Iara M. S. De Luca

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.

Effect of arginine analogues on rat hind paw oedema and mast cell activation in vitro.

Nitro-L-arginine methyl ester (0.15 mumol/paw) significantly reduced both bradykinin- and 5-hydroxytryptamine-induced rat paw oedema. At this dose, L-arginine (L-Arg), D-Arg and nitro-D-arginine methyl ester had no effect on the oedematogenic responses induced by these agents. Nitro-L-arginine methyl ester, nitro-D-arginine methyl ester, L-Arg, D-Arg, L-arginine methyl ester and L-arginine ethyl ester, at the dose of 15 mumol/paw, significantly potentiated both bradykinin- and 5-hydroxytryptamine-induced oedema. This potentiation was not observed in animals treated with both mepyramine and methysergide or in animals chronically treated with compound 48/80. Nitro-L-arginine methyl ester (0.3-3 mM) and L-Arg (0.3-3 mM) released small amounts (< 10%) of histamine from rat peritoneal mast cells when compared to compound 48/80-induced degranulation (> 40%). Histamine release was quantified by radioimmunoassay since nitro-L-arginine methyl ester and L-Arg interfere with the fluorometric assay. The potentiation of paw oedema observed with higher doses of all arginine analogues is caused by in vivo mast cell degranulation and is probably due to the cationic charge of these substances.

Attenuation of hypertension, cardiomyocyte hypertrophy, and myocardial fibrosis by β-adrenoceptor blockers in rats under long-term blockade of nitric oxide synthesis

The effects of propranolol and atenolol were investigated on arterial hypertension, cardiomyocyte hypertrophy, and ventricular ischaemic lesions induced by an 8-week treatment with the nitric oxide synthase inhibitor N ω -nitro- l -arginine methyl ester ( l -NAME; 20 mg/rat per day) in Wistar rats. Propranolol and atenolol (30 mg/rat per day each) were given in the drinking water concomitantly to l -NAME. Treatment with l -NAME induced marked arterial hypertension and cardiomyocyte hypertrophy, both of which were significantly reduced by propranolol and atenolol. A marked repairing fibrosis was also observed in l -NAME-treated rats and this was significantly attenuated in animals receiving the &bgr;-blockers. In l -NAME group, 33% mortality was observed, whereas all the animals from the other groups survived. Our study demonstrates that propranolol and atenolol reduce arterial hypertension, cardiomyocyte hypertrophy and myocardial fibrosis induced by l -NAME, suggesting that &bgr;-blockers are of beneficial value in treatment of vascular and cardiac alterations caused by chronic nitric oxide deficiency.

Development of cardiomyocyte hypotrophy in rats under prolonged treatment with a low dose of a nitric oxide synthesis inhibitor

Chronic administration of the nitric oxide (NO) synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) to rats causes hypertension and morphological abnormalities in the heart, consisting mainly of ventricular hypertrophy and foci of necrosis and fibrosis. Since these phenomena have usually been described with high (or moderate) doses of L-NAME, this study was undertaken to evaluate the effects of a low dose of L-NAME on arterial blood pressure, heart weight index, left ventricular weight index, amount of ventricular fibrosis, and cardiomyocyte size. Male Wistar rats received L-NAME (7.5 mg/kg per day) in the drinking water for 2, 4, and 6 months, whereas control animals received tap water alone. At this dose, L-NAME caused 90% inhibition (P<0.001) of brain NO synthase (NOS) activity. The chronic L-NAME treatment caused an approximately 15% reduction in body weight of the animals, and no death was observed. The tail-cuff pressure was markedly (P<0.01) elevated in L-NAME-treated rats. A significant (P<0.05) reduction in both heart weight index (13-20% decrease) and left ventricular weight index (20-34% decrease) at 2, 4, and 6 months of treatment was observed in L-NAME-treated rats. The cardiomyocyte size in subendocardial, subepicardial, and midmyocardial regions of the left ventricles was time-dependently reduced, irrespective of the region studied, as measured at 2 (11% decrease), 4 (28% decrease, P<0.05), and 6 (45% decrease, P<0.05) months of chronic L-NAME treatment. The amount of fibrous tissue was unaltered at 2 and 4 months, but a small (but significant) increase in the amount of fibrous tissue was detected at 6 months (7.1+/-0.2 %, P<0.05) compared to that of control animals (5.9+/-0.2%). Our results show that chronic treatment of rats with a low dose of L-NAME for prolonged periods (up to 6 months) causes arterial hypertension accompanied by significant reductions in heart weight, left ventricular weight indexes, and cardiomyocyte size.

