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Find video protocols related to scientific articles indexed in Pubmed.
Oxidative stress associated with middle aging leads to sympathetic hyperactivity and downregulation of soluble guanylyl cyclase in corpus cavernosum.
Am. J. Physiol. Heart Circ. Physiol.
PUBLISHED: 09-12-2014
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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 ?1/?1-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 ?1A-adrenoceptor remained unchanged among groups. Protein levels of ?1/?1-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 ?1/?1-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.
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Prolonged Therapy with the Soluble Guanylyl Cyclase Activator BAY 60-2770 Restores the Erectile Function in Obese Mice.
J Sex Med
PUBLISHED: 09-05-2014
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Cardiovascular and endocrine-metabolic diseases associated with increased oxidative stress such as obesity lead to erectile dysfunction (ED). Activators of soluble guanylyl cyclase (sGC) such as BAY 60-2770 reactivate the heme-oxidized sGC in vascular diseases.
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The soluble guanylyl cyclase activator BAY 60-2770 potently relaxes the pulmonary artery on congenital diaphragmatic hernia rabbit model.
Pediatr. Surg. Int.
PUBLISHED: 07-16-2014
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Congenital diaphragmatic hernia (CDH) is associated with pulmonary hypertension which is often difficult to manage, and a significant cause of morbidity and mortality. In this study, we have used a rabbit model of CDH to evaluate the effects of BAY 60-2770 on the in vitro reactivity of left pulmonary artery.
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L-Carnitine supplementation impairs endothelium-dependent relaxation in mesenteric arteries from rats.
Arch. Physiol. Biochem.
PUBLISHED: 06-23-2014
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L-Carnitine (L-Car) is taken as fat burner. The risks of L-Car supplementation for the cardiovascular system are unclear. We evaluated the relaxing responses of the mesenteric and aorta rings from rats after four weeks of L-Car supplementation and/or physical training. Concentration response curves to acetylcholine (ACh) and sodium nitroprusside (SNP), as well as cyclic GMP levels, superoxide dismutase (SOD) activity and malondialdehyde (MDA) were evaluated. Physical training decreased body weight gain that was potentiated by L-Car. In mesenteric rings, L-Car impaired endothelium-dependent relaxation whereas endothelium independent relaxation was increased. In aorta, exercise improved endothelium-dependent relaxation; however, it was partially inhibited by L-Car. SNP-induced relaxation was similar in aorta of all groups. Basal cGMP were increased in aorta of exercised rats. SOD activity and MDA levels were unaltered. In conclusion, L-Car and physical exercise promotes body weight loss; however, it impairs endothelium-dependent vaso-relaxation possibly involving alterations in muscarinic receptors/eNOS/NO signalling pathway in mesenteric artery.
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Blockade of renin-angiotensin system prevents micturition dysfunction in renovascular hypertensive rats.
Eur. J. Pharmacol.
PUBLISHED: 03-24-2014
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Association between hypertension and bladder symptoms has been described. We hypothesized that micturition dysfunction may be associated with renin-angiotensin system (RAS) acting in urethra. The effects of the anti-hypertensive drugs losartan (AT1 antagonist) and captopril (angiotensin-converting enzyme inhibitor) in comparison with atenolol (?1-adrenoceptor antagonist independently of RAS blockade) have been investigated in bladder and urethral dysfunctions during renovascular hypertension in rats. Two kidney-1 clip (2K-1C) rats were treated with losartan (30 mg/kg/day), captopril (50mg/kg/day) or atenolol (90 mg/kg/day) for eight weeks. Cystometric study, bladder and urethra smooth muscle reactivities, measurement of cAMP levels and p38 MAPK phosphorylation in urinary tract were determined. Losartan and captopril markedly reduced blood pressure in 2K-1C rats. The increases in non-voiding contractions, voiding frequency and bladder capacity in 2K-1C rats were prevented by treatments with both drugs. Likewise, losartan and captopril prevented the enhanced bladder contractions to electrical-field stimulation (EFS) and carbachol, along with the impaired relaxations to ?-adrenergic-cAMP stimulation. Enhanced neurogenic contractions and impaired nitrergic relaxations were observed in urethra from 2K-1C rats. Angiotensin II also produced greater urethral contractions that were accompanied by higher phosphorylation of p38 MAPK in urethral tissues of 2K-1C rats. Losartan and captopril normalized the urethral dysfunctions in 2K-1C rats. In contrast, atenolol treatment largely reduced the blood pressure in 2K-1C rats but failed to affect the urinary tract smooth muscle dysfunction. The urinary tract smooth muscle dysfunction in 2K-1C rats takes place by local RAS activation irrespective of levels of arterial blood pressure.
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Soluble guanylyl cyclase (sGC) degradation and impairment of nitric oxide-mediated responses in urethra from obese mice: reversal by the sGC activator BAY 60-2770.
J. Pharmacol. Exp. Ther.
PUBLISHED: 01-13-2014
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Obesity has emerged as a major contributing risk factor for overactive bladder (OAB), but no study examined urethral smooth muscle (USM) dysfunction as a predisposing factor to obesity-induced OAB. This study investigated the USM relaxant machinery in obese mice and whether soluble guanylyl cyclase (sGC) activation with BAY 60-2770 [acid 4-({(4-carboxybutyl) [2-(5-fluoro-2-{[4-(trifluoromethyl) biphenyl-4-yl] methoxy} phenyl) ethyl] amino} methyl) benzoic] rescues the urethral reactivity through improvement of sGC-cGMP (cyclic guanosine monophosphate) signaling. Male C57BL/6 mice were fed for 12 weeks with a high-fat diet to induce obesity. Separate groups of animals were treated with BAY 60-2770 (1 mg/kg per day for 2 weeks). Functional assays and measurements of cGMP, reactive-oxygen species (ROS), and sGC protein expression in USM were determined. USM relaxations induced by NO (acidified sodium nitrite), NO donors (S-nitrosoglutathione and glyceryl trinitrate), and BAY 41-2272 [5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine] (sGC stimulator) were markedly reduced in obese compared with lean mice. In contrast, USM relaxations induced by BAY 60-2770 (sGC activator) were 43% greater in obese mice (P < 0.05), which was accompanied by increases in cGMP levels. Oxidation of sGC with ODQ [1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one] (10 ?M) potentiated BAY 60-2770-induced USM responses in the lean group. Long-term oral BAY 60-2770 administration fully prevented the impairment of USM relaxations in obese mice. Reactive-oxygen species (ROS) production was enhanced, but protein expression of ?1 second guanylate cyclase subunit was reduced in USM from obese mice, both of which were restored by BAY 60-2770 treatment. In conclusion, impaired USM relaxation in obese mice is associated with ROS generation and down-regulation of sGC-cGMP signaling. Prevention of sGC degradation by BAY 60-2770 ameliorates the impairment of urethral relaxations in obese mice.
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Menthol Inhibits Detrusor Contractility Independently of TRPM8 Activation.
PLoS ONE
PUBLISHED: 01-01-2014
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Agonists such as icilin and menthol can activate the cool temperature-sensitive ion channel TRPM8. However, biological responses to menthol may occur independently of TRPM8 activation. In the rodent urinary bladder, menthol facilitates the micturition reflex but inhibits muscarinic contractions of the detrusor smooth muscle. The site(s) of TRPM8 expression in the bladder are controversial. In this study we investigated the regulation of bladder contractility in vitro by menthol. Bladder strips from wild type and TRPM8 knockout male mice (25-30 g) were dissected free and mounted in organ baths. Isometric contractions to carbachol (1 nM-30 µM), CaCl2 (1 µM to 100 mM) and electrical field stimulation (EFS; 8, 16, 32 Hz) were measured. Strips from both groups contracted similarly in response to both carbachol and EFS. Menthol (300 µM) or nifedipine (1 µM) inhibited carbachol and EFS-induced contractions in both wild type and TRPM8 knockout bladder strips. Incubation with the sodium channel blocker tetrodotoxin (1 µM), replacement of extracellular sodium with the impermeant cation N-Methyl-D-Glucamine, incubation with a cocktail of potassium channel inhibitors (100 nM charybdotoxin, 1 µM apamin, 10 µM glibenclamide and 1 µM tetraethylammonium) or removal of the urothelium did not affect the inhibitory actions of menthol. Contraction to CaCl2 was markedly inhibited by either menthol or nifedipine. In cultured bladder smooth muscle cells, menthol or nifedipine abrogated the carbachol or KCl-induced increases in [Ca2+]i. Intravesical administration of menthol increased voiding frequency while decreasing peak voiding pressure. We conclude that menthol inhibits muscarinic bladder contractions through blockade of L-type calcium channels, independently of TRPM8 activation.
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The Soluble Guanylyl Cyclase Activator BAY 60-2770 Ameliorates Overactive Bladder in Obese Mice.
J. Urol.
PUBLISHED: 09-10-2013
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Activators of soluble guanylyl cyclase are of potential interest as treatment for cardiovascular diseases but to our knowledge they have never been proposed to treat overactive bladder. We evaluated the effects of the soluble guanylyl cyclase activator BAY 60-2270 on voiding dysfunction and detrusor overactivity in a mouse model of obesity associated overactive bladder.
