small GTPase RhoA and its effector, Rho-kinase in regulating cavernosal smooth muscle tone. The RhoA/Rho-kinase pathway modulates the level of phosphorylation of the myosin light chain, mainly through inhibition of myosin phosphatase, and contributes to agonist-induced Ca(2+)-sensitization in smooth muscle contraction. Changes in this pathway may contribute to ED in various patient subgroups (e.g. hypertension, diabetes, hypogonadism). This review summarizes the importance of Rho-kinase signaling in the erectile response and introduces the evidence pointing to RGS-containing Rho-guanine nucleotide exchange factors (GEFs) as critical mediators of RhoA-GTPase activation in cavernosal smooth muscle and its possible compartmentalization in the caveolae. In addition, we suggest that the design of selective inhibitors of these GEFs might represent a novel class of pharmacological agents to treat ED.

This study was performed to characterize the beta-adrenoceptor population in rabbit isolated corpus cavernosum (RbCC) by using nonselective and selective beta-adrenoceptor agonists and antagonists in functional assays. Metaproterenol, ritodrine, fenoterol, and 8-hydroxy-5-[(1R)-1-hydroxy-2-[N-[(1R)-2-(rho-methoxyphenyl)-1-methylethyl]amino]ethyl]carbostyril (TA 2005) (3-100 nmol each) dose dependently relaxed the RbCC preparations. These relaxations were markedly reduced by N(omega)-nitro-L-arginine methyl ester (L-NAME; 10 microM) and 1H-[1,2,4]-oxadiazolo-[4,3,-a]quinoxalin-1-one (ODQ) (10 microM), whereas the adenylyl cyclase inhibitor SQ 22,536 [9-(2-tetrahydrofuryl) adenine] (10 microM) had no effect. In contrast, neither L-NAME nor ODQ affected the isoproterenol-induced RbCC relaxations, but SQ 22,536 abolished this response. Sildenafil (1 microM) significantly potentiated the relaxations induced by beta(2)-agonists without affecting the isoproterenol-evoked relaxations. Rolipram (10 microM) enhanced the relaxations elicited by isoproterenol but had no effect on those induced by the selective beta(2) agonists. Propranolol and (+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol hydrochloride (ICI 118,551) determined a rightward shift in the concentration-response curves to isoproterenol in a noncompetitive manner with a reduction of maximum response at the highest antagonist concentration, with the slope values significantly different from unity. Propranolol and ICI 118,551 had no effect on the relaxations elicited by fenoterol, TA 2005, metaproterenol, and ritodrine. Atenolol and 1-[2-((3-carbamoyl-4-hydroxy)phenoxy) ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)-phenoxy]-2-propanol methanesulfonate (CGP 20712A) (0.1-10 microM) failed to affect the relaxations induced by all tested beta-adrenoceptor agonists. Our study revealed the existence of two atypical beta-adrenoceptors in the rabbit erectile tissue. Isoproterenol relaxes the rabbit cavernosal tissue by activating atypical beta-adrenoceptors coupled to adenylyl cyclase pathway, whereas the selective beta(2)-adrenoceptor agonists relax the RbCC tissue through another atypical beta-adrenoceptor subtype coupled to nitric oxide release from the sinusoidal endothelium.

