Acute hypobaric hypoxia and cardiac energetic response in prepubertal rats: Role of nitric oxide.

NEW FINDINGS: What is the central question of this study? In adult rat hearts, exposure to hypobaric hypoxia increases tolerance to hypoxia-reoxygenation, termed endogenous cardioprotection. The mechanism involves the nitric oxide system and modulation of mitochondrial oxygen consumption. What is the cardiac energetic response in prepubertal rats exposed to hypobaric hypoxia? What is the main finding and its importance? Prepubertal rats, unlike adult rats, did not increase tolerance to hypoxia-reoxygenation in response acute exposure to hypobaric hypoxia, which impaired cardiac contractile economy. This finding could be related to a failure to increase nitric oxide synthase expression, hence modulation of mitochondrial oxygen consumption and ATP production. ABSTRACT: Studies in our laboratory showed that exposure of rats to hypobaric hypoxia (HH) increased the tolerance of the heart to hypoxia-reoxygenation (H/R), involving mitochondrial and cytosolic nitric oxide synthase (NOS) systems. The objective of the present study was to evaluate how the degree of somatic maturation could alter this healthy response. Prepubertal male rats were exposed for 48 h to a simulated altitude of 4400 m in a hypobaric chamber. The mechanical energetic activity in perfused hearts and the contractile functional capacity of NOS in isolated left ventricular papillary muscles were evaluated during H/R. Cytosolic nitric oxide (NO), production of nitrites/nitrates (Nx), expression of NOS isoforms, mitochondrial O2 consumption and ATP production were also evaluated. The left ventricular pressure during H/R was not improved by HH. However, the energetic activity was increased. Thus, the contractile economy (left ventricular pressure/energetic activity) decreased in HH. Nitric oxide did not modify papillary muscle contractility after H/R. Cytosolic p-eNOS-Ser1177 and inducible NOS expression were decreased by HH, but no changes were observed in NO production. Interestingly, HH increased Nx levels, but O2  consumption and ATP production in mitochondria were not affected by HH. Prepubertal rats exposed to HH preserved cardiac contractile function, but with a high energetic cost, modifying contractile economy. Although this could be related to the decreased NOS expression detected, cytosolic NO production was preserved, maybe through the Nx metabolic pathway, without modification of mitochondrial ATP production and O2  consumption. In this scenario, the treatment was unable to increase tolerance to H/R as observed in adult animals.

Cardioprotection after acute exposure to simulated high altitude in rats. Role of nitric oxide.

AIM: In previous studies, upregulation of NOS during acclimatization of rats to sustained hypobaric hypoxia was associated to cardioprotection, evaluated as an increased tolerance of myocardium to hypoxia/reoxygenation. The objective of the present work was to investigate the effect of acute hypobaric hypoxia and the role of endogenous NO concerning cardiac tolerance to hypoxia/reoxygenation under ß-adrenergic stimulation. METHODS: Rats were submitted to 58.7 kPa in a hypopressure chamber for 48 h whereas their normoxic controls remained at 101.3 kPa. By adding NOS substrate L-arg, or blocker L-NNA, isometric mechanical activity of papillary muscles isolated from left ventricle was evaluated at maximal or minimal production of NO, respectively, under ß-adrenergic stimulation by isoproterenol, followed by 60/30 min of hypoxia/reoxygenation. Activities of NOS and cytochrome oxidase were evaluated by spectrophotometric methods and expression of HIF1-α and NOS isoforms by western blot. Eosin and hematoxiline staining were used for histological studies. RESULTS: Cytosolic expression of HIF1-α, nNOS and eNOS, and NO production were higher in left ventricle of hypoxic rats. Mitochondrial cytochrome oxidase activity was decreased by hypobaric hypoxia and this effect was reversed by L-NNA. After H/R, recovery of developed tension in papillary muscles from normoxic rats was 51-60% (regardless NO modulation) while in hypobaric hypoxia was 70% ±â€¯3 (L-arg) and 54% ±â€¯1 (L-NNA). Other mechanical parameters showed similar results. Preserved histological architecture was observed only in L-arg papillary muscles of hypoxic rats. CONCLUSION: Exposure of rats to hypobaric hypoxia for only 2 days increased NO synthesis leading to cardioprotection.