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The renin-angiotensin system plays a major role in voiding dysfunction of ovariectomized rats.
Life Sci.
PUBLISHED: 06-25-2013
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The renin-angiotensin system (RAS) plays a major role in cardiovascular diseases in postmenopausal women, but little is known about its importance to lower urinary tract symptoms. In this study we have used the model of ovariectomized (OVX) estrogen-deficient rats to investigate the role of RAS in functional and molecular alterations in the urethra and bladder.
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Obesity increases eosinophil activity in asthmatic children and adolescents.
BMC Pulm Med
PUBLISHED: 06-10-2013
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BACKGROUND: A clear relationship between asthma and obesity has been reported, but the mechanisms remain unclear. The aim of this study was to evaluate the influence of obesity on eosinophil activity (chemotaxis and adhesion) in asthmatic children and adolescents compared with cells from healthy volunteers. METHODS: Asthmatic obese (AO), asthmatic non-obese (ANO), non-asthmatic obese (NAO) and non-asthmatic non-obese (NANO) individuals were included in the present study. The chemotaxis of eosinophils after stimulation with eotaxin (300 ng/ml), platelet-activating factor (10 muM; PAF) and RANTES (100 ng/ml) was performed using a microchemotaxis chamber. The eosinophil peroxidase activity was measured to determine the adhesion activity of eosinophils cultivated on fibronectin-coated plates. The serum leptin, adiponectin, TNF-alpha and IgE levels were quantified using ELISA assays. RESULTS: The serum IgE levels and eosinophil counts were significantly higher in asthmatic (obese and non-obese) individuals compared with non-asthmatic individuals (obese and non-obese). Spontaneous eosinophil chemotaxis was greater in the AO group compared with either the ANO or NANO groups. The activation of eosinophils using eotaxin and PAF increased eosinophil chemotaxis in the AO group. RANTES treatment increased eosinophil chemotaxis in the NAO group compared with the NANO or ANO groups. The activation of eosinophils using eotaxin significantly increased eosinophil adhesion in the AO group compared with other groups. The serum leptin and TNF-alpha levels were higher in obese subjects (asthmatic and non-asthmatic), whereas the levels of adiponectin did not significantly differ among these groups. CONCLUSION: This study is the first to show increased eosinophilic activity (chemotaxis and adhesion) associated with high serum leptin and TNF-alpha levels in atopic asthmatic obese children and adolescents compared with non-obese healthy volunteers.
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Rapid purification and procoagulant and platelet aggregating activities of Rhombeobin: a thrombin-like/gyroxin-like enzyme from Lachesis muta rhombeata snake venom.
Biomed Res Int
PUBLISHED: 06-04-2013
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We report a rapid purification method using one-step chromatography of SVSP Rhombeobin (LMR-47) from Lachesis muta rhombeata venom and its procoagulant activities and effects on platelet aggregation. The venom was fractionated by a single chromatographic step in RP-HPLC on a C8 Discovery BIO Wide Pore, showing high degree of molecular homogeneity with molecular mass of 47035.49?Da. Rhombeobin showed amidolytic activity upon BA ? NA, with a broad optimum pH (7-10) and was stable in solution up to 60°C. The amidolytic activity was inhibited by serine proteinase inhibitors and reducing agents, but not chelating agents. Rhombeobin showed high coagulant activity on mice plasma and bovine fibrinogen. The deduced amino acid sequence of Rhombeobin showed homology with other SVSPs, especially with LM-TL (L. m. muta) and Gyroxin (C. d. terrificus). Rhombeobin acts, in vitro, as a strong procoagulant enzyme on mice citrated plasma, shortening the APTT and PT tests in adose-dependent manner. The protein showed, "ex vivo", a strong defibrinogenating effect with 1?µg/animal. Lower doses activated the intrinsic and extrinsic coagulation pathways and impaired the platelet aggregation induced by ADP. Thus, this is the first report of a venom component that produces a venom-induced consumptive coagulopathy (VICC).
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Simvastatin attenuates neutrophil recruitment in one-lung ventilation model in rats.
Acta Cir Bras
PUBLISHED: 03-19-2013
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To investigate the anti-inflammatory effects of simvastatin in rats undergoing one-lung ventilation (OLV) followed by lung re-expansion.
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Insulin relaxes bladder via PI3K/AKT/eNOS pathway activation in mucosa: unfolded protein response-dependent insulin resistance as a cause of obesity-associated overactive bladder.
J. Physiol. (Lond.)
PUBLISHED: 03-11-2013
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We aimed to investigate the role of insulin in the bladder and its relevance for the development of overactive bladder (OAB) in insulin-resistant obese mice. Bladders from male individuals who were involved in multiple organ donations were used. C57BL6/J mice were fed with a high-fat diet for 10 weeks to induce insulin-resistant obesity. Concentration-response curves to insulin were performed in human and mouse isolated mucosa-intact and mucosa-denuded bladders. Cystometric study was performed in terminally anaesthetized mice. Western blot was performed in bladders to detect phosphorylated endothelial NO synthase (eNOS) (Ser1177) and the phosphorylated protein kinase AKT (Ser473), as well as the unfolded protein response (UPR) markers TRIB3, CHOP and ATF4. Insulin (1-100 nm) produced concentration-dependent mouse and human bladder relaxations that were markedly reduced by mucosal removal or inhibition of the PI3K/AKT/eNOS pathway. In mouse bladders, insulin produced a 3.0-fold increase in cGMP levels (P < 0.05) that was prevented by PI3K/AKT/eNOS pathway inhibition. Phosphoinositide 3-kinase (PI3K) inhibition abolished insulin-induced phosphorylation of AKT and eNOS in bladder mucosa. Obese mice showed greater voiding frequency and non-voiding contractions, indicating overactive detrusor smooth muscle. Insulin failed to relax the bladder or to increase cGMP in the obese group. Insulin-stimulated AKT and eNOS phosphorylation in mucosa was also impaired in obese mice. The UPR markers TRIB3, CHOP and ATF4 were increased in the mucosa of obese mice. The UPR inhibitor 4-phenyl butyric acid normalized all the functional and molecular parameters in obese mice. Our data show that insulin relaxes human and mouse bladder via activation of the PI3K/AKT/eNOS pathway in the bladder mucosa. Endoplasmic reticulum stress-dependent insulin resistance in bladder contributes to OAB in obese mice.
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Airway exposure to staphylococcal enterotoxin A potentiates allergen-induced bone marrow eosinophilia and trafficking to peripheral blood and airways.
Am. J. Physiol. Lung Cell Mol. Physiol.
PUBLISHED: 03-08-2013
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Bone marrow (BM) eosinopoiesis is a common feature during allergen exposure in atopic individuals. Airway exposure to staphylococcal superantigens aggravates allergic airway disease and increases the output of BM eosinophils. However, the exact mechanisms regulating eosinophil mobilization and trafficking to the peripheral circulation and airways remain to be elucidated. Therefore, this study aimed to investigate the mechanisms determining the BM eosinopoiesis in allergic mice under exposure to staphylococcal enterotoxin A (SEA). Ovalbumin (OVA)-sensitized male BALB/C mice were intranasally exposed to SEA (1 ?g), and at 4, 12, 24, and 48 h later animals were challenged with OVA (10 ?g, twice a day). Measurement of IL-5, eotaxin, and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels, flow cytometry for CCR3(+), VLA4(+), and CCR3(+)VLA4(+), as well as adhesion assays to VCAM-1 were performed in BM. Prior airway exposure to SEA time dependently increased the BM eosinophil number in OVA-challenged mice. Eosinophils gradually disappear from peripheral blood, being recruited over time to the airways, where they achieve a maximal infiltration at 24 h. SEA exposure increased the levels of IL-5 and eotaxin (but not GM-CSF) in BM of OVA-challenged mice. Marked increases in CCR3(+) and CCR3(+)VLA4(+) expressions in BM eosinophils of OVA-challenged mice were observed, an effect largely reduced by prior exposure to SEA. Adhesion of BM eosinophils to VCAM-1 was increased in OVA-challenged mice, but prior SEA exposure abrogated this enhanced cell adhesion. Accumulation of BM eosinophils by airway SEA exposure takes place through IL-5- and CCR3-dependent mechanisms, along with downregulation of CCR3/VL4 and impaired cell adhesion to VCAM-1.
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Effect of acute administration of sildenafil to rats with detrusor overactivity induced by chronic deficiency of nitric oxide.
Int Braz J Urol
PUBLISHED: 02-20-2013
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Recently, the effect of phosphodiesterase inhibitors (PDE5i) in the lower urinary tract symptoms (LUTS) associated to benign prostatic hyperplasia have been studied thoroughly. However, it remains unclear how the PDE5i improve LUTS. Therefore, the aim of the present study was to evaluate the potential of acute administration of the PDE5i sildenafil to improve detrusor overactivity (DO) induced by N?-nitro-L-arginine methyl ester hydrochloride (L-NAME), an nitric oxide sinthase (NOS) inhibitor, in rats.