Effect of Ca2+ channel blockers on arterial hypertension and heart ischaemic lesions induced by chronic blockade of nitric oxide in the rat

The effects of the Ca2+ channel blockers diltiazem, nifedipine and amlodipine were investigated on both arterial hypertension and myocardial changes induced by chronic blockade of nitric oxide synthesis. Control male Wistar rats received Nomega-nitro-L-arginine methyl ester (L-NAME; 20 mg rat(-1) day(-1)) in the drinking water for 8 weeks; blood pressure and body weight were monitored weekly. The Ca2+ channel blockers were given concomitantly to L-NAME, as follows: diltiazem (13.5 mg rat(-1) day(-1)) and amlodipine (6.25 mg rat(-1) day(-1)) were administered in the drinking water whereas nifedipine (6.25 mg rat(-1) day(-1)) was given in the chow. Nomega-nitro-L-arginine methyl ester induced a time-dependent increase in blood pressure which was significantly attenuated by diltiazem (154+/-1.6 vs. 139+/-1.6 mm Hg, p < 0.05), nifedipine (166+/-2.7 vs. 150+/-2.1 mm Hg, p < 0.05) and amlodipine (208+/-5.8 vs. 158+/-1.8 mm Hg, p < 0.05) at the last week of the treatment. Rats treated with the L-NAME also developed myocardial ischaemia, as indicated by the increased percentage of fibrous tissue found in the left ventricles of these animals (10.9+/-0.1%, p < 0.01) when compared to control ones (6.3+/-0.1%). Neither diltiazem (14.9+/-1.2%) nor nifedipine (11.1+/-1.5%) prevented this effect whereas amlodipine (6.9+/-1.1%, p < 0.01) virtually abolished the increase in fibrous tissue induced by L-NAME. The plasma concentration of the Ca2+ channel blockers was measured by liquid chromatography coupled to mass spectrometry at two different time points (morning and afternoon). Only amlodipine treatment was able to maintain constant levels (186+/-46 ng ml(-1) in the morning and 110+/-19 ng ml(-1) in the evening) compared to nifedipine (3003+/-578 ng ml(-1) in the morning and 436+/-100 ng ml(-1) in the evening) and diltiazem (77+/-51 ng ml(-1) in the morning and not detectable in the evening). In conclusion, our results indicate that amlodipine (but not diltiazem and nifedipine) can efficiently control myocardial ischaemia in nitric oxide deficient rats, probably due to its intrinsically long half-life.

Moulting behavior in leaf-frogs of the genus phyllomedusa (Anura : hylidae)

Abstract The species belonging to the subfamily Phyllomedusinae presents physiological and behavioral adaptations to economise water such as secretion of lipids coupled with its spreading onto the skin surface through a complex behavior, adoption of diurnal torpor and secretion of uric acid. Here we describe other adaptation probably involved with water economy, registered in Phyllomedusa distincta , P . tarsius , P . tetraploidea and natural hybrids of P . distincta and P . tetraploidea — at dusk, these amphibians perform an elaborate moulting behavior that encompasses the cleaning of the body with the limbs plus gaping and body contractions. Our data show the daily moulting in P. distincta and suggest that the same mechanism occurs for the other species studied. We suppose that daily moulting improves the skin permeability to water being a characteristic of the phyllomedusines which spread lipids onto the skin surface.

Contribution of C-fibers to leucocyte recruitment in bronchoalveolar lavage fluid and pleural cavity in the rat.

The effect of neonatal capsaicin (8 methyl-N-vanillyl-6-nonenamide) treatment on the leucocyte infiltration into the airways and pleural cavity was investigated in rats actively sensitized with ovalbumin. The animals were neonatally injected with either capsaicin (50 mg/kg, s.c., 2nd day of life) or vehicle (10% ethanol and 10% Tween 80). At adult ages, the animals were actively sensitized with ovalbumin (200 microg, s.c.) and 14 days later they were intratracheally (or intrapleurally) challenged with ovalbumin. The substance P level in bronchoalveolar lavage fluid of the capsaicin group was reduced by >90% compared to control group (vehicle), confirming the efficacy of capsaicin treatment. In the capsaicin group, the number of neutrophils (but not of eosinophils and mononuclear cells) in bronchoalveolar lavage fluid of sensitized animals was significantly higher than the control group. Intrapleural injection of ovalbumin in sensitized rats caused a significant neutrophil influx at 6 h that was markedly increased in the capsaicin-pretreated animals compared to control group. The counts of eosinophils and mononuclear cells in the pleural exudates did not differ significantly between capsaicin and control groups. The increased levels of immunoglobulin (Ig)E, IgG1 and IgG2a anti-ovalbumin antibodies in serum of sensitized rats did not differ between capsaicin and control groups. In conclusion, the exacerbated pulmonary neutrophil recruitment caused by the capsaicin neonatal treatment is unrelated to increase in serum immunoglobulin antibodies, and suggests a protective role for C-fibers in attenuating the allergic neutrophil infiltration.