Objectives: To evaluate the contractile and relaxing responses in rat corpus cavernosum (RCC) from rats after 8 weeks of run training, because erectile function is highly dependent on nitric oxide (NO) from nitrergic fibers or endothelium. Physical activity enhances NO production and improves endothelial function, with beneficial effects on cardiovascular disease., Methods: The training program consisted of 8 weeks of run training, 5 days/wk, and each session lasted 60 minutes. The RCC was isolated, and concentration-response curves to NO, acetylcholine, sodium nitroprusside, phenylephrine, and endothelin were obtained. The excitatory and inhibitory effects of electrical field stimulation (2 to 32 Hz) were also evaluated., Results: NO (0.1 to 100 microM) and sodium nitroprusside (0.01 to 1000 microM) produced a relaxing effect in RCC in a dose-dependent manner, with the maximal responses to NO (control 62% +/- 4%, trained 88% +/- 3%) and sodium nitroprusside (control 83% +/- 3%, trained 95% +/- 2%) significantly enhanced after 8 weeks of run training. However, acetylcholine-induced relaxations were not affected by exercise. Similarly, electrical field stimulation-induced relaxations were significantly increased in RCC from trained rats at 2 Hz (control 2.4% +/- 0.3%, trained 4.2% +/- 0.5%) and 4 Hz (control 5.3% +/- 1.2%, trained 12.5% +/- 1.7%). The contractile sensitivity of RCC to phenylephrine (0.01 to 100 microM) and endothelin (0.01 to 100 nM) was not modified by training exercise., Conclusions: Our findings suggest that run training enhances functional responses in rat RCC that involves increases in the NO-cyclic guanosine monophosphate signaling pathway by endothelium-independent mechanisms that is not accompanied by changes in contractile sensitivity.

Objectives: To investigate the effects of a purified scorpion toxin (Ts3) on human corpus cavernosum (HCC) in vitro. Scorpion venoms cause a massive release of neurotransmitters that contribute to the clinical symptoms resulting from envenomation., Methods: HCC strips were mounted in organ baths containing Krebs solution. After equilibration, the tissues were precontracted with phenylephrine (10 micromol/L). The relaxations caused by Ts3 (30 nmol/L) were compared with those induced by electrical field stimulation (1 to 20 Hz) and nitric oxide (NO, 1 to 100 micromol/L)., Results: The addition of Ts3 evoked long-lasting relaxations of precontracted HCC strips, and exogenously applied NO and electrical field stimulation caused short-lived responses. The NO synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 100 micromol/L) reduced by 87% +/- 2% the Ts3-induced relaxations; this inhibition was reversed by pretreating the tissues with L-arginine (1 mmol/L). The relaxant responses mediated by Ts3 were blocked to a similar degree by the soluble guanylyl cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3,-alquinoxalin-1-one] (10 micromol/L). In contrast, the addition of the phosphodiesterase type 5 inhibitor sildenafil (0.1 micromol/L) significantly enhanced Ts3-evoked relaxations by 78% +/- 4%. The sodium channel blocker tetrodotoxin (1 micromol/L) completely blocked the relaxant responses elicited by both Ts3 and electrical field stimulation, without significantly affecting those elicited by NO., Conclusions: The results indicate that Ts3 relaxes the HCC through the release of NO from nitrergic nerves. The elucidation of this mechanism is useful for the development of new therapeutic strategies to treat priapism after scorpion envenomation or to modulate sodium channel activity in the case of penile dysfunction.

Phoneutria nigriventer venom (PNV) contracts vascular tissues and increases arterial blood pressure. This study aimed to investigate the mechanisms involved on PNV-induced contractions of rabbit mesenteric and celiac arteries. Strips of mesenteric and celiac arteries were suspended in a cascade system and superfused with warmed and oxygenated Krebs solution. PNV was dialyzed in order to exclude the participation of biogenic amines in the contractions elicited by the venom. Noradrenaline (NA, 30-300 pmol), PNV (1-10 microg), Bay K-8644 (0.3-3 nmol) and KCl (10-100 micromol) dose-dependently contracted the preparations. Ca(2+)-free solution reduced by 38 and 83% the PNV-induced contractions of mesenteric and celiac arteries, respectively. Subsequent infusion of EGTA (0.2 mM) suppressed the residual contractions. Nifedipine (1 microM) and verapamil (10 microM) abolished PNV- and Bay K-8644-evoked contractions, whereas those induced by NA were reduced to a lesser extent. Lanthanum chloride (0.2 mM) inhibited by 75-90% the mesenteric and celiac contractions mediated by PNV. Caffeine (2 mM) fully blocked contractions induced by NA (95% mean inhibition), but only partly reduced those induced by PNV (35% mean inhibition). Ryanodine (10 microM) inhibited by 50% the contractions evoked by NA, but had no effect on the PNV-induced contractions in both tissues. Our findings indicate that PNV contracts vascular smooth muscle mainly due to increased influx of Ca(2+) from extracellular sources.