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Superoxide anion production by NADPH oxidase plays a major role in erectile dysfunction in middle-aged rats: prevention by antioxidant therapy.
J Sex Med
PUBLISHED: 01-24-2013
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INTRODUCTION.: Prevalence of erectile dysfunction (ED) increases progressively with aging, but the ED pathophysiology at its early stages is still poorly investigated. AIM.: This study aimed to evaluate the functional and molecular alterations of erectile function at middle age, focusing on the contribution of oxidative stress in erectile tissue for the ED. METHODS.: Young (3.5-month) and middle-aged (10-month) male Wistar rats were used. Rat corpus cavernosum (RCC) was dissected free and mounted in 10-mL organ baths containing Krebs solution. Intracavernosal pressure (ICP) in anesthetized rats was evaluated. MAIN OUTCOME MEASURES.: Concentration-response curves to endothelium-dependent and endothelium-independent agents, as well as to electrical field stimulation (EFS), were obtained in RCC strips. Measurement of cyclic guanosine monophosphate (cGMP) and expressions of neuronal nitric oxide synthase (nNOS) and endothelial NOS (eNOS), gp91(phox) and superoxide dismutase-1 (SOD-1) expressions in RCC were evaluated. RESULTS.: ICP was significantly reduced in middle-aged compared with young rats. RCC relaxations to acetylcholine (10(-8) to 10(-2) ?M), sodium nitroprusside (10(-8) to 10(-2) ?M), sildenafil (10(-9) to 10(-5) ?M), BAY 41-2272 (10(-9) to 10(-5) ?M), and EFS (4-32?Hz) were decreased in middle-aged group, which were nearly normalized by apocynin (NADPH oxidase inhibitor; 10(-4) ?M) or SOD (75?U/mL). Prolonged treatment with apocynin (85?mg/rat/day, 4 weeks) also restored the impaired relaxations in middle-aged rats. Relaxations to 8-bromoguanosine 3,5-cyclic monophosphate sodium salt (8-Br-cGMP; 10(-8) to 3?×?10(-4) ?M) remained unchanged between groups. Basal and stimulated cGMP production were lower in middle-aged group, an effect fully restored by apocynin and SOD. Protein expression of nNOS and phosphorylated eNOS (p-eNOS) (Ser-1177) reduced, whereas gp(91phox) mRNA expression increased in RCC from middle-aged rats. CONCLUSIONS.: ED in middle-aged rats is associated with decreased NO bioavailability in erectile tissue due to upregulation of NADPH oxidase subunit gp91(phox) and downregulation of nNOS/p-eNOS. Antioxidant therapies may be a good pharmacological approach to prevent ED at its early stages.
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Mechanisms involved in abdominal nociception induced by either TRPV1 or TRPA1 stimulation of rat peritoneum.
Eur. J. Pharmacol.
PUBLISHED: 01-22-2013
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Abdominal pain is a frequent symptom of peritoneal cavity irritation, but little is known about the role of the receptors for irritant substances, transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1), in this painful condition. Thus, we investigated the abdominal nociception caused by peritoneal stimulation with TRPV1 (capsaicin) and TRPA1 (allyl isothiocyanate, AITC) agonists and their mechanisms in rats. The intraperitoneal (i.p.) injection of either capsaicin or AITC (0.03-10 mg/kg) induced short-term (up to 20 min) and dose-dependent abdominal nociception, and also produced c-fos expression in spinal afferents of the dorsal horn. TRPV1 antagonism prevented (94 ± 4% inhibition) nociception induced by capsaicin but not by AITC. In contrast, the TRPA1 antagonism almost abolished AITC-induced nociception (95 ± 2% inhibition) without altering the capsaicin response. Moreover, nociception induced by either capsaicin or AITC was reduced by the desensitisation of TRPV1-positive sensory fibres with resiniferatoxin (73 ± 18 and 76 ± 15% inhibitions, respectively) and by the NK1 receptor antagonist aprepitant (56 ± 5 and 53 ± 8% inhibitions, respectively). Likewise, the i.p. injections of capsaicin or AITC increased the content of substance P in the peritoneal fluid. Nevertheless, neither the mast cell membrane stabiliser cromoglycate, nor the H1 antagonist promethazine, nor depletion of peritoneal macrophages affected abdominal nociception induced either by capsaicin or AITC. Accordingly, neither capsaicin nor AITC increased the histamine content in the peritoneal fluid or provoked peritoneal mast cell degranulation in vitro. Collectively, our findings suggest that TRPV1 and TRPA1 stimulation in the peritoneum produces abdominal nociception that is mediated by sensory fibres activation.
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Metformin attenuates the exacerbation of the allergic eosinophilic inflammation in high fat-diet-induced obesity in mice.
PLoS ONE
PUBLISHED: 01-01-2013
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A positive relationship between obesity and asthma has been well documented. The AMP-activated protein kinase (AMPK) activator metformin reverses obesity-associated insulin resistance (IR) and inhibits different types of inflammatory responses. This study aimed to evaluate the effects of metformin on the exacerbation of allergic eosinophilic inflammation in obese mice. Male C57BL6/J mice were fed for 10 weeks with high-fat diet (HFD) to induce obesity. The cell infiltration and inflammatory markers in bronchoalveolar lavage (BAL) fluid and lung tissue were evaluated at 48 h after ovalbumin (OVA) challenge. HFD obese mice displayed peripheral IR that was fully reversed by metformin (300 mg/kg/day, two weeks). OVA-challenge resulted in higher influx of total cell and eosinophils in lung tissue of obese mice compared with lean group. As opposed, the cell number in BAL fluid of obese mice was reduced compared with lean group. Metformin significantly reduced the tissue eosinophil infiltration and prevented the reduction of cell counts in BAL fluid. In obese mice, greater levels of eotaxin, TNF-? and NOx, together with increased iNOS protein expression were observed, all of which were normalized by metformin. In addition, metformin nearly abrogated the binding of NF-?B subunit p65 to the iNOS promoter gene in lung tissue of obese mice. Lower levels of phosphorylated AMPK and its downstream target acetyl CoA carboxylase (ACC) were found in lung tissue of obese mice, which were restored by metformin. In separate experiments, the selective iNOS inhibitor aminoguanidine (20 mg/kg, 3 weeks) and the anti-TNF-? mAb (2 mg/kg) significantly attenuated the aggravation of eosinophilic inflammation in obese mice. In conclusion, metformin inhibits the TNF-?-induced inflammatory signaling and NF-?B-mediated iNOS expression in lung tissue of obese mice. Metformin may be a good pharmacological strategy to control the asthma exacerbation in obese individuals.
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The Evolutionary Implications of Hemipenial Morphology of Rattlesnake Crotalus durissus terrificus (Laurent, 1768) (Serpentes: Viperidae: Crotalinae).
PLoS ONE
PUBLISHED: 01-01-2013
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Most amniotes vertebrates have an intromittent organ to deliver semen. The reptile Sphenodon and most birds lost the ancestral penis and developed a cloaca-cloaca mating. Known as hemipenises, the copulatory organ of Squamata shows unique features between the amniotes intromittent organ. They are the only paired intromittent organs across amniotes and are fully inverted and encapsulated in the tail when not in use. The histology and ultrastructure of the hemipenes of Crotalus durissus rattlesnake is described as the evolutionary implications of the main features discussed. The organization of hemipenis of Crotalus durissus terrificus in two concentric corpora cavernosa is similar to other Squamata but differ markedly from the organization of the penis found in crocodilians, testudinata, birds and mammals. Based on the available data, the penis of the ancestral amniotes was made of connective tissue and the incorporation of smooth muscle in the framework of the sinusoids occurred independently in mammals and Crotalus durissus. The propulsor action of the muscle retractor penis basalis was confirmed and therefore the named should be changed to musculus hemipenis propulsor.The retractor penis magnus found in Squamata has no homology to the retractor penis of mammals, although both are responsible for the retraction of the copulatory organ.
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Effects of acute inflammation induced in the rat paw on the deep digital flexor tendon.
Connect. Tissue Res.
PUBLISHED: 12-05-2011
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The tendon is commonly affected by inflammation, and in such situations, the tissue undergoes a process of reorganization of the extracellular matrix to improve and regenerate the affected region. Little is known about the mechanisms that trigger inflammation in the tissues surrounding the affected area. The objective of this study was to biochemically and morphologically analyze the deep digital flexor tendon at the peak of acute inflammation in the rat paw. Wistar rats were divided into the following three groups: those that received injection of 1% carrageenan, those that received 0.9% NaCl, and those that received nothing. The deep digital flexor tendon was divided into the distal, proximal, and intermediate regions. For biochemical analysis, the tendons were treated with guanidine hydrochloride and analyzed by sodium dodecyl sulfate-polyacrilamide gel electrophoresis. Proteins, glycosaminoglycans (GAGs), and hydroxyproline were quantified, and metalloproteinases were analyzed. The GAGs were analyzed by agarose gel electrophoresis. Tissue sections were stained with hematoxylin-eosin, toluidine blue, and Ponceau SS. The content of proteins and GAGs was smaller in the group receiving the application of carrageenan. The concentration of hydroxyproline in the two tendon regions that respond to tension forces was higher in the inflammation group. The metalloproteinase-9 was detected in the distal region, and a thicker epitenon with cellular infiltrate was observed in the groups with inflamed paws. Meanwhile, a better organization of collagen bundles was observed in the two tension regions of that same group. Our results show that although the tendon was not directly inflamed, changes in the surrounding structural and biochemical parameters were observed.