Mechanism of action of novel naphthofuranquinones on rat liver microsomal peroxidation.

In order to elucidate the effect on mammal systems of new derivatives from 2-hydroxy-3-allyl-naphthoquinone, alpha-iodinated naphthofuranquinone (NPPN-3223), beta-iodinated naphthofuranquinone (NPPN-3222) and beta-methyl naphthofuranquinone (NPPN-3226) synthesized as possible trypanocidal agents, their effect on rat liver microsomal lipid peroxidation was investigated. They (a) inhibited NADPH-dependent, iron-catalyzed microsomal rat liver lipid peroxidation; (b) did not inhibit the tert-butyl hydroperoxide-dependent lipid peroxidation; (c) did not inhibit ascorbate-lipid peroxidation with the exception of NPPN-3226 which did inhibit it; (d) stimulated NADPH oxidation and microsomal oxygen uptake; (e) increased superoxide anion formation by NADPH-supplemented microsomes and (f) stimulated ascorbate oxidation. The three drugs were reduced to their seminaphthofuranquinone radical by the liver NADPH-P450 reductase system, as detected by ESR measurements. These results support the hypothesis that naphthofuranquinones reduction by microsomal NADPH-P450 reductase and semiquinone oxidation by molecular oxygen diverts electrons, preventing microsomal lipid peroxidation. In addition, hydroquinones and/or semiquinones formed by naphthofuranquinones reduction would be capable of lipid peroxidation inhibition and on interacting with the lipid peroxide radicals can lead to an antioxidant effect as we suggested for NPPN-3226 in close agreement to the inhibition of ascorbate-lipid peroxidation. Due to the properties of these molecules and their incoming structure developments, naphthofuranquinones would be considered as potentially promising therapeutic agents, mainly against Chagas disease.

Time course of regression of the protection conferred by simulated high altitude to rat myocardium: correlation with mtNOS.

During acclimatization to sustained hypobaric hypoxia, retardation of age-associated decline in left ventricle mechanical activity and improved posthypoxic recovery were accompanied by upregulation of mitochondrial nitric oxide synthase (mtNOS). To evaluate the time course of regression of these effects on deacclimatization, rats exposed to 53.8 kPa in a hypopressure chamber for 5 mo were returned to 101.3 kPa, whereas controls remained at 101.3 kPa throughout the study. At three time points, contractile function in response to calcium and to hypoxia-reoxygenation (H/R) were determined in papillary muscle, and NOS activity and expression were determined in mitochondria isolated from left ventricle. Developed tension was, before H/R, 65, 58, and 40%, and, after H/R, 129, 107, and 71% higher than in controls at 0.4, 2, and 5 mo of normoxia, respectively. Maximal rates of contraction and relaxation followed a similar pattern. All three parameters showed a linear decline during deacclimatization, with mean half-time (t(1/2)) of 5.9 mo for basal mechanical activity and 5.3 mo for posthypoxic recovery. Left ventricle mtNOS activity was 42, 27, and 20% higher than in controls at 0.4, 2, and 5 mo, respectively (t(1/2) = 5.0 mo). The expression of mtNOS showed similar behavior. The correlation of mtNOS activity with muscle contractility sustained a biphasic modulation, suggesting an optimal mtNOS activity. This experimental model would provide the most persistent effect known at present on preservation of myocardial mechanical activity and improved tolerance to O(2) deprivation. Results support the putative role of mtNOS in the mechanism involved.

Effect of sustained hypobaric hypoxia during maturation and aging on rat myocardium. I. Mechanical activity.

Long-lasting cardioprotection may be attained by chronic hypoxia. The basal parameters of contractile function and their response to hypoxia/reoxygenation were measured under isometric conditions, in papillary muscles isolated from left ventricle of rats that were submitted to 53.8 kPa in a hypobaric chamber from 7 wk of age and for their lifetime and of their siblings kept at 101.3 kPa. During acclimatization, hematocrit increased, body weight gain decreased, and heart weight increased with right ventricle hypertrophy. Papillary muscle cross-sectional area was similar in both control and hypoxic groups up to 45 wk of exposure. Developed tension (DT) was 34-64% higher in rats exposed to hypoxia for 10, 26, and 45 wk than in their age-matched controls, whereas resting tension was unchanged. Maximal rates of contraction and relaxation showed a similar pattern of changes as DT. Recovery of DT and maximal rates of contraction and relaxation after 60-min hypoxia and 30-min reoxygenation was also improved in adult hypoxic rats to values similar to those of young rats. Heart acclimatization was lost after 74 wk of exposure. Results are consistent with the development of cardioprotection during high-altitude acclimatization and provide an experimental model to study the mechanisms involved, which are addressed in the accompanying paper.

