Cleber E. Teixeira

Nitric oxide modulates eosinophil infiltration in antigen-induced airway inflammation in rats.

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.

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.

Vasorelaxing effect of BAY 41-2272 in rat basilar artery : Involvement of cGMP-dependent and independent mechanisms

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 &mgr;mol/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.

Vardenafil, but not sildenafil or tadalafil, has calcium-channel blocking activity in rabbit isolated pulmonary artery and human washed platelets

Phosphodiesterase type‐5 (PDE5) inhibitors constitute a novel and important therapeutic option for the treatment of pulmonary hypertension. The effects of the PDE5 inhibitors sildenafil, tadalafil and vardenafil on rabbit isolated pulmonary artery ring preparations and on intracellular Ca2+ concentration of thrombin‐stimulated human platelets were investigated.

Pharmacological characterization of a novel phosphodiesterase type 5 (PDE5) inhibitor lodenafil carbonate on human and rabbit corpus cavernosum

Nitrergic nerves and endothelial cells release nitric oxide (NO) in the corpus cavernosum, a key mediator that stimulates soluble guanylyl cyclase to increase cGMP levels causing penile erection. Phosphodiesterase 5 (PDE5) inhibitors, such as sildenafil, prolong the NO effects by inhibiting cGMP breakdown. Here, we report a novel PDE5 inhibitor, lodenafil carbonate, (Bis-(2-{4-[4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-benzenesulfonyl]piperazin-1-yl}-ethyl)carbonate) that is a dimer of lodenafil. We therefore aimed to compare the effects of sildenafil, lodenafil and lodenafil carbonate on in vitro human and rabbit cavernosal relaxations, activity of crude PDE extracts from human platelets, as well as stability and metabolic studies in rat, dog and human plasma. Pharmacokinetic evaluations after intravenous and oral administration were performed in male beagles. Functional experiments were conducted using organ bath techniques. Pharmacokinetics was studied in beagles by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), following oral or intravascular administration. All PDE5 inhibitors tested concentration-dependently relaxed (0.001-100 microM) phenylephrine-precontracted rabbit and human corpus cavernosum. The cavernosal relaxations evoked by either acetylcholine (0.01-100 microM) or electrical field stimulation (EFS, 1-20 Hz) were markedly potentiated by sildenafil, lodenafil and lodenafil carbonate. Lodenafil carbonate was more potent to inhibit the cGMP hydrolysis in PDE extracts compared with lodenafil and sildenafil. Following intravascular and single oral administration of lodenafil carbonate, only lodenafil and norlodenafil were detected in vivo. These results indicate that lodenafil carbonate works as a prodrug, being lodenafil the active moiety of lodenafil carbonate.

Effect of Tityus serrulatus scorpion venom on the rabbit isolated corpus cavernosum and the involvement of NANC nitrergic nerve fibres

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. Tityus serrulatus venom (3–100 μg), 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 μM). The selective soluble guanylate cyclase inhibitor 1H‐[1,2,4] oxadiazolo [4,3,‐alquinoxalin‐1‐one] (ODQ; 30 μM) increased the basal tone of the rabbit isolated corpus cavernosum and abolished the relaxations induced by the agents mentioned above. Methylene blue (30 μM) also inhibited the relaxations induced by Tityus serrulatus venom but, in contrast to ODQ, the inhibition was irreversible. The non‐selective NO synthase (NOS) inhibitors NΩ‐nitro‐L‐arginine methyl ester (L‐NAME; 10 μM) and NG‐iminoethyl‐L‐ornithine (L‐NIO; 30 μM) 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 μM), but not D‐arginine (300 μM). The neuronal NOS inhibitor 1‐(2‐trifluoromethylphenyl) imidazole (TRIM, 100 μM) 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. The protease inhibitor aprotinin (Trasylol; 10 μg 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 μM) and the Ca2+‐activated K+  channel antagonists apamin (0.1 μM) and charybdotoxin (0.1 μM) also failed to affect the venom‐induced relaxations. Similarly, the K+ channel blocker tetraethylammonium (TEA; 10 μM) had no effect on the venom‐induced relaxations. Capsaicin (3 and 10 nmol) relaxed the rabbit isolated corpus cavernosum in a dose‐dependent and non‐tachyphylactic manner. Ruthenium red (30 μM), 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 μM) had no effect on the capsaicin‐induced relaxations of the rabbit isolated corpus cavernosum. The sodium channel blocker tetrodotoxin (TTX; 1 μM) 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 μM) also promptly reversed the response to the venom when infused during the relaxation phase. 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 μM) or L‐NAME (10 μM). 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.

Oxidative Stress Impairs Vasorelaxation Induced by the Soluble Guanylyl Cyclase Activator BAY 41-2272 in Spontaneously Hypertensive Rats

