Edson Antunes

Chronic inhibition of nitric oxide synthesis. A new model of arterial hypertension.

Recent studies have indicated that acute inhibition of nitric oxide biosynthesis in the rat promotes arterial hypertension and renal vasoconstriction. We evaluated the renal and systemic effects of 4-6 weeks of nitric oxide blockade in Munich-Wistar rats receiving the nitric oxide inhibitor nitro-L-arginine orally. Age-matched untreated rats were used as controls. In an additional seven rats, nitric oxide blockade was carried out in conjunction with oral administration of the novel angiotensin II antagonist losartan potassium. Tail-cuff pressure rose progressively in nitro-L-arginine-treated rats, reaching 164 +/- 6 mm Hg at 4-6 weeks, compared with 108 +/- 3 mm Hg in controls. In rats concomitantly receiving losartan, tail-cuff pressure reached 125 +/- 6 mm Hg, still elevated compared with rats receiving losartan alone (98 +/- 3 mm Hg). Nitro-L-arginine-treated rats presented marked renal vasoconstriction and hypoperfusion, as well as a 30% fall in glomerular filtration rate and a 39% increase in filtration fraction. Treatment with Losartan normalized glomerular filtration rate, but not filtration fraction or renal vascular resistance. Plasma renin activity was elevated after nitro-L-arginine treatment. Renal histological examination revealed widespread arteriolar narrowing, focal arteriolar obliteration, and segmental fibrinoid necrosis in the glomeruli. In a separate group of rats, nitro-L-arginine administered for 1 week induced hypertension that was partially reversed by acute L-arginine, but not D-arginine or L-glycine, infusions. We conclude that chronic nitric oxide blockade may constitute a new model of severe arterial hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Mast cell degranulation induced by two phospholipase A2 homologues : Dissociation between enzymatic and biological activities

Bothropstoxin-I and bothropstoxin-II are phospholipase A2 homologues isolated from Bothrops jararacussu snake venom. The former is devoid of phospholipase A2 activity whereas the latter has very low enzymatic activity. In this study, we have investigated the in vivo (rat paw and skin oedema) and in vitro (mast cell degranulation) inflammatory effects caused by bothropstoxin-I and bothropstoxin-II. Bothropstoxin-I (25-100 microg/paw) and bothropstoxin-II (12.5-50 microg/paw) caused dose-dependent rat paw oedema. The intradermal injection of bothropstoxin-I (0.125-5 microg/site) and bothropstoxin-II (0.125-5 microg/site) into rat skin also resulted in dose-dependent oedema formation. These oedematogenic activities were largely reduced in animals pretreated with the histamine/5-hydroxytryptamine (5-HT) receptor antagonist cyproheptadine (2 mg/kg, i.p. 0.5 h before). Similarly, p-bromophenacyl bromide, a compound known to inhibit phospholipase A2 activity, significantly inhibited rat paw and skin oedema induced by both phospholipase A2 homologues. The polyanion heparin (5 IU/site) significantly reduced the rat skin oedema induced by either bothropstoxin-I or bothropstoxin-II as well as the paw oedema (50 IU/site) induced by the former. When assayed in the rat peritoneal mast cells in vitro, both bothropstoxin-I (10 and 100 microg/ml) and bothropstoxin-II (3 and 10 microg/ml) significantly caused [14C]5-HT release. The [14C]5-HT release caused by these phospholipase A2 homologues were reduced by p-bromophenacyl bromide and heparin (50 IU/ml). Our results indicate that oedema formation induced by bothropstoxin-I and bothropstoxin-II is mostly dependent on in vivo mast cell degranulation. Since heparin greatly reduced the oedematogenic activity of these phospholipase A2 homologues, it is likely that the cationic charge of these substances plays a major role in the mast cell activation. Our results also indicate that p-bromophenacyl bromide may not be a suitable pharmacological tool to investigate the correlation between enzymatic activity and the inflammatory effects of phospholipases A2.