Scorpion venoms are known to cause peripheral nerve stimulation with enhanced autonomic responses. This study, therefore, examined the effects of Tityus serrulatus venom (TSV) on adrenergic, cholinergic and nitrergic nerve fibers using the rat anococcygeus muscle. The contractile effects of TSV (1 microg/ml) and electrical field stimulation were markedly reduced by phentolamine (5 microM), prazosin (0.1 microM), guanethidine (30 microM) and tetrodotoxin (TTX, 1 microM), whereas imipramine (3 microM) enhanced these responses. The responses to tyramine (10 microM) were partially reduced by guanethidine and completely blocked by phentolamine, prazosin and imipramine. Atropine (1 microM) fully prevented carbachol (CCh, 30 microM)-induced contractions without affecting those mediated by TSV. Neostigmine significantly potentiated TSV-and ACh-evoked contractions, whereas hexamethonium had no effect. The relaxant responses induced by EFS and TSV (3 microg/ml) were completely blocked by L-NAME (100 microM), ODQ (1 microM) or TTX (1 microM). Addition of L-arginine (1 mM) reversed the effect of L-NAME. Thus, the motor and inhibitory responses of TSV in the rat anococcygeus muscle are mediated by prejunctional mechanisms dependent on Na(+) channel activation, causing the stimulation of NA and NO release from adrenergic and nitrergic nerve fibers, respectively.

5-Cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272) is a potent soluble guanylyl cyclase stimulator in a nitric oxide (NO)-independent manner. The relaxant effect of BAY 41-2272 was investigated in rabbit and human corpus cavernosum in vitro. BAY 41-2272 (0.01-10 microM) relaxed both rabbit (pEC(50)=6.82+/-0.06) and human (pEC(50)=6.12+/-0.10) precontracted cavernosal strips. The guanylyl cyclase inhibitor (ODQ, 10 microM) caused significant rightward shifts in the concentration-response curves for BAY 41-2272 in rabbit (4.7-fold) and human (2.3-fold) tissues. The NO synthesis inhibitor (N-nitro-L-arginine methyl ester (L-NAME), 100 microM) also produced similar rightward shifts, revealing that BAY 41-2272 acts synergistically with endogenous NO to elicit its relaxant effect. The results also indicate that ODQ is selective for the NO-stimulated enzyme, since relaxations evoked by BAY 41-2272 were only partly attenuated by ODQ. The present study shows that both BAY 41-2272 and sildenafil evoke relaxations independent of inhibition of haem in soluble guanylate cyclase. Moreover, there is no synergistic effect of the two compounds in corpus cavernosum.