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Immunohistochemical and functional characterization of nitric oxide signaling pathway in isolated aorta from Crotalus durissus terrificus.
Comp. Biochem. Physiol. C Toxicol. Pharmacol.
PUBLISHED: 09-06-2011
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We characterized the nitric oxide (NO)-cyclic GMP-phosphodiesterase-5 (PDE5) pathway in Crotalus durissus terrificus aorta. Concentration responses curves to acetylcholine (ACh), sodium nitroprusside (SNP), BAY41-2272 (soluble guanylyl cyclase [sGC] stimulator), BAY60-2770 (sGC activator) and tadalafil (PDE5 inhibitor) were constructed in phenylephrine (10 ?M)-precontracted tissues with intact (E(+)) or denuded (E(-)) endothelium. ACh (0.0001-10 ?M) and SNP (0.0001-10 ?M) relaxed aorta, which were reduced by the NO synthase (L-NAME,100 ?M) or the sGC inhibitors (ODQ, 10 ?M). Tadalafil (0.0001-10 ?M) relaxed E(+) rings with potency (pEC(50)) and maximal response (E(max)) values of 7.34±0.02 and 105±8%, respectively. E(-) or ODQ treatment significantly (P<0.05) reduced tadalafil relaxations (66±18% and 71±7%, respectively). BAY41-2272 (0.0001-300 nM) produced concentration-dependent relaxations in E(+) rings, which were reduced by addition of either ODQ or L-NAME (16.0- and 5.2-fold rightward shifts, respectively). The relaxation of BAY60-2770 was markedly potentiated by ODQ and L-NAME (41.0- and 9.7-fold leftward shifts, respectively), whereas in E(-) the pEC(50) values were shifted by 7-fold to the right. Immunohistochemistry, followed validation by transcriptomic analysis, revealed the presence of eNOS in endothelium, whereas nNOS was observed only in perivascular nerves. sGC and PDE5 were expressed in smooth muscle. Thus, NO-sGC-PDE5 pathway is evolutionarily present in Crotalus sp. vessels, and has a remarkable degree of functional similarity to mammalian vessels.
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Lipopolysaccharide treatment reduces rat platelet aggregation independent of intracellular reactive-oxygen species generation.
Platelets
PUBLISHED: 08-02-2011
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High production of reactive-oxygen species (ROS) by blood cells is involved in damage of the vascular endothelium and multiple organ dysfunction in sepsis. However, little is known about the intraplatelet ROS production in sepsis and its consequences on platelet reactivity. In this study, we evaluated whether the treatment of rats with lipopolysaccharide (LPS) affects platelet aggregation through intraplatelet ROS generation. Rats were injected with LPS (1?mg/kg, i.p.), and at 2 to 72?h thereafter, adenosine diphosphate (ADP) (3-10?µM) induced platelet aggregation was evaluated. Production of ROS in platelets was measured by flow cytometry using 2,7-dichlorofluorescein diacetate (DCFH-DA). Treatment of rats with LPS time-dependently inhibited ADP-induced platelet aggregation within 72?h. The inhibitory effect of LPS on platelet aggregation was further increased when the platelets were incubated with polyethylene glycol-superoxide dismutase (PEG-SOD; 30?U/mL), polyethylene glycol-catalase (PEG-CAT; 1000?U/mL) or the NADPH oxidase inhibitor diphenyleneiodonium (DPI; 10?µM). The ROS production in non-stimulated platelets did not differ between control and LPS-treated rats. However, in ADP-activated platelets, generation of ROS was increased by 3.0- and 7.0-fold, as evaluated at 8 and 48?h after LPS injection, respectively. This increased ROS production was significantly reduced when platelets were incubated in vitro with DPI, PEG-SOD or PEG-CAT. In contrast, treatment of rats with N-acetylcysteine (150?mg/kg, i.p.) significantly reduced the inhibitory effect of LPS on platelet aggregation, and prevented the increased ROS production by in vivo LPS. Our results indicate that the increased intraplatelet ROS production does not contribute to the inhibitory effect of LPS on platelet aggregation; however, the maintenance of redox balance in LPS-treated rats is fundamental to restore the normal platelet response in these animals.
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Characterization of pulmonary and systemic inflammatory responses produced by lung re-expansion after one-lung ventilation.
J. Cardiothorac. Vasc. Anesth.
PUBLISHED: 06-13-2011
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To characterize the pulmonary and systemic inflammatory responses of rats undergoing 1-hour or 3-hour one-lung ventilation (OLV) with subsequent 1-hour lung re-expansion.
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The cholinergic response is increased in isolated ileum from gastroschisis rat model.
Pediatr. Surg. Int.
PUBLISHED: 05-04-2011
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Babies with gastroschisis (G) have high morbidity rate and long hospital stay due to bowel hypomotility caused by chronic exposure of the bowel to the amniotic fluid. Our aim was to evaluate the reactivity of isolated ileum in fetal rats selected for experimental gastroschisis.
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Interaction between serotoninergic-and ?-adrenergic receptors signaling pathways in rat femoral artery.
Arq. Bras. Cardiol.
PUBLISHED: 04-28-2011
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Coronary heart disease has been widely studied in cardiovascular research. However, patients with peripheral artery disease (PAD) have worst outcomes compared to those with coronary artery disease. Therefore, pharmacological studies using femoral artery are highly relevant for a better understanding of the pathophysiologic responses of the PAD.
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Reactive oxygen and nitrogen species modulate the ex-vivo effects of LPS on platelet adhesion to fibrinogen.
Life Sci.
PUBLISHED: 03-01-2011
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Excessive production of nitric oxide (NO) and reactive oxygen species (ROS) in sepsis modulates different cell functions. Since the sepsis severity is associated with the degree of platelet activation, we decided to investigate the role of systemic generation of NO and ROS in modulating the platelet adhesion of lipopolysaccharide (LPS)-treated rats.
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Standardization of a method of prolonged thoracic surgery and mechanical ventilation in rats to evaluate local and systemic inflammation.
Acta Cir Bras
PUBLISHED: 01-29-2011
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To evaluate the immediate pulmonary and systemic inflammatory response after a long-term operative period.
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Characterization of the urinary bladder dysfunction in renovascular hypertensive rats.
Neurourol. Urodyn.
PUBLISHED: 01-24-2011
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Association between arterial hypertension and urinary bladder dysfunction has been reported in humans and spontaneously hypertensive rats. However, no study exists evaluating the bladder dysfunction in conditions of renovascular hypertension. The purpose of this study was to characterize the bladder dysfunction in two kidney-one clip (2K-1C) hypertensive rats.
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Enhancement of the pulmonary allergic granulocyte recruitment in rats exposed to DMTI-II, a Kunitz-type inhibitor isolated from Dimorphandra mollis seeds.
Int. Immunopharmacol.
PUBLISHED: 01-20-2011
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DMTI-II (23-kDa trypsin inhibitor purified from Dimorphandra mollis seeds) promotes acute inflammation accompanied by an early infiltration of eosinophils, a critical cell type involved in allergic diseases. We have evaluated here the capacity of DMTI-II to enhance the allergic pulmonary inflammation, looking over time to the leukocyte trafficking from bone marrow to peripheral blood, and their recruitment into the allergic airways. Male Wistar rats were sensitized and challenged with ovalbumin (OVA). At 2 to 16h prior to OVA challenge, animals were exposed to DMTI-II (10?g). Bronchoalveolar lavage fluid (BAL), circulating blood and bone marrow were examined at 24h post-OVA challenge. Challenge with OVA significantly increased the influx of total inflammatory cells, neutrophils and eosinophils in BAL and lung tissue. Pre-exposure to DMTI-II potentiated total inflammatory cell and neutrophil recruitment (p<0.05). Neutropoiesis and neutrophilia accompanied pulmonary cell influx. Pre-exposure to DMTI-II also significantly increased eosinophil recruitment to BAL, an effect starting at 4h, remaining markedly elevated at 16h (p<0.05). Eosinopoiesis and eosinophilia (seen within 2 to 4h) were also observed. Exposure to DMTI-II alone increased the IL-4 levels, and further increased the IL-4 levels in OVA-challenged rats. The levels of IgE, LTB(4) and eotaxin in OVA-challenged rats were greater compared with non-sensitized rats, but DMTI-II exposure failed to further enhance such levels. In summary, our study shows that DMTI-II itself presents granulocytopoietic activity, and enhances allergen-induced neutrophil and eosinophil mobilization from bone marrow to lung tissues that is accompanied by enhanced IL-4 production.
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In vitro evaluation of the safe margin, antithrombotic and antiproliferative actions for the treatment of restenosis: Nitric oxide donor and polymers.
Cell Biochem. Funct.