Exercise training improves relaxation response and SOD-1 expression in aortic and mesenteric rings from high caloric diet-fed rats

BackgroundObesity has been associated with a variety of disease such as type II diabetes mellitus, arterial hypertension and atherosclerosis. Evidences have shown that exercise training promotes beneficial effects on these disorders, but the underlying mechanisms are not fully understood. The aim of this study was to investigate whether physical preconditioning prevents the deleterious effect of high caloric diet in vascular reactivity of rat aortic and mesenteric rings.MethodsMale Wistar rats were divided into sedentary (SD); trained (TR); sedentary diet (SDD) and trained diet (TRD) groups. Run training (RT) was performed in sessions of 60 min, 5 days/week for 12 weeks (70–80% VO2max). Triglycerides, glucose, insulin and nitrite/nitrate concentrations (NOx-) were measured. Concentration-response curves to acetylcholine (ACh) and sodium nitroprusside (SNP) were obtained. Expression of Cu/Zn superoxide dismutase (SOD-1) was assessed by Western blotting.ResultsHigh caloric diet increased triglycerides concentration (SDD: 216 ± 25 mg/dl) and exercise training restored to the baseline value (TRD: 89 ± 9 mg/dl). Physical preconditioning significantly reduced insulin levels in both groups (TR: 0.54 ± 0.1 and TRD: 1.24 ± 0.3 ng/ml) as compared to sedentary animals (SD: 0.87 ± 0.1 and SDD: 2.57 ± 0.3 ng/ml). On the other hand, glucose concentration was slightly increased by high caloric diet, and RT did not modify this parameter (SD: 126 ± 6; TR: 140 ± 8; SDD: 156 ± 8 and TRD 153 ± 9 mg/dl). Neither high caloric diet nor RT modified NOx- levels (SD: 27 ± 4; TR: 28 ± 6; SDD: 27 ± 3 and TRD: 30 ± 2 μM). Functional assays showed that high caloric diet impaired the relaxing response to ACh in mesenteric (about 13%), but not in aortic rings. RT improved the relaxing responses to ACh either in aortic (28%, for TR and 16%, to TRD groups) or mesenteric rings (10%, for TR and 17%, to TRD groups) that was accompanied by up-regulation of SOD-1 expression and reduction in triglycerides levels.ConclusionThe improvement in endothelial function by physical preconditioning in mesenteric and aortic arteries from high caloric fed-rats was directly related to an increase in NO bioavailability to the smooth muscle mostly due to SOD-1 up regulation.

Chronic nitric oxide inhibition as a model of hypertensive heart muscle disease

We have compared the myocardial alterations in rats made hypertensive by the chronic inhibition of nitric oxide biosynthesis with those having renal hypertension (two kidney-one clip model). Male Wistar rats were chronically administered the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) for 2, 4 and 8 weeks. Both groups initially developed a similar increase in blood pressure but only the 2K-1C rats developed myocardial hypertrophy after 2–4 weeks. L-NAME-treated animals developed a similar degree of hypertrophy following 8 weeks of treatment. As observed by light microscopy, the myocardial alterations in the latter animals consisted of extensive areas of fibrosis and myocardial necrosis, especially in regions of the subendocardium. The histological alterations induced by L-NAME were not caused by the accompanying hypertension, since the 2K-1C animals had a similar increase in arterial blood pressure without any significant alterations in the heart morphology. 2K-1C rats treated chronically with L-NAME behaved in a manner similar to the L-NAME-treated animals with regard to both the blood pressure increases and cardiac morphological alterations. Animals which received the inactive enantiomer D-NAME did not develop hypertension nor did they have any morphological abnormalities. Both the coronary flow and the contractile capacity of hearts isolated from rats treated with L-NAME for 8 weeks were impaired compared to control animals. These results indicate that the chronic inhibition of NO biosynthesis causes cardiac ischemia associated with a mechanical dysfunction that is unrelated to cardiac hypertrophy which is similar to those seen in some patients suffering from chronic arterial hypertension.

Inhibition of carrageenin‐induced rat paw oedema by crotapotin, a polypeptide complexed with phospholipase A2