Objectives: To investigate the capacity of voltage-gated Na(+) channel activators such as batrachotoxin, aconitine, veratridine, Ts1 (formerly Tityus gamma-toxin), and brevetoxin-3 to induce relaxation of rabbit isolated corpus cavernosum (RbCC) and the pharmacologic mechanisms underlying this phenomenon. The voltage-gated Na(+) channels of the corpus cavernosum are essential for erectile function. A number of biologic toxins exert their effects by modifying the properties of these channels., Methods: Male New Zealand white rabbits were anesthetized with pentobarbital sodium. Strips of RbCC were transferred to 10-mL organ baths containing oxygenated and warmed Krebs solution. The RbCC strips were connected to force-displacement transducers, and changes in isometric force were recorded using a PowerLab 400 data acquisition system. Corporeal smooth muscle was precontracted submaximally with phenylephrine (10 micromol/L)., Results: The binding site-2 (batrachotoxin, aconitine, and veratridine) and binding site-5 (brevetoxin-3) voltage-gated Na(+) channel activators caused slow-onset RbCC relaxations, and the binding site-4 activator Ts1 produced transitory relaxations followed by a return to baseline. The Na(+)channel blockers tetrodotoxin and saxitoxin (0.1 micromol/L each) abolished the relaxations induced by these agonists. Similarly, the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester (100 micromol/L) markedly reduced the relaxations and l-arginine (1 mmol/L) restored the relaxations. The soluble guanylyl cyclase inhibitor 1H-[1,2,4] oxidiazolo[4,3-alpha] quinoxalin-1-one (10 micromol/L) reduced the relaxations, and the phosphodiesterase type 5 inhibitor sildenafil (100 nmol/L) significantly potentiated the relaxations by all activators., Conclusions: Our results indicate that the relaxations evoked by selective activators of voltage-gated Na(+) channels are mediated by the release of nitric oxide from nitrergic nerves and the activation of the nitric oxide-cyclic guanosine monophosphate pathway in the smooth muscle cells of erectile tissue.

An alpha-toxin responsible for nitric oxide (NO) release in rabbit corpus cavernosum (RbCC) was isolated from Tityus serrulatus venom (TSV). The isolated peptide (molecular mass of 7427.66+/-0.15 Da) was identified as Ts3 after determination of Cys residues, N-terminal amino acid analysis, and proteolytic peptide mapping. Ts3 (30 nM) markedly relaxed the RbCC; this response was blocked by the NO synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (100 microM) and the Na+ channel blocker tetrodotoxin (100 nM). Synthetic peptides based on either Ts3 (P1-16, P17-32, P33-48, P49-64, P9-24, P25-40, P41-56, YGLPDKVPTKT) or Bukatoxin (isolated from Buthus martensi Karsch scorpion venom) sequence (Buka11, Buka11-B, PDKVP, PDSEP) were assayed. These peptides slightly relaxed the RbCC, and such an effect was independent of Na+ channel activation or NO release. Our results indicate that Ts3 exerts nitrergic actions and contributes to the relaxing activity of TSV in RbCC, thus providing a valuable tool to investigate the mechanisms underlying nerve activation in erectile tissues, because NO released from nitrergic fibers plays a key role in the erectile process. Our findings revealed the key importance of the Ts3 structure three-dimensional conformation maintenance for biological activity, because linear peptide sequences neither presented substantial relaxations nor was this effect related to nitrergic activity.

The effects of the Brazilian herbal medicine Catuama and each of its plant constituents (Paullinia cupana, Trichilia catigua, Zingiber officinalis and Ptychopetalum olacoides) were investigated on rabbit corpus cavernosum (RbCC) using a bioassay cascade. Catuama caused short-lived and dose-dependent relaxations (11% +/- 7%, 26% +/- 5% and 82% +/- 9%, at doses of 1, 3 and 10 mg, respectively). Neither the nitric oxide synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 10 microM) nor the soluble guanylate cyclase inhibitor ODQ (10 microM) significantly affected the Catuama-induced relaxations. Similarly, the selective ATP-dependent K(+) channel (K(ATP)) blocker glibenclamide (10 microM), the muscarinic receptor antagonist atropine (1 microM) and the voltage-dependent Na(+) channel blocker tetrodotoxin (1 microM) all failed to affect significantly the Catuama-induced relaxations. These results indicate that the relaxations induced by Catuama involve neither nitric oxide release nor K(ATP) channel activation. The extracts of P. cupana, Z. officinalis and P. olacoides caused short-lived and dose-dependent RbCC relaxations, whereas T. catigua evoked long-lasting relaxations which were occasionally preceded by a brief contractile effect. The extract of P. cupana was the most active in relaxing RbCC strips. The relaxations induced by all extracts were not significantly affected by L-NAME (10 microM). The infusion of ODQ (10 microM) had no significant effect on the P. cupana- and Z. officinalis-induced relaxations but reduced by >50% (p < 0.05) those evoked by P. olacoides and T. catigua. Incubations of RbCC with Catuama(10 mg/mL for 0.25 to 5 min) caused increases of cAMP levels (143% increase at 5 min of incubation). Incubations of RbCC with P. cupana extract (1 mg/mL) increased the cAMP levels by 200% whereas higher doses (10 and 100 mg/mL) caused smaller increases in the nucleotide levels (150% and 89%, respectively). The extracts of Z. officinalis and P. olacoides (same doses) caused smaller increases of the cAMP levels compared with the P. cupana extract, whereas T. catigua (1-100 mg) did not increase the levels of this nucleotide above the basal values. Our results show that of the four extracts assayed, P. cupana was the most effective, indicating that it is the main extract responsible for the relaxing effect of Catuama on rabbit cavernosal tissue., (Copyright 2001 John Wiley & Sons, Ltd.)