PUBLISHED: 01-18-2011
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Drug-eluting stents (DES) were developed to combat the problem of in-stent restenosis, and evaluating the biological activity from DES systems is critical for its safety and efficacy. To test the cytotoxicity of nitric oxide (NO) donor-containing polymers for their potential use in DES applications, S-nitrosoglutathione (GSNO) or in combination with poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP) in an aqueous polymeric solution (PVA/PVP/GSNO) was investigated using Balb/c 3T3 and Rabbit arterial smooth muscle (RASM) cells. The sensitivity of 3T3 cells to the cytotoxicity effects induced by GSNO was higher than that of RASM cells, while RASM cells were more susceptible to alterations in membrane permeability. Cell growth assays showed that GSNO and PVA/PVP/GSNO induced antiproliferative effects in RASM cells. Moreover, the presence of polymers can reduce the cytotoxicity and enhance the antiproliferative effects of GSNO. Dose-dependent inhibition of platelet aggregation was similar for both PVA/PVP/GSNO (EC50 of 3.4?±?2.3?µM) and GSNO (EC50 of 2.8?±?1.1?µM) solutions. Platelet adhesion assays showed that the inhibition caused by GSNO (EC50 of 5.0?mM) was dependent on the presence of plasma. These results demonstrate that the methodology adopted here is suitable to establish safety margins and evaluate the antithrombotic potential and antiproliferative effects of NO-eluting biomaterials and polymeric solutions for the new cardiovascular devices, and also to emphasize the importance of using more specific cell lines in these evaluations.
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Long-term oral treatment with BAY 41-2272 ameliorates impaired corpus cavernosum relaxations in a nitric oxide-deficient rat model.
BJU Int.
PUBLISHED: 10-15-2010
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• To investigate the potential beneficial effects of 4-week oral treatment with 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1Hpyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272), a nitric oxide (NO)-independent soluble guanylate cyclase activator, on impaired rat corpus cavernosum relaxations in NO-deficient rats.
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High-fat diet associated with obesity induces impairment of mouse corpus cavernosum responses.
BJU Int.
PUBLISHED: 10-13-2010
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• Obesity induced by high-fat diet (HFD) is one of the most important risk factor for the development of erectile dysfunction (ED) in man. This study aimed to characterize the ED resulting from obesity associated with HFD in mice.
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Exercise training ameliorates the impairment of endothelial and nitrergic corpus cavernosum responses in diabetic rats.
Life Sci.
PUBLISHED: 07-22-2010
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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.
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Quercetin as an inhibitor of snake venom secretory phospholipase A2.
Chem. Biol. Interact.
PUBLISHED: 07-15-2010
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As polyphenolic compounds isolated from plants extracts, flavonoids have been applied to various pharmaceutical uses in recent decades due to their anti-inflammatory, cancer preventive, and cardiovascular protective activities. In this study, we evaluated the effects of the flavonoid quercetin on Crotalus durissus terrificus secretory phospholipase A2 (sPLA2), an important protein involved in the release of arachidonic acid from phospholipid membranes. The protein was chemically modified by treatment with quercetin, which resulted in modifications in the secondary structure as evidenced through circular dichroism. In addition, quercetin was able to inhibit the enzymatic activity and some pharmacological activities of sPLA2, including its antibacterial activity, its ability to induce platelet aggregation, and its myotoxicity by approximately 40%, but was not able to reduce the inflammatory and neurotoxic activities of sPLA2. These results suggest the existence of two pharmacological sites in the protein, one that is correlated with the enzymatic site and another that is distinct from it. We also performed molecular docking to better understand the possible interactions between quercetin and sPLA2. Our docking data showed the existence of hydrogen-bonded, polar interactions and hydrophobic interactions, suggesting that other flavonoids with similar structures could bind to sPLA2. Further research is warranted to investigate the potential use of flavonoids as sPLA2 inhibitors.
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Systemic inflammation caused by chronic periodontite in patients victims of acute ischemic heart attack.
Rev Bras Cir Cardiovasc
PUBLISHED: 06-22-2010
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Infectious and inflammatory processes mediated by bacteria in distant sites have been described as a risk factor for acute ischemic heart disease (AIHD).
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Long-term administration of BAY 41-2272 prevents bladder dysfunction in nitric oxide-deficient rats.
Neurourol. Urodyn.
PUBLISHED: 04-11-2010
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Chronic blockade of nitric oxide (NO) synthesis leads to detrusor smooth muscle overactivity. This study aimed to evaluate the protective effects of BAY 41-2272, a soluble guanlylate cyclase activator, on changes in cystometric parameters in NO-deficient rats.
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Mechanisms of relaxant activity of the nitric oxide-independent soluble guanylyl cyclase stimulator BAY 41-2272 in rat tracheal smooth muscle.
Eur. J. Pharmacol.
PUBLISHED: 03-22-2010
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The soluble guanylyl cyclase is expressed in airway smooth muscle, and agents that stimulate this enzyme activity cause airway smooth muscle relaxation and bronchodilation. The compound 5-Cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272) is a potent nitric oxide (NO)-independent soluble guanylyl cyclase stimulator, but little is known about its effects in airway smooth muscle. Therefore, this study aimed to investigate the mechanisms underlying the relaxations of rat tracheal smooth muscle induced by BAY 41-2272. Tracheal rings were mounted in 10-ml organ baths for isometric force recording. BAY 41-2272 concentration-dependently relaxed carbachol-precontracted tracheal rings (pEC(50)=6.68+/-0.14). Prior incubation with the NO synthesis inhibitor l-NAME (100 microM) or the soluble guanylyl cyclase inhibitor ODQ (10 microM) caused significant rightward shifts in the concentration-response curves to BAY 41-2272. Sodium nitroprusside caused concentration-dependent relaxations, which were greatly potentiated by BAY 41-2272 and completely inhibited by ODQ. In addition, BAY 41-2272 shifted to the right the tracheal contractile responses to either carbachol (0.01-1 microM) or electrical field stimulation (EFS, 1-32 Hz). BAY 41-2272 (1 microM) also caused a marked rightward shift and decreased the maximal contractile responses to extracellular CaCl2, and such effect was not modified by pretreatment with ODQ. In addition, BAY 41-2272 (up to 1 microM) significantly increased the cGMP levels, and that was abolished by ODQ. Our results indicate that BAY 41-2272 causes cGMP-dependent rat tracheal smooth muscle relaxations in a synergistic fashion with exogenous NO. BAY 41-2272 has also an additional mechanism independently of soluble guanylyl cyclase activation possibly involving Ca(2+) entry blockade.
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Mechanisms underlying the inhibitory effects of lipopolysaccharide on human platelet adhesion.
Platelets
PUBLISHED: 03-12-2010
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Alterations in platelet aggregation in septic conditions are well established. However, little is known about the effects of lipopolysaccharide (LPS) on platelet adhesion. We have therefore investigated the effects of LPS in human platelet adhesion, using an in vitro model of platelet adhesion to fibrinogen-coated wells. Microtiter plates were coated with human fibrinogen, after which washed platelets (6 x 10(8) platelets/ml) were allowed to adhere. Adherent platelets were quantified through measurement of acid phosphatase activity. Calcium mobilization in Fura2-AM-loaded platelets was monitored with a spectrofluorimeter. Platelet flow cytometry in thrombin-stimulated platelets was performed using monoclonal mouse anti-platelet GPIIb/IIIa antibody (PAC-1). Prior incubation of washed platelets with LPS (0.01-300 microg/ml) for 5 to 60 min concentration- and time-dependently inhibited non-activated platelet adhesion. In thrombin-activated (50 mU/ml) platelets, LPS inhibited the adhesion to a significantly lesser extent than non-activated platelets. Cyclohexamide, superoxide dismutase polyethylene glycol (PEG-SOD) or catalase polyethylene glycol did not affect the LPS responses. No alterations in cyclic GMP levels were seen after platelet incubation with LPS, except with the highest concentration employed (300 microg/ml) where an increase of 36% (P < 0.05) was observed. Thrombin increased by 7.5-fold the internal Ca(2+) platelet levels, an effect markedly inhibited by LPS. Thrombin induced concentration-dependent platelet GPIIb/IIIa activation, but LPS failed to affect the activation state of this membrane glycoprotein. In conclusion, LPS inhibits human platelet adhesion to fibrinogen by mechanisms involving blockade of external Ca(2+), independently of cGMP generation and activation of GPIIb/IIIa complex.
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Evaluation of the relaxant effect of the nitric oxide-independent soluble guanylyl cyclase stimulator BAY 41-2272 in isolated detrusor smooth muscle.
Eur. J. Pharmacol.