1 The effect of purified crotapotin, a non‐toxic non‐enzymatic chaperon protein normally complexed to a phospholipase A2 (PLA2) in South America rattlesnake venom, was studied in the acute inflammatory response induced by carrageenin (1 mg/paw), compound 48/80 (3 μg/paw) and 5‐hydroxytryptamine (5‐HT) (3 μg/paw) in the rat hind‐paw. The effects of crotapotin on platelet aggregation, mast cell degranulation and eicosanoid release from guinea‐pig isolated lung were also investigated. 2 Subplantar co‐injection of crotapotin (1 and 10 μg/paw) with carrageenin or injection of crotapotin (10 μg/paw) into the contralateral paw significantly inhibited the carrageenin‐induced oedema. This inhibition was also observed when crotapotin (10–30 μg/paw) was administered either intraperitoneally or orally. Subplantar injection of heated crotapotin (15 min at 60°C) failed to inhibit carrageenin‐induced oedema. Subplantar injection of crotapotin (10 μg/paw) also significantly inhibited the rat paw oedema induced by compound 48/80, but it did not affect 5‐HT‐induced oedema. 3 In adrenalectomized animals, subplantar injection of crotapotin markedly inhibited the oedema induced by carrageenin. The inhibitory effect of crotapotin was also observed in rats depleted of histamine and 5‐HT stores. 4 Crotapotin (30 μg/paw) had no effect on either the histamine release induced by compound 48/80 in vitro or on the platelet aggregation induced by both arachidonic acid (1 mm) and platelet activating factor (1 μm) in human platelet‐rich plasma. The platelet aggregation and thromboxane B2 (TXB2) release induced by thrombin (100 mu ml−1) in washed human platelets were also not affected by crotapotin. In addition, crotapotin (10 μg/paw) did not affect the release of 6‐oxo‐prostaglandin F1α and TXB2 induced by ovalbumin in sensitized guinea‐pig isolated lungs. 5 Our results indicate that the anti‐inflammatory activity of crotapotin is not due to endogenous corticosteroid release or inhibition of cyclo‐oxygenase activity. It is possible that crotapotin may interact with extracellular PLA2 generated during the inflammatory process thereby reducing its hydrolytic activity.

Obesity enhances eosinophilic inflammation in a murine model of allergic asthma.

Background and purpose:  Obesity is associated with deterioration in asthma outcomes. Although airways eosinophil accumulation is characteristic of lung allergic diseases, little is known about the influence of obesity on the allergic eosinophil trafficking from bone marrow to lung tissues, and recruitment to airways lumen. Here, we have assessed the effects of diet‐induced obesity on allergic eosinophilic inflammation in mice, examining eosinophil trafficking from bone marrow to airways, and production of TH1/TH2 cytokines.

Involvement of kinins, mast cells and sensory neurons in the plasma exudation and paw oedema induced by staphylococcal enterotoxin B in the mouse.

Intraplantar injection of staphylococcal enterotoxin B induces long-lasting oedema mediated by both cyclooxygenase and lipoxygenase products as well as by neuropeptides from sensory nerves. This study was undertaken to further clarify the role of peripheral primary afferent sensory nerves in staphylococcal enterotoxin B (25 microg/paw)-induced plasma extravasation and oedema formation. The tachykinin NK(1) receptor antagonist (S)-1-[2-[3-(3, 4-dichlorophenyl)-1 (3-isopropoxyphenylacetyl)piperidin-3-yl] ethyl]-4-phenyl-1 azoniabicyclo [2.2.2]octane cloride (SR140333; 120 nmol/kg, s.c.+120 nmol/kg, i.v.) significantly inhibited plasma exudation and paw oedema evoked by staphylococcal enterotoxin B. The tachykinin NK(2) receptor antagonist (S)-N-methyl-N[4-(4-acetylamino-4-phenyl piperidino)-2-(3, 4-dichlorophenyl)butyl]-benzamide (SR48968) had no effect on the staphylococcal enterotoxin B-induced responses. The bradykinin B(2) receptor antagonist D-Arg-[Hyp(3),Thi(5),D-Tic(7),Oic(8)]bradykinin (Hoe 140; 400 nmol/kg, i.v.) significantly reduced staphylococcal enterotoxin B-induced responses. The magnitude of the inhibition observed with Hoe 140 alone was similar to that caused by concomitant treatment of animals with SR140333 and Hoe 140, suggesting that there is a final common pathway. Additionally, SR140333 given alone reduced bradykinin (3 nmol/paw)-induced paw oedema. The vanilloid receptor antagonist N-[2-(4-chlorophenyl) ethyl]-1,3,4,5-tetrahydro-7, 8-dihydroxy-2H-2-benzazepine-2-carbothioamide (capsazepine; 100 micromol/kg) significantly reduced staphylococcal enterotoxin B-induced responses. The 5-HT receptor antagonist methysergide (10 mg/kg, i.v.) and the histamine H(1) receptor antagonist mepyramine (10 mg/kg, i.v.) produced a significant reduction in paw oedema whereas plasma exudation was reduced only by methysergide. In diabetic mice, exudation and oedema evoked by staphylococcal enterotoxin B were markedly reduced. Acute administration of insulin (20 UI/kg, s.c., 30 min before) did not restore the increased permeability induced by staphylococcal enterotoxin B. We conclude that plasma exudation and paw oedema in response to staphylococcal enterotoxin B are a consequence of a complex neurogenic response involving direct activation of vanilloid receptors on sensory nerves, release of kinins and subsequent activation of bradykinin B(2) receptors at a prejunctional level, and direct or indirect degranulation of mast cells.