In this study, we have investigated the relaxing effects of both Androctonus australis venom (AAV) and Buthotus judaicus venom (BJV) on the rabbit corpus cavernosum (RbCC) smooth muscle strips. The RbCC strips were mounted in a cascade system and superfused with warmed and gassed Krebs solution. The nitric oxide (NO) synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME, 10microM), but not D-NAME (10microM), significantly inhibited the RbCC relaxations induced by acetylcholine (ACh, 0.6nmol), AAV (30microg) and BJV (30microg). Subsequent infusion of L-arginine (300microM), but not of D-arginine (300microM), partially restored the relaxations evoked by these agents. The brain NO synthase inhibitor 7-nitroindazole (7-NI, 10microM) also inhibited the relaxant responses elicited by the scorpion venoms. The guanylyl cyclase inhibitors methylene blue (MB, 30microM) and 1H-[1,2,4] oxadiazolo [4,3,-alquinoxalin-1-one] (ODQ, 10microM) virtually abolished the relaxations induced by either AAV or BJV. The infusion of muscarinic receptor antagonists such as scopolamine and atropine (1microM, each) completely abolished the ACh-induced relaxations but had no effect on those evoked by the scorpion venoms. The Na(+) channel blocker tetrodotoxin (1microM) prevented the relaxations evoked by both AAV and BJV. Thus, NO released from nitrergic nerve fibres mediates the relaxations elicited by AAV and BJV in the rabbit cavernosal tissue.

Objectives: To examine the effects of Tityus serrulatus scorpion venom (TSV) on human corpus cavernosum (HCC) using a bioassay cascade. Priapism is occasionally observed in scorpion envenomation, mostly in children., Methods: HCC strips were suspended in a cascade system and superfused with aerated and warmed Krebs' solution at 5 mL/min. Noradrenaline (3 micromol/L) was infused to induce a submaximal contraction of the HCC strips. The release of cyclooxygenase products was prevented by infusing indomethacin (6 micromol/L)., Results: N(omega)-nitro-L-arginine methyl ester (10 micromol/L; n = 10) increased the tone of the preparations and significantly reduced (P <0.01) the acetylcholine (ACh) and TSV-induced relaxations. Subsequent infusion of L-arginine (300 micromol/L) partially reversed the increased tone and significantly restored the relaxations induced by TSV and ACh (P <0.01). The soluble guanylyl cyclase inhibitor ODQ (10 micromol/L; n = 8) markedly reduced (P <0.01) the relaxations induced by TSV, ACh, glyceryl trinitrate, and bradykinin. 7-Nitroindazole (10 micromol/L; n = 8) inhibited the relaxations induced by TSV by 84% (P <0.01) and also caused small, but significant, reductions in the ACh and bradykinin-induced HCC relaxations (P <0.05). Atropine (1 micromol/L; n = 6) abolished the relaxations evoked by ACh (P <0.01), but had no effect on those elicited by TSV. Tetrodotoxin (1 micromol/L; n = 6) abolished the relaxations induced by TSV (P <0.01) and also reversed the established TSV-induced relaxation (n = 4)., Conclusions: Our results indicate that TSV relaxes HCC through the release of nitric oxide from nonadrenergic, noncholinergic (NANC) nerves. The elucidation of the mechanism responsible for the TSV-induced relaxations might be useful for a better understanding of the development of priapism in cases of scorpion envenomation.