PUBLISHED: 03-09-2010
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The nitric oxide (NO)-independent soluble guanylyl cyclase stimulator stimulator BAY 41-2272 was reported to produce relaxant response in different types of smooth muscle. However no study was carried out to investigate the effects of BAY 412282 in detrusor smooth muscle. Thus, this study aimed to evaluate the relaxant effects of BAY 41-2272, in isolated mouse, rat and rabbit detrusor smooth muscle. Mouse, rat and rabbit were anesthetized, and urinary bladder removed. Detrusor smooth muscle was transferred to 10-mL organ baths containing oxygenated and warmed Krebs-Henseleit solution. Tissues were connected to force-displacement transducers and changes in isometric force were recorded. BAY 41-2272 (0.001-100 microM) produced concentration-dependent detrusor smooth muscle relaxations in mouse, rat and rabbit with maximal responses of 61.3+/-6.6%, 95.1+/-9.9% and 91.7+/-5.9%, respectively. Sodium nitroprusside and glyceryl trinitrate, as well as 8-bromo-cGMP also produced detrusor relaxations, but to a much lesser extent than BAY 41-2272. The NO synthesis inhibitor L-NAME and the phosphodiesterase-5 inhibitor sildenafil had no effect in BAY 41-2272-induced responses. However, the soluble guanylyl cyclase inhibitor ODQ significantly reduced BAY 41-2272-induced relaxations. BAY 41-2272 increased the bladder cGMP levels by about of 14- and 20-fold for 10 and 100 microM, respectively, which were markedly reduced by ODQ. The cAMP levels were unaffected by BAY 41-2272. Moreover, BAY 41-2272 significantly reduced the contractile responses to extracellular Ca(2+) in an ODQ-insensitive manner. In conclusion, rabbit detrusor smooth muscle relaxations by BAY 41-2272 involve mainly cGMP production, but an additional mechanism involving Ca(2+) influx blockade independently of cGMP production appears to be involved.
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Platelet adhesion and intracellular calcium levels in antigen-challenged rats.
Pulm Pharmacol Ther
PUBLISHED: 02-24-2010
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There is considerable evidence that platelet activation occurs in allergic airways diseases. In this study we aimed to investigate platelet adhesion to immobilized fibrinogen and intracellular calcium levels in a rat model of allergic inflammation. Male Wistar rats were challenged with ovalbumin (OVA). At 30 min to 24h after OVA-challenge, assays of platelet adhesion to immobilized fibrinogen and intracellular calcium levels using fura 2-AM loaded platelets were performed. The serum levels of IgE were approximately 5-fold greater in OVA-sensitized rats. A marked eosinophil influx in bronchoalveolar lavage (BAL) fluid of OVA-challenged rats at 24h after OVA-challenge was also seen. OVA-challenge resulted in a marked thrombocytopenia, as observed within 12h after OVA-challenge. The agonists ADP (0.5-50 microM) and thrombin (30-100 mU/ml) concentration-dependently increased platelet adhesion to immobilized fibrinogen. At an early time after OVA-challenge (30 min), platelets exhibited greater platelet adhesion compared with the non-sensitized group, whereas at a late time (24h) they exhibited lower platelet adhesion to both agonists. Moreover, at 30 min after OVA-challenge, intracellular calcium levels to ADP (20 microM) and thrombin (100 mU/ml)-activated platelets were greater compared with non-challenged rats. As opposed, at 24h after OVA challenge, a lower intracellular calcium level to ADP- and thrombin-activated platelets was observed. In conclusion, OVA-challenge in rats promotes a biphasic response in platelet adhesion consisting of an increased adhesion and intracellular calcium levels at an early phase (30 min), which progress to a reduction in adhesion and intracellular calcium levels at a late time (24h) after antigen challenge.
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Chronic alcoholism associated with diabetes impairs erectile function in rats.
BJU Int.
PUBLISHED: 02-02-2010
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To investigate the effects of chronic ethanol consumption and diabetes on nitric oxide (NO)-mediated relaxation of cavernosal smooth muscle (CSM).
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Effect of the phosphodiesterase 5 inhibitors sildenafil, tadalafil and vardenafil on rat anococcygeus muscle: functional and biochemical aspects.
Clin. Exp. Pharmacol. Physiol.
PUBLISHED: 12-16-2009
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1. The anococcygeus muscle is part of the erectile machinery in male rodents. Phosphodiesterase (PDE) 5 inhibitors enhance and prolong the effects of cGMP, which has a key role in penile erection. The aim of the present study was to provide a functional and biochemical comparison of the three PDE5 inhibitors, namely sildenafil, tadalafil and vardenafil, in the rat anococcygeus muscle. 2. Muscle strips were mounted in 4 mL organ baths and isometric force recorded. Levels of cGMP were measured using an enzyme immunoassay kit. Western blots were used to determine PDE5 protein expression. 3. The PDE5 inhibitors concentration-dependently relaxed carbachol-precontracted anococcygeus muscle; however, vardenafil was more potent (pEC(50) = 8.11 +/- 0.05) than sildenafil (7.72 +/- 0.06) or tadalafil (7.69 +/- 0.05). Addition of N(G)-nitro-l-arginine methyl ester (100 micromol/L) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10 micromol/L) to the organ baths caused significant rightward shifts in concentration-response curves for all PDE5 inhibitors. 4. Sildenafil, tadalafil and vardenafil (all at 0.1 micromol/L) caused leftward shifts in the glyceryl trinitrate (GTN) concentration-response curves (by 4.0-, 3.7- and 5.5-fold, respectively). In addition, all three PDE5 inhibitors significantly potentiated relaxation responses to both GTN (0.01-10 micromol/L) and electrical field stimulation (EFS; 1-32 Hz), with vardenafil having more pronounced effects. 5. All three PDE5 inhibitors reduced EFS-evoked contractions in a concentration-dependent manner over the concentration range 0.001-1 micromol/L. There were no significant differences between the effects of the three PDE5 inhibitors. 6. Vardenafil (0.01-0.1 micromol/L) was more potent in preventing cGMP degradation in vitro than sildenafil (0.01-0.1 micromol/L) and tadalafil (0.01-0.1 micromol/L). 7. Under control conditions, the expression of PDE5 was higher in the anococcygeus muscle than in the corpus cavernosum. 8. In conclusion, PDE5 inhibitors enhance exogenous and endogenous nitric oxide-mediated relaxation in the rat anococcygeus muscle. The potency of vardenafil was greater than that of either sildenafil or tadalafil.
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The role of superoxide anion in the inhibitory effect of SIN-1 in thrombin-activated human platelet adhesion.
Eur. J. Pharmacol.
PUBLISHED: 07-15-2009
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Reactive oxygen species have an important role in the control of platelet activity. Superoxide anion (O(2)(-)) is a free radical that can be converted into other reactive oxygen species such as peroxynitrite (ONOO(-)) that is formed from the reaction between O(2)(-) and nitric oxide (NO). There are conflicting data on ONOO(-) effects in platelets because it presents pro- or anti-aggregatory actions. 3-morpholinosydnonimine (SIN-1) co-generates NO and O(2)(-), yielding ONOO(-). Therefore, the present study aimed to investigate the mechanisms involved in the inhibition of human platelet adhesion by SIN-1. Microtiter plates were coated with human fibrinogen, after which washed platelets (6 x 10(8)platelets/ml) were added to adhere. Exposure of non-activated and thrombin-activated platelets to SIN-1 (0.001-100 microM) concentration-dependently inhibited adhesion, which was accompanied by marked increases in the cyclic GMP levels. In non-activated platelets, the soluble guanylate cyclase inhibitor ODQ prevented the SIN-1-induced cGMP elevations and adhesion inhibition. In thrombin-activated platelets, ODQ fully prevented the SIN-1-induced cGMP elevations, but only partly prevented the adhesion inhibition. The O(2)(-) and ONOO(-) scavengers superoxide dismutase (SOD) and -(-)epigallocatechin gallate, respectively, had minimal effects in non-activated platelets. The inhibition of activated platelets by SIN-1 was reversed by SOD and partly reduced by ECG. Western blot analysis of SIN-1-treated platelets showed a single 105 kDa-nitrated band. Nanospray LC-MS-MS identified the protein containing 3-nitrotyrosine residues as human alpha-actinin-1-cytoskeletal isoform. Our data show that platelet adhesion inhibition by SIN-1 in activated platelets involves cGMP-independent mechanism through O(2)(-) generation. Superoxide anion signaling pathway includes ONOO(-) formation and alpha-actinin nitration.
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Pre-exposure to Staphylococcal enterotoxin A exacerbates the pulmonary allergic eosinophil recruitment in rats.
Int. Immunopharmacol.
PUBLISHED: 07-01-2009
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Gram-positive Staphylococcus aureus releases classical enterotoxins which aggravates allergic airway diseases. However, little is known about the mechanisms underlying the cell influx exacerbation in asthmatic individuals under exposure to Staphylococcal enterotoxins. We therefore aimed to investigate the effects of airways exposure to Staphylococcal enterotoxin A (SEA) to pulmonary leukocyte recruitment in rats sensitized and challenged with ovalbumin (OVA). Rats were exposed to SEA at 4h prior to OVA challenge or at 4h post-OVA challenge. Bronchoalveolar lavage (BAL) fluid, bone marrow and lung tissue were obtained at 24h after OVA challenge. Pre-exposure to SEA markedly enhanced the eosinophil counts in both BAL fluid and pulmonary tissue in OVA-challenged rats, whereas neutrophil and mononuclear cell counts remained unchanged. In bone marrow, pre-exposure to SEA alone significantly increased the number of eosinophils, and that was further increased in OVA-challenged rats. Exposure to SEA post-OVA challenge did not affect the number of eosinophils, neutrophils and mononuclear cells in BAL fluid. Pre-exposure to the endotoxin lipopolyssacharide (LPS) in OVA-challenged animals rather enhanced the neutrophil number in BAL fluid. In rats pre-exposed to SEA and OVA-challenged, a marked elevation in the levels of TNF-alpha and eotaxin (but not of IL-10) in BAL fluid was observed. The eotaxin levels increased by about of 3-fold in alveolar macrophages treated with SEA in vitro. In conclusion, airways pre-exposure to SEA causes a selective increase in eosinophil number in BAL fluid and bone marrow of OVA-challenged rats by mechanisms involving enhancement of TNF-alpha and eotaxin synthesis.