BACKGROUND BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine) relaxes mesenteric arteries (MA) in a synergistic fashion with nitric oxide (NO). We hypothesized that the relaxation to BAY 41-2272 is decreased in spontaneously hypertensive rats (SHR) because of the reduced NO bioavailability in this strain and that relaxation would be improved by inhibiting the oxidative stress. We aimed to evaluate the influence of oxidative stress in BAY 41-2272-induced vasorelaxation in isolated MA from SHR. METHODS MA function was evaluated by concentration-response curves to BAY 41-2272. We measured protein expression of endothelial NO synthase (eNOS), soluble guanylyl cyclase (sGC) and human-antigen R (HuR) (sGC mRNA-stabilizing protein), sGC activity and plasma levels of superoxide dismutase (SOD), and total antioxidant status (TAS). RESULTS Cyclic guanosine monophosphate (cGMP)-dependent and -independent relaxation induced by BAY 41-2272 (0.0001-1 micromol/l) was impaired in SHR compared with Wistar-Kyoto (WKY). We observed reduced expression of eNOS, sGC and HuR, and decreased sGC activity in SHR. Plasma levels of SOD and TAS were also diminished in SHR. Incubation with SOD or indomethacin increased relaxation to BAY 41-2272 in SHR. Furthermore, acetylcholine (ACh)-induced relaxation was increased in the presence of BAY 41-2272 or SOD, apocynin, or indomethacin. CONCLUSION Augmented oxidative stress in SHR impaired cGMP-dependent and -independent relaxation induced by BAY 41-2272, by decreasing NO bioavailability and sGC expression and by increasing contractile activity. Inhibiton of oxidative stress improved the relaxation of BAY 41-2272 in SHR. BAY 41-2272 might be an alternative therapeutic tool for hypertension if administrated with antioxidant compounds.

Nitric oxide release from human corpus cavernosum induced by a purified scorpion toxin.

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.

Up-regulation of the RhoA/Rho-kinase signaling pathway in corpus cavernosum from endothelial nitric-oxide synthase (NOS), but not neuronal NOS, null mice.

We tested the hypothesis that the basal release of nitric oxide (NO) from endothelial cells modulates contractile activity in the corpus cavernosum (CC) via inhibition of the RhoA/Rho-kinase signaling pathway. Cavernosal strips from wild-type (WT), endothelial nitric-oxide synthase knockout [eNOS(−/−)], and neuronal nitric-oxide synthase knockout [nNOS(−/−)] mice were mounted in myographs, and isometric force was recorded. mRNA and protein expression of key molecules in the RhoA/Rho-kinase pathway were analyzed by real-time polymerase chain reaction and Western blot, respectively. The cGMP levels were determined. The Rho-kinase inhibitors (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) and (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl] homopiperazine (H-1152) reduced cavernosal contractions evoked by phenylephrine or electrical field stimulation (EFS) in a concentration-dependent manner, although this inhibition was less effective in tissues from eNOS(−/−) mice. Y-27632 enhanced relaxations induced by sodium nitroprusside, EFS, and NO (administered as acidified NaNO2) without affecting the cGMP content of the cavernosal strips. This enhancement was less prominent in CC from eNOS(−/−). The protein expression of RhoA, Rho-guanine dissociation inhibitor, and Rho-kinase β did not differ among the strains. However, in eNOS(−/−) CC, the protein expression of Rho-kinase α and both mRNA and protein expression of p115-Rho-associated guanine exchange factor (RhoGEF), PDZ-RhoGEF, and leukemia-associated RhoGEF were up-regulated. Phosphorylation of MYPT1 at Thr696 was higher in tissues from eNOS(−/−) mice. A high concentration of Y-27632 significantly enhanced NO release in CC stimulated by EFS. These results suggest a basal release of NO from endothelial cells, which inhibits contractions mediated by the RhoA/Rho-kinase pathway and modulates the expression of proteins related to this pathway in mouse CC. It indicates that endothelial integrity is essential to the maintenance of erectile function.

Effects of 5-Cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4-ylamine (BAY 41-2272) on Smooth Muscle Tone, Soluble Guanylyl Cyclase Activity, and NADPH Oxidase Activity/Expression in Corpus Cavernosum from Wild-Type, Neuronal, and Endothelial Nitric-Oxide Synthase Null Mice

We aimed to characterize the relaxation induced by the soluble guanylyl cyclase (sGC) stimulator 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]pyrimidin-4-ylamine (BAY 41-2272) and its pharmacological interactions with nitric oxide (NO) in the corpus cavernosum (CC) from wild-type (WT), endothelial nitric-oxide synthase (eNOS)–/–, and neuronal (n)NOS–/– mice. The effect of BAY 41-2272 on superoxide formation and NADPH oxidase expression was also investigated. Tissues were mounted in myographs for isometric force recording. Enzyme immunoassay kits were used for cGMP determination. sGC activity was determined in the supernatant fractions of the cavernosal samples by the conversion of GTP to cGMP. Superoxide formation and expression of NADPH oxidase subunits were studied using the reduction of ferricytochrome c and Western blot analysis, respectively. BAY 41-2272 (0.01–10 μM) relaxed CC with pEC50 values of 6.36 ± 0.07 (WT), 6.27 ± 0.06 (nNOS–/–), and 5.88 ± 0.07 (eNOS–/–). The relaxations were accompanied by increases in cGMP levels. Nω-Nitro-l-arginine methyl ester inhibited BAY 41-2272-evoked responses in CC from WT and nNOS–/–, but not eNOS–/–.1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one reduced and sildenafil potentiated the relaxations induced by BAY 41-2272 in all groups. BAY 41-2272 enhanced NO (endogenous and exogenous)-induced relaxations in a concentration-dependent manner. Expression and activity of sGC was similar among the different groups. Superoxide formation was reduced by BAY 41-2272 (0.1–1 μM). The compound also inhibited p22phox and gp91phox expression induced by 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F2α (U46619). Our results demonstrated that sGC activation in the penis by BAY 41-2272 directly or via enhancement of NO effects may provide a novel treatment for erectile dysfunction, particularly in the event of an increased intrapenile oxidative stress.