Myelomeningocele occurs in 0.4 for 1000 neonates and is associated with hydrocephalus in 85-90%, and reports on cognition are sparsely found in literature. Forty five children with treated hydrocephalus and myelomeningocele were studied in regard of IQ, and statistically correlated to functional motor level, age of the first shunt, number of revisions of shunt, infection of the shunt and circumference of the head. The medium age was of 7.5 years (3-15 years), 16 males and 29 females. Three (6.6%) had a IQ score > 110, 11 (24.4%) had a score between 100-110, 8 between 85-100 (17.7%), 16 (35.5%) between 85-100 (17.7%) and 7 (15.5%) between 50-70. IQ directly correlated with motor level, having better cognitive results the children with minor functional motor disabilities. Cognition was best in children operated until the seven day of life (t 0.0099), with progressive worse results in children operated after the first month of life, no significance was observed in children operated in the period 7 to 31 days (t 0.1013). Worse results were observed in the group of patients with infection of shunts (t 0.0146). Results were progressively worse with reoperations. The best results in relation of the circumference of the head were seen with children in the medium range (t 0.0115); intermediate results were seen in patients between the medium range and-1SD (t 0.00130) and medium range and +1SD. The worse results were seen in patients at the extremes of > 1SD (t 0.0269) and < ISD (t 0.0042). According to cognitive results the surgical treatment of hydrocephalus have to be done until the first month of life, avoiding reoperations and infections that have unfavorable impact in IQ.

The influence of nitric oxide (NO) on eosinophil infiltration into the airways was investigated in rats actively sensitized with ovalbumin. The animals were treated chronically with the NO synthase inhibitor, N omega-Nitro-L-arginine methyl ester (L-NAME; 75 mumol rat-1 day-1), for 4 weeks. Bronchoalveolar lavage was performed at 6, 24, 48 and 72 h after intratracheal injection of ovalbumin. Intratracheal challenge of the sensitized rats with ovalbumin caused a significant increase in total leucocyte infiltration in bronchoalveolar lavage fluid both 24 and 48 h post-ovalbumin injection. Neutrophils and eosinophils peaked, respectively, at 24 h (29%) and 48 h (30%) in bronchoalveolar lavage fluid whereas the mononuclear cell did not differ significantly from the counts in non-sensitized rats at any time. At both 6 and 24 h post-ovalbumin injection, the chronic treatment of the animals with L-NAME affected neither the total nor the differential leucocyte content. However, at 48 h post-ovalbumin challenge, the total cell count was reduced by approximately 48% in the L-NAME-treated animals and this was associated with a marked inhibition (81%) of the eosinophil influx. Histological examination of the lungs from these animals (48 h post-ovalbumin challenge) also showed a prominent reduction (69.5%; P < 0.05) of the eosinophil infiltration in the respiratory segments. Our results demonstrate that NO plays a pivotal role in the eosinophil infiltration in airways of actively sensitized rats.