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Increased cavernosal relaxations in sickle cell mice priapism are associated with alterations in the NO-cGMP signaling pathway.
J Sex Med
PUBLISHED: 06-02-2009
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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).
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Role of exercise training on pulmonary ischemia/reperfusion and inflammatory response.
Rev Bras Cir Cardiovasc
PUBLISHED: 05-21-2009
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Advances in new technologies associated with improvement of knowledge in medicine have promoted important development in therapeutic and preventive approaches in an attempt to diminish complications following cardiothoracic process involving ischemia/ reperfusion (IR). Nevertheless, postoperative pulmonary injuries remain high and are considered one of the most frequent complications after cardiothoracic surgery. Thus, new strategies with prophylactic actions are crucial in cardiovascular area in an attempt to reduce complications and to improve patient life. It is well documented that exercise training is a non-pharmacological tool to prevent and/or treat cardiovascular and endocrine-metabolic diseases. The aim of this review was to provide an update of several studies pulmonary IR process and its local and systemic complications and the role of inflammatory response. Furthermore, this review focused on the effects of exercise training on the pulmonary IR as an important strategy to diminish its complications. This review shows that few studies exist regarding the health-promoting physical exercise in cardiothoracic surgery and how important is necessary to increase studies in this area. Recently, studies from our laboratory showed beneficial effects of exercise training in experimental model of pulmonary IR. Collectively, data show that physical preconditioning for patients is very important approach to reduce postsurgical complications as well as diminish the time of hospitalization which includes a specialized personal trainer in the health team. Moreover, this preventive strategy might improve patient recovery and would lead to consuming less resources of the health care system. This review included experimental studies in English and Portuguese found in SciELO and MEDLINE (from 1987 to 2008) and also classics texts related to the title.
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Different mechanisms underlie the effects of acute and long-term inhibition of nitric oxide synthases in antigen-induced pulmonary eosinophil recruitment in BALB/C mice.
Pulm Pharmacol Ther
PUBLISHED: 05-01-2009
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Nitric oxide synthase (NOS) inhibitors are largely used to evaluate the NO contribution to pulmonary allergy, but contrasting data have been reported. In this study, pharmacological, biochemical and pharmacokinetic assays were performed to compare the effects of acute and long-term treatment of BALB/C mice with the non-selective NOS inhibitor L-NAME in ovalbumin (OVA)-challenged mice. Acute L-NAME treatment (50 mg/kg, gavage) significantly reduced the eosinophil number in bronchoalveolar lavage fluid (BALF). The inducible NOS (iNOS) inhibitor aminoguanidine (20 mg/kg/day in the drinking water) also significantly reduced the eosinophil number in BALF. In contrast, 3-week L-NAME treatment (50 and 150 mg/kg/day in the drinking water) significantly increased the pulmonary eosinophil influx. The constitutive NOS (cNOS) activity in brain and lungs was reduced by both acute and 3-week L-NAME treatments. The pulmonary iNOS activity was reduced by acute L-NAME (or aminoguanidine), but unaffected by 3-week L-NAME treatment. Acute L-NAME (or aminoguanidine) treatment was more efficient to reduce the NOx- levels compared with 3-week L-NAME treatment. The pharmacokinetic study revealed that L-NAME is not bioavailable when given orally. After acute L-NAME intake, serum concentrations of the metabolite Nomega-nitro-L-arginine decreased from 30 min to 24 h. In the 3-week L-NAME treatment, the Nomega-nitro-L-arginine concentration was close to the detection limit. In conclusion, 3-week treatment with l-NAME yields low serum Nomega-nitro-L-arginine concentrations, causing preferential inhibition of cNOS activity. Therefore, eosinophil influx potentiation by 3-week L-NAME treatment may reflect removal of protective cNOS-derived NO, with no interference on the ongoing inflammation due to iNOS-derived NO.
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Mechanisms involved in the rat peritoneal leukocyte migration induced by a Kunitz-type inhibitor isolated from Dimorphandra mollis seeds.
Toxicon
PUBLISHED: 04-28-2009
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DMTI-II is a Kunitz-type inhibitor isolated from Dimorphandra mollis seeds that causes rat inflammatory edema by mechanisms involving activation of mast cells and sensory C-fibers. The present study aimed to further explore the inflammatory mechanisms involved in DMTI-II-induced inflammation, focusing to the leukocyte migration in vivo. Male Wistar rats (250-280 g) were injected with DMTI-II (1-100microg/cavity), and at 4-24h thereafter the leukocyte counts in peritoneal lavage were evaluated. DMTI-II caused dose- and time-dependent accumulation of neutrophils and eosinophils. The peritoneal neutrophil influx initiated at 4h, achieving maximal responses at 16 h after DMTI-II injection (16- and 22-fold increase, respectively). The DMTI-II-induced eosinophil recruitment was observed as early as 4h achieving the maximal responses at 16 h (12- and 17-fold increase, respectively). The mononuclear cell number increased at 4h and 16 h (1.5-fold and 1.6-increase, respectively). Prior treatments with dexamethasone, the cyclooxygenase (COX) inhibitors indomethacin and celecoxib, as well as the PAF receptor antagonist PCA4248 largely reduced the neutrophil and eosinophil accumulation. The selective lypoxygenase inhibitor AA861, the tachykinin NK(1) antagonist SR-140333 and the nitric oxide inhibitor L-NAME reduced only the eosinophil number. The eotaxin levels were significantly higher in DMTI-II-injected rats compared with control animals. In conclusion, DMTI-II causes an early migration of eosinophils and neutrophils by mechanisms involving COX-2- and lipoxygenase-derived metabolites, PAF, substance P and NO. The capacity of DMTI-II to recruit eosinophils at early times is likely to reflect the allergen properties of proteinase inhibitors belonging to Kunitz family.
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Comparative relaxing effects of sildenafil, vardenafil, and tadalafil in human corpus cavernosum: contribution of endogenous nitric oxide release.
Urology
PUBLISHED: 04-15-2009
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To compare the direct relaxant activity of sildenafil, vardenafil, and tadalafil in the human corpus cavernosum (HCC) and to investigate their modulatory effects on nitric oxide (NO)-mediated responses. Phosphodiesterase (PDE)-5 inhibitors cause cavernosal smooth muscle relaxation and penile erection.
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Chronic ethanol consumption induces cavernosal smooth muscle dysfunction in rats.
Urology
PUBLISHED: 04-08-2009
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To investigate the effects of chronic ethanol consumption on nitric oxide (NO)-mediated relaxation in rat cavernosal smooth muscle (CSM).
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Inflammatory oedema induced by Lachesis muta muta (Surucucu) venom and LmTX-I in the rat paw and dorsal skin.
Toxicon
PUBLISHED: 04-08-2009
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The ability of crude venom and a basic phospholipase A(2) (LmTX-I) from Lachesis muta muta venom to increase the microvascular permeability in rat paw and skin was investigated. Crude venom or LmTX-I were injected subplantarly or intradermally and rat paw oedema and dorsal skin plasma extravasation were measured. Histamine release from rat peritoneal mast cell was also assessed. Crude venom or LmTX-I induced dose-dependent rat paw oedema and dorsal skin plasma extravasation. Venom-induced plasma extravasation was inhibited by the histamine H(1) antagonist mepyramine (6mg/kg), histamine/5-hydroxytriptamine antagonist cyproheptadine (2mg/kg), cyclooxygenase inhibitor indomethacin (5mg/kg), nitric oxide synthesis inhibitor l-NAME (100nmol/site), tachykinin NK(1) antagonist SR140333 (1nmol/site) and bradykinin B(2) receptor antagonist Icatibant (0.6mg/kg). Platelet-activating factor (PAF) antagonist PCA4248 (5mg/kg) had no effect. LmTX-I-induced skin extravasation was inhibited by cyproheptadine, mepyramine, indomethacin and PCA4248, while l-NAME and SR140333 had no effect. Additionally, both Lachesis muta muta venom and LmTX-I concentration-dependently induced histamine release from rat mast cells. In conclusion, Lachesis muta muta venom and LmTX-I increase microvascular permeability by mechanisms involving in vivo mast cell activation and arachidonic acid metabolites. Additionally, crude venom-induced responses also involve substance P, nitric oxide and bradykinin release, whether LmTX-I-induced responses involve PAF.