Effect of sustained hypobaric hypoxia during maturation and aging on rat myocardium. II. mtNOS activity.

Mitochondrial nitric oxide (NO) production was assayed in rats submitted to hypobaric hypoxia and in normoxic controls (53.8 and 101.3 kPa air pressure, respectively). Heart mitochondria from young normoxic animals produced 0.62 and 0.37 nmol NO.min(-1).mg protein(-1) in metabolic states 4 and 3, respectively. This production accounts for a release to the cytosol of 29 nmol NO.min(-1).g heart(-1) and for 55% of the NO generation. The mitochondrial NO synthase (mtNOS) activity measured in submitochondrial membranes at pH 7.4 was 0.69 nmol NO.min(-1).mg protein(-1). Rats exposed to hypobaric hypoxia for 2-18 mo showed 20-60% increased left ventricle mtNOS activity compared with their normoxic siblings. Left ventricle NADH-cytochrome-c reductase and cytochrome oxidase activities decreased by 36 and 12%, respectively, from 2 to 18 mo of age, but they were not affected by hypoxia. mtNOS upregulation in hypoxia was associated with a retardation of the decline in the mechanical activity of papillary muscle upon aging and an improved recovery after anoxia-reoxygenation. The correlation of left ventricle mtNOS activity with papillary muscle contractility (determined as developed tension, maximal rates of contraction and relaxation) showed an optimal mtNOS activity (0.69 nmol.min(-1).mg protein(-1)). Heart mtNOS activity is regulated by O(2) in the inspired air and seems to play a role in NO-mediated signaling and myocardial contractility.

Heart mitochondrial nitric oxide synthase. Effects of hypoxia and aging.

The production of NO by heart mitochondria was 0.7-1.1 nmol NO/min.mg protein, an activity similar to the ones observed in mitochondrial membranes from other organs. Heart mtNOS seems to contribute with about 56% of the total cellular NO production. The immunological nature of the mtNOS isoform of cardiac tissue remains unclear; in our laboratory, heart mtNOS reacted with an anti-iNOS anti-body. Heart mtNOS expression and activity are regulated by physiological and pharmacological effectors. The state 4/state 3 transition regulates heart mtNOS activity and NO release in intact respiring mitochondria: NO production rates in state 3 were 40% lower than in state 4. Heart mtNOS expression was selectively regulated by O(2) availability in hypobaric conditions and the activity was 20-60% higher in hypoxic rats than in control animals, depending on age. In contrast, NADH-cytochrome c reductase and cytochrome oxidase activities were not affected by hypoxia. The activity of rat heart mtNOS decreased 20% on aging from 12 to 72 weeks of age. On the pharmacological side, mitochondrial NO production was increased after enalapril treatment (the inhibitor of the angiotensin converting enzyme) with modification of heart mtNOS functional activity in the regulation of mitochondrial O(2) uptake and H(2)O(2) production. Thus, heart mtNOS is a highly regulated mitochondrial enzyme, which in turn, plays a regulatory role through mitochondrial NO steady state levels that modulate O(2) uptake and O(2)(-) and H(2)O(2) production rates. Nitric oxide and H(2)O(2) constitute signals for metabolic control that are involved in the regulation of cellular processes, such as proliferation and apoptosis.

Enalapril increases mitochondrial nitric oxide synthase activity in heart and liver.