Long-Term Nitric Oxide Inhibition and Chronotropic Responses in Rat Isolated Right Atria

The long-term administration of nitric oxide synthesis inhibitors induces arterial hypertension accompanied by left ventricular hypertrophy and myocardial ischemic lesions. Because the enhancement of sympathetic drive has been implicated in these phenomena, the current study was performed to determine the potency of beta-adrenoceptor agonists and muscarinic agonists on the spontaneous rate of isolated right atria from rats given long-term treatment with the nitric oxide inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). Atrial lesions induced by long-term treatment with L-NAME were also evaluated. Long-term L-NAME treatment caused a time-dependent, significant (P<0.05) increase in tail-cuff pressure compared with control animals. Our results showed that the potency of isoproterenol, norepinephrine, carbachol, and pilocarpine in isolated right atria from rats given long-term treatment with L-NAME for 7, 15, 30, and 60 days was not affected as compared with control animals. Addition of L-NAME in vitro (100 microl/L) affected neither basal rate nor chronotropic response for isoproterenol and norepinephrine in rat heart. Stereological analysis of the right atria at 15 and 30 days revealed a significant increase on amount of fibrous tissues in L-NAME-treated groups (27+/-2.3% and 28+/-1.3% for 15 and 30 days, respectively; P<0.05) as compared with the control group (22+/-1.1%). Our results indicate that nitric oxide does not to interfere with beta-adrenoceptor-mediated and muscarinic receptor-mediated chronotropic responses.

Modulation of Coronary Flow and Cardiomyocyte Size by Sensory Fibers

Cardiac tissue is densely innervated by sensory neurons that are believed to play important modulatory roles in cardiac functions. In this study, pretreatment of neonate rats with capsaicin was performed. In adult rats, cardiomyocyte size and amount of fibrous tissue in left ventricles as well as in vitro coronary flow were evaluated. The chronotropic and inotropic responses to beta-adrenoceptor agonists (norepinephrine and isoproterenol), muscarinic agonists (carbachol and pilocarpine), and calcitonin gene-related peptide (CGRP) were also investigated with the use of the isolated right atria preparation. Capsaicin pretreatment significantly (P<0.05) reduced both basal coronary flow (18% reduction) and cardiomyocyte size (34% reduction) without affecting the amount of fibrous tissues in the left ventricles. The positive inotropic and chronotropic effects in response to norepinephrine in the isolated rat heart did not significantly differ between control and capsaicin-treated rats. Similarly, the positive chronotropic effects in response to norepinephrine, isoproterenol, and CGRP as well as the negative chronotropic responses to carbachol and pilocarpine in the isolated right atria were not affected by capsaicin pretreatment. Our data are consistent with the suggestion that reductions of both basal coronary flow and cardiomyocyte size seen in hearts from capsaicin-pretreated rats may be consequences of CGRP depletion. The cardiomyocyte size reduction produced by capsaicin treatment may be related to a modulatory role of CGRP as a growth factor.

Enhanced airways responsiveness in rats depleted of sensory neuropeptides by neonatal capsaicin treatment

This study aimed to investigate the in vivo and in vitro reactivity of airway smooth muscle in rats depleted of sensory neuropeptides by treatment with capsaicin at neonatal stage. Wistar rats were neonatally injected with either capsaicin (50 mg/kg, s.c., 2nd day of life) or its vehicle (10% ethanol and 10% Tween 80, in 0.9% w/v NaCl solution) and used at adult ages (60-70 days later). Analysis of the lungs showed a higher number of infiltrating neutrophils, eosinophils and mononuclear cells into the peribronchiolar regions of capsaicin-pretreated rats compared to vehicle group. This was associated with a higher contraction index of bronchiolar wall in the capsaicin group. The in vitro tracheal reactivity in response to methacholine (full muscarinic agonist) and pilocarpine (partial muscarinic agonist) was also significantly higher in capsaicin-pretreated rats compared to vehicle group. In conclusion, the neuropeptide depletion by capsaicin neonatal treatment lead to marked contraction of the rat airways at adult age, suggesting a protective role for C fibers in the lungs.