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Role of sensory innervation in the rat pulmonary neutrophil recruitment induced by staphylococcal enterotoxins type A and B.
Eur. J. Pharmacol.
PUBLISHED: 03-27-2009
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Rat airways exposure to Staphylococcal enterotoxin A (SEA) and B (SEB) induces marked neutrophil influx. Since sensory neuropeptides play important roles in cell infiltration, in this study we have investigated its contribution in triggering SEA- and SEB-induced pulmonary neutrophil infiltration. Male Wistar rats were exposed intratracheally with SEA (3 ng/trachea) or SEB (250 ng/trachea). Animals received different in vivo pretreatments, after which the neutrophil counts and levels of substance P and IL-1 in bronchoalveolar lavage fluid were evaluated. Alveolar macrophages and peritoneal mast cells were incubated with SEA and SEB to determine the IL-1 and TNF-alpha levels. Capsaicin pretreatment significantly reduced SEA- and SEB-induced neutrophil influx in bronchoalveolar lavage fluid, but this treatment was more effective to reduce SEA responses. Treatments with SR140333 (tachykinin NK(1) receptor antagonist) and SR48968 (tachykinin NK(2) receptor antagonist) decreased SEA-induced neutrophil influx, whereas SEB-induced responses were inhibited by SR140333 only. Cyproheptadine (histamine/5-hydroxytriptamine receptor antagonist) and MD 7222 (5-HT(3) receptor antagonist) reduced SEA- and SEB-induced neutrophil influx. The substance P and IL-1 levels in bronchoalveolar lavage fluid of SEA-exposed rats were significantly higher than SEB. In addition, SEA (but not SEB) significantly released mast cell TNF-alpha. Increased production of TNF-alpha and IL-1 in alveolar macrophages was observed in response to SEA and SEB. In conclusion, sensory neuropeptides contribute significantly to SEA- and SEB-induced pulmonary neutrophil recruitment, but SEA requires in a higher extent the airways sensory innervation, and participation of mast cells and alveolar macrophage products.
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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.
Urology
PUBLISHED: 02-19-2009
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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.
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Involvement of sensory nerves and TRPV1 receptors in the rat airway inflammatory response to two environment pollutants: diesel exhaust particles (DEP) and 1,2-naphthoquinone (1,2-NQ).
Arch. Toxicol.
PUBLISHED: 02-03-2009
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The environmental chemical 1,2-naphthoquinone (1,2-NQ) is implicated in the exacerbation of airways diseases induced by exposure to diesel exhaust particles (DEP), which involves a neurogenic-mediated mechanism. Plasma extravasation in trachea, main bronchus and lung was measured as the local (125)I-bovine albumin accumulation. RT-PCR quantification of TRPV1 and tachykinin (NK(1) and NK(2)) receptor gene expression were investigated in main bronchus. Intratracheal injection of DEP (1 and 5 mg/kg) or 1,2-NQ (35 and 100 nmol/kg) caused oedema in trachea and bronchus. 1,2-NQ markedly increased the DEP-induced responses in the rat airways in an additive rather than synergistic manner. This effect that was significantly reduced by L-732,138, an NK(1) receptor antagonist, and in a lesser extent by SR48968, an NK(2) antagonist. Neonatal capsaicin treatment also markedly reduced DEP and 1,2-NQ-induced oedema. Exposure to pollutants increased the TRPV1, NK(1) and NK(2) receptors gene expression in bronchus, an effect was partially suppressed by capsaicin treatment. In conclusion, our results are consistent with the hypothesis that DEP-induced airways oedema is highly influenced by increased ambient levels of 1,2-NQ and takes place by neurogenic mechanisms involving up-regulation of TRPV1 and tachykinin receptors.
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Interaction between advanced glycation end products formation and vascular responses in femoral and coronary arteries from exercised diabetic rats.
PLoS ONE
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The majority of studies have investigated the effect of exercise training (TR) on vascular responses in diabetic animals (DB), but none evaluated nitric oxide (NO) and advanced glycation end products (AGEs) formation associated with oxidant and antioxidant activities in femoral and coronary arteries from trained diabetic rats. Our hypothesis was that 8-week TR would alter AGEs levels in type 1 diabetic rats ameliorating vascular responsiveness.
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Increased contractility and impaired relaxation of the left pulmonary artery in a rabbit model of congenital diaphragmatic hernia.
Pediatr. Surg. Int.
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Congenital diaphragmatic hernia (CDH) is associated with pulmonary hypertension which is often difficult to manage and a significant cause of morbidity and mortality. Our aim was to study the pulmonary artery reactivity in an animal model of CDH.
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Role of PKC and CaV1.2 in detrusor overactivity in a model of obesity associated with insulin resistance in mice.
PLoS ONE
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Obesity/metabolic syndrome are common risk factors for overactive bladder. This study aimed to investigate the functional and molecular changes of detrusor smooth muscle (DSM) in high-fat insulin resistant obese mice, focusing on the role of protein kinase C (PKC) and Ca(v)1.2 in causing bladder dysfunction. Male C57BL/6 mice were fed with high-fat diet for 10 weeks. In vitro functional responses and cystometry, as well as PKC and Ca(v)1.2 expression in bladder were evaluated. Obese mice exhibited higher body weight, epididymal fat mass, fasting glucose and insulin resistance. Carbachol (0.001-100 µM), ?,?-methylene ATP (1-10 µM), KCl (1-300 mM), extracellular Ca(2+) (0.01-100 mM) and phorbol-12,13-dibutyrate (PDBu; 0.001-3 µM) all produced greater DSM contractions in obese mice, which were fully reversed by the Ca(v)1.2 blocker amlodipine. Cystometry evidenced augmented frequency, non-void contractions and post-void pressure in obese mice that were also prevented by amlodipine. Metformin treatment improved the insulin sensitivity, and normalized the in vitro bladder hypercontractility and cystometric dysfunction in obese mice. The PKC inhibitor GF109203X (1 µM) also reduced the carbachol induced contractions. PKC protein expression was markedly higher in bladder tissues from obese mice, which was normalized by metformin treatment. The Ca(v)1.2 channel protein expression was not modified in any experimental group. Our findings show that Ca(v)1.2 blockade and improvement of insulin sensitization restores the enhanced PKC protein expression in bladder tissues and normalizes the overactive detrusor. It is likely that insulin resistance importantly contributes for the pathophysiology of this urological disorder in obese mice.
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Activation of haem-oxidized soluble guanylyl cyclase with BAY 60-2770 in human platelets lead to overstimulation of the cyclic GMP signaling pathway.
PLoS ONE
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Nitric oxide-independent soluble guanylyl cyclase (sGC) activators reactivate the haem-oxidized enzyme in vascular diseases. This study was undertaken to investigate the anti-platelet mechanisms of the haem-independent sGC activator BAY 60-2770 in human washed platelets. The hypothesis that sGC oxidation potentiates the anti-platelet activities of BAY 60-2770 has been tested.
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Allergen-induced bone marrow eosinophilopoiesis and airways eosinophilic inflammation in leptin-deficient ob/ob mice.
Obesity (Silver Spring)
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Asthma and obesity are growing epidemics in the world. It is well established that obesity worsens the asthma outcomes. High-fat diet-induced obesity in mice exacerbates the pulmonary eosinophilic inflammation. We have used wild-type (WT) and ob/ob mice to further explore the mechanisms by which obesity aggravates the pulmonary eosinophilic inflammation. The eosinophil (EO) number in bronchoalveolar lavage (BAL) fluid, lung tissue, blood, and bone marrow were evaluated at 24, 48, and 72 h after ovalbumin (OVA) challenge in sensitized mice. The basal EO number (phosphate-buffered saline (PBS)-instilled mice) in lung tissue was about 3.5-fold greater in ob/ob compared with WT mice. OVA challenge in ob/ob mice promoted an EO accumulation into the lung that was accompanied by a lower emigration to airways lumen (BAL fluid) in comparison with WT mice. OVA challenge also markedly elevated the number of mature and immature EO in bone marrow of ob/ob mice at 24 h compared with WT group. Blood EO at 48 h was markedly greater in ob/ob mice. Tumor necrosis factor (TNF)-? and interleukin (IL)-10 levels in BAL fluid were significantly higher in ob/ob mice, whereas no changes for IL-5 and eotaxin were found. The IL-6 levels were significantly lower in ob/ob mice. In conclusion, OVA challenge in ob/ob obese mice potentiates eosinophilopoiesis and promotes an accumulation of EO into the lung tissue, delaying their transit to airways lumen. The longer EO remain into the lung tissue is likely to contribute, at least in part, to the asthma worsened by obesity.
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Platelet hyperaggregability in high-fat fed rats: a role for intraplatelet reactive-oxygen species production.
Cardiovasc Diabetol
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Adiposity greatly increases the risk of atherothrombotic events, a pathological condition where a chronic state of oxidative stress is reported to play a major role. This study aimed to investigate the involvement of (NO)-soluble guanylyl cyclase (sGC) signaling pathway in the platelet dysfunction from high fat-fed (HFF) rats.
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