Objective: To characterize the kinin receptor subtype involved in the relaxation of human isolated corpus cavernosum (HCC) induced by bradykinin (BK), Lys-bradykinin (Lys-BK), Met-Lys-bradykinin (Met-Lys-BK) and des-Arg9-bradykinin, and to investigate whether the kinin-induced relaxation of HCC results from the stimulation of nonadrenergic, noncholinergic (NANC) neurons supplying the cavernosal tissue., Materials and Methods: Excised HCC tissues were immediately placed in Krebs solution and kept at 4 degrees C until use (never > 24 h after removal). HCC was cut in strips of approximately 2 cm, suspended in a cascade system and superfused with oxygenated and warmed Krebs solution at 5 mL/min. After equilibration for approximately 90 min, noradrenaline (3 micromol/L) was infused to induce a submaximal contraction of the HCC strips. The release of cyclo-oxygenase products was prevented by infusing indomethacin (6 micromol/L). HCC strips were calibrated by injecting a single bolus of the nitrovasodilator glyceryl trinitrate (GTN) and the sensitivity of the tissues adjusted electronically to be similar. The agonists (kinins, histamine and acetylcholine) were injected as a single bolus (up to 100 microL) and the relaxation of HCC expressed as a percentage of the submaximal relaxation induced by GTN., Results: Bradykinin, Lys-BK and Met-Lys-BK significantly relaxed the HCC tissues; on a molar basis, there was no statistical difference among the degrees of relaxation induced by these peptides. The B1 kinin receptor agonist des-Arg9-bradykinin had no effect on the HCC. The infusion of the B2 kinin receptor antagonist Hoe 140 (50 nmol/L) virtually abolished the relaxation induced by BK, Lys-BK and Met-Lys-BK without affecting those induced by acetylcholine and histamine. The infusion of the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester increased the tone of the HCC tissues and significantly reduced (P < 0.01) the relaxation induced by BK (74%), Lys-BK (90%), Met-Lys-BK (87%) and acetylcholine (89%) without affecting those induced by GTN. The subsequent infusion of L-arginine (300 micromol/L) partially reversed the increased tone and significantly (P < 0.01) restored the relaxation induced by BK, Lys-BK and Met-Lys-BK. The results were similar with the novel guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo[4,3,-alquinoxalin-1-one] which reduced by > 95% (P < 0.01) the relaxation induced by BK, Lys-BK, Met-Lys-BK, acetylcholine and GTN. The infusion of the sodium-channel blocker tetrodotoxin had no significant effect on the BK-, GTN- and acetylcholine-induced relaxation of HCC., Conclusion: This study clearly showed the existence of functional B2 kinin receptors in human erectile tissues that when activated lead to the release of NO and hence relaxation of the HCC tissues. As tetrodotoxin failed to affect the kinin-induced relaxation of HCC strips, it is likely that these peptides release NO from the endothelium of sinusoidal capillaries rather than from neuronal sources supplying the cavernosal tissue. Although tissue kallikreins and their components have been found in the male reproductive system, the physiopathological importance of these findings has yet to be elucidated.