Heart and liver mitochondria isolated from rats treated with enalapril, 3-30 mg/kg/day in the drinking water for 7-120 days, showed a time- and dose-dependent increased nitric oxide (NO) production in the range of 14-250%. Heart and liver mitochondria from control rats produced 0.69 and 0.50 nmol of NO/min/mg of protein, respectively, as determined by dual wavelength spectrophotometry (577-591 nm) following hemoglobin oxidation to methemoglobin. The response to enalapril treatment, attributed to a gene-mediated up-regulation of mitochondrial nitric oxide synthase (mtNOS) activity, was half-maximal at 5-6 days and was maintained up to 120 days. Enalapril-treated animals showed an increased mtNOS functional activity in heart mitochondria that inhibited state 3 O(2) uptake (from 22% in control rats to 43%) and increased state 4 hydrogen peroxide (H(2)O(2)) production (from 30% in control rats to 52%). Calculated heart intramitochondrial NO and H(2)O(2) steady-state concentrations were increased 66% and 20%, respectively, by enalapril treatment. Signaling pathways dependent on mitochondrial NO and H(2)O(2) may account for the beneficial effects of enalapril in aging mammals.

Long-term angiotensin II inhibition increases mitochondrial nitric oxide synthase and not antioxidant enzyme activities in rat heart.

OBJECTIVE: To provide insight into the subcellular mechanisms involved in the improvement of cardiovascular structure and function by long-term inhibition of the renin-angiotensin system. DESIGN: The activities of antioxidant enzymes and mitochondrial free radical production were determined in the heart of control (C), enalapril-treated (E), and losartan-treated (L) rats to test the hypothesis of increased antioxidant enzyme activities and participation of mitochondria in the effects of chronic treatments with angiotensin II inhibitors. METHODS: At 6 and 18 months of treatment, superoxide dismutases (SOD), Se-glutathione peroxidase, and catalase activities were determined in left ventricle homogenates by spectrophotometric methods and nitric oxide (NO) production in submitochondrial membranes by the oxyhemoglobin oxidation assay. The maximal rate of hydrogen peroxide (H2O2) production by submitochondrial membranes was also evaluated at 18 months by the scopoletin-horseradish peroxidase method. RESULTS: No significant increase was found in the antioxidant enzymes measured. At 6 months, Mn-SOD was actually decreased in E and catalase in both E and L, whereas at 18 months Se-glutathione peroxidase was decreased in L. Production of NO by submitochondrial particles was 64% higher at 6 months in E and 105% higher at 18 months in E and L. Maximal hydrogen peroxide production was lower at 18 months in both groups. CONCLUSIONS: Results do not support the hypothesis of an increase in antioxidant enzyme activity by long-term treatment with angiotensin II inhibitors as previously suggested and point towards a role for the NO produced by mitochondrial nitric oxide synthase (mtNOS) in the protective effect of these drugs.

Metahemoglobinemia en lactante pequeños / Methemoglobinemia in small infants

Se analizaron 40 casos consecutivos asistidos durante los dos últimos años, en el Hospital de Niños "Sor María Ludovica" de La Plata, que presentaron metahemoglobinemia (MHb). El 75% de los casos eran menores de 3 meses de vida. El motivo de consulta fueron manifestaciones gastrointestinales (diarrea, vómitos) en el 80% de los casos, cianosis en 17%, enfermedad respiratoria en el 12% y signos neurológicos en el 10%. Analizando los datos se vio que el 70% de los niños no tenían en sus hogares ningún servicio sanitario. El valor medio de MHb fue de 2,1 g/dl (rango 0,4-5,5 g/dl). Todos los niños, con excepción de uno, habían sido destetados. El 100% de los casos fue tratado exitosamente con vitamina C, como única medicación para su MHb

Metahemoglobinemia en lactante pequeños / Methemoglobinemia in small infants

Se analizaron 40 casos consecutivos asistidos durante los dos últimos años, en el Hospital de Niños "Sor María Ludovica" de La Plata, que presentaron metahemoglobinemia (MHb). El 75% de los casos eran menores de 3 meses de vida. El motivo de consulta fueron manifestaciones gastrointestinales (diarrea, vómitos) en el 80% de los casos, cianosis en 17%, enfermedad respiratoria en el 12% y signos neurológicos en el 10%. Analizando los datos se vio que el 70% de los niños no tenían en sus hogares ningún servicio sanitario. El valor medio de MHb fue de 2,1 g/dl (rango 0,4-5,5 g/dl). Todos los niños, con excepción de uno, habían sido destetados. El 100% de los casos fue tratado exitosamente con vitamina C, como única medicación para su MHb (AU)