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.

Inflammatory oedema induced by the Lys-49 phospholipase A2 homologue piratoxin-i in the rat and rabbit: Effect of polyanions and p-bromophenacyl bromide

Piratoxin-I (PrTX-I) is a Lys-49 phospholipase (PLA(2)) homologue, isolated from Bothrops pirajai snake venom, that has no phospholipase activity. In this study, we investigated the in vivo oedematogenic activity of PrTX-I in both the rat and the rabbit as well as the ability of PrTX-I to activate rat mast cells in vitro. In the rat paw and skin, PrTX-I (3-100 microg/paw) induced a dose-dependent oedema that was associated with extensive mast cell degranulation. The involvement of mast cells in PrTX-I-mediated oedema formation in the rat was further confirmed by the findings that this protein significantly activated rat peritoneal mast cells in vitro, causing the release of [(14)C]5-hydroxytryptamine ([(14)C]5-HT; 51 +/- 1%). In the rabbit, PrTX-I (10-100 microg/site) also induced dose-dependent skin oedema formation that was not affected by either mepyramine (a histamine H(1) receptor antagonist) or cyproheptadine (1.0 microg/site), indicating that mast cells do not play a role in this animal species. The bradykinin B(2) receptor antagonist Hoe 140 (0.5 microg/site) and the platelet-activating factor (PAF) receptor antagonist WEB 2086 (200 microg/site) also failed to affect the PrTX-I-induced rabbit skin oedema, ruling out the involvement of kinins and PAF. The PLA(2) inhibitor p-bromophenacyl bromide greatly reduced the PrTX-I-induced oedema in both the rat and the rabbit, and also inhibited the rat in vitro mast cell activation induced by this PLA(2) homologue. The polyanions heparin and dermatan sulphate efficiently prevented oedema formation in both species, and heparin inhibited PrTX-I-induced rat mast cell degranulation. Our results are consistent with the suggestion that the cationic charge of PrTX-I plays a major role in the inflammatory responses induced by this PLA(2) homologue.

Defective Insulin and Acetylcholine Induction of Endothelial Cell–Nitric Oxide Synthase Through Insulin Receptor Substrate/Akt Signaling Pathway in Aorta of Obese Rats

The actions of acetylcholine (ACh) on endothelium mainly are mediated through muscarinic receptors, which are members of the G protein–coupled receptor family. In the present study, we show that ACh induces rapid tyrosine phosphorylation and activation of Janus kinase 2 (JAK2) in rat aorta. Upon JAK2 activation, tyrosine phosphorylation of insulin receptor substrate (IRS)-1 is detected. In addition, ACh induces JAK2/IRS-1 and IRS-1/phosphatidylinositol (PI) 3-kinase associations, downstream activation of Akt/protein kinase B, endothelial cell–nitric oxide synthase (eNOS), and extracellular signal–regulated kinase (ERK)-1/2. The pharmacological blockade of JAK2 or PI 3-kinase reduced ACh-stimulated eNOS phosphorylation, NOS activity, and aorta relaxation. These data indicate a new signal transduction pathway for IRS-1/PI 3-kinase/Akt/eNOS activation and ERK1/2 by means of JAK2 tyrosine phosphorylation stimulated by ACh in vessels. Moreover, we demonstrate that in aorta of obese rats (high-fat diet), there is an impairment in the insulin- and ACh-stimulated IRS-1/PI 3-kinase pathway, leading to reduced activation with lower protein levels of eNOS associated with a hyperactivated ERK/mitogen-activated protein kinase pathway. These results suggest that in aorta of obese rats, there not only is insulin resistance but also ACh resistance, probably mediated by a common signaling pathway that controls the activity and the protein levels of eNOS.