1. The effect of Tityus serrulatus scorpion venom and its toxin components on the rabbit isolated corpus cavernosum was investigated by use of a bioassay cascade. 2. Tityus serrulatus venom (3-100 microg), acetylcholine (ACh; 0.3-30 nmol) and glyceryl trinitrate (GTN; 0.5-10 nmol) dose-dependently relaxed rabbit isolated corpus cavernosum preparations precontracted with noradrenaline (3 microM). The selective soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3,-alquinoxalin-1-one] (ODQ; 30 microM) increased the basal tone of the rabbit isolated corpus cavernosum and abolished the relaxations induced by the agents mentioned above. Methylene blue (30 microM) also inhibited the relaxations induced by Tityus serrulatus venom but, in contrast to ODQ, the inhibition was irreversible. 3. The non-selective NO synthase (NOS) inhibitors Nomega-nitro-L-arginine methyl ester (L-NAME; 10 microM) and NG-iminoethyl-L-ornithine (L-NIO; 30 microM) also increased the tone of the rabbit isolated corpus cavernosum and markedly reduced both ACh- and Tityus serrulatus venom-induced relaxations without affecting those evoked by GTN. The inhibitory effect was reversed by infusion of L-arginine (300 microM), but not D-arginine (300 microM). The neuronal NOS inhibitor 1-(2-trifluoromethylphenyl) imidazole (TRIM, 100 microM) did not affect either the tone of the rabbit isolated corpus cavernosum or the relaxations induced by ACh, bradykinin (Bk), Tityus serrulatus venom and GTN. TRIM was approximately 1,000 times less potent than L-NAME in inhibiting rabbit cerebellar NOS in vitro, as measured by the conversion of [3H]-L-arginine to [3H]-L-citrulline. 4. The protease inhibitor aprotinin (Trasylol; 10 microg ml[-1]) and the bradykinin B2 receptor antagonist Hoe 140 (D-Arg-[Hyp3,Thi5,D-Tic7, Oic8]-BK; 50 nM) did not affect the rabbit isolated corpus cavernosum relaxations induced by Tityus serrulatus venom. The ATP-dependent K+ channel antagonist glibenclamide (10 microm) and the Ca2+-activated K+ channel antagonists apamin (0.1 microM) and charybdotoxin (0.1 microM) also failed to affect the venom-induced relaxations. Similarly, the K+ channel blocker tetraethylammonium (TEA; 10 microM) had no effect on the venom-induced relaxations. 5. Capsaicin (3 and 10 nmol) relaxed the rabbit isolated corpus cavernosum in a dose-dependent and non-tachyphylactic manner. Ruthenium red (30 microM), an inhibitor of capsaicin-induced responses, markedly reduced the relaxations caused by capsaicin, but failed to affect those induced by Tityus serrulatus venom. L-NAME (10 microM) had no effect on the capsaicin-induced relaxations of the rabbit isolated corpus cavernosum. 6. The sodium channel blocker tetrodotoxin (TTX; 1 microM) abolished the relaxations of the rabbit isolated corpus cavernosum induced by Tityus serrulatus venom without affecting those evoked by capsaicin, ACh and GTN. Tetrodotoxin (1 microM) also promptly reversed the response to the venom when infused during the relaxation phase. 7. The bioassay cascade of the toxin components purified from Tityus serrulatus venom revealed that only fractions X, XI and XII caused dose-dependent relaxations of the rabbit isolated corpus cavernosum and these were markedly reduced by either TTX (1 microM) or L-NAME (10 microM). 8. Our results indicate that Tityus serrulatus scorpion venom (and the active fractions X, XI and XII) relaxes rabbit corpus cavernosum via the release of NO. This release is specifically triggered by the activation of capsaicin-insensitive cavernosal non-adrenergic non-cholinergic (NANC) fibres, that may possibly be nitrergic neurones. Tityus serrulatus venom may therefore provide an important tool for understanding further the mechanism of NANC nitrergic nerve activation.

Paw edema was induced in male Wistar rats (200-250 g) by intraplantar (ipl) administration of 2.5 micrograms endotoxin (Etx). Etx, like carrageenin, produced two distinct edema formation phases, an early phase (75 min) followed by a late phase (7 h). We showed that the edema formation in the early phase was antagonized by dipyrone (80 mg/kg, i.p.) and indomethacin (1 mg/kg, i.p.) by 52% and 55%, respectively, and that the late phase was resistant to these drugs. These results suggest that in the early phase prostaglandins appear to be involved in the process. However, the activation of the kinin cascade leading to the release of other mediators may be involved in the increase of edema in the late phase. To test this hypothesis, we investigated whether the release of nitric oxide (NO) is involved in the mechanism of endotoxin-induced rat paw edema during the late phase, using N omega-nitro-L-arginine methyl ester (L-NAME) (50 micrograms, ipl) as inhibitor of NO synthase and L-arginine (1 mg, ipl) as substrate of NO synthase. The paw edema induced by Etx was inhibited by L-NAME by 56% and increased by L-arginine by 81%. Furthermore, L-arginine given in combination with L-NAME completely reversed the inhibition of Etx-induced edema produced by L-NAME. These results support the hypothesis that in the late phase NO production is associated with the edema evoked by Etx.