Effect of 7-Nitroindazole Sodium on the Cellular Distribution of Neuronal Nitric Oxide Synthase in the Cerebral Cortex of Hypoxic Newborn Piglets

Cerebral hypoxia results in generation of nitric oxide (NO) free radicals by Ca⁺⁺-dependent activation of neuronal nitric oxide synthase (nNOS). The present study tests the hypothesis that the hypoxia-induced increased expression of nNOS in cortical neurons is mediated by NO. To test this hypothesis the cellular distribution of nNOS was determined immunohistochemically in the cerebral cortex of hypoxic newborn piglets with and without prior exposure to the selective nNOS inhibitor 7-nitroindazole sodium (7-NINA). Studies were conducted in newborn piglets, divided into normoxic (n = 6), normoxic treated with 7-NINA (n = 6), hypoxic (n = 6) and hypoxic pretreated with 7-NINA (n = 6). Hypoxia was induced by lowering the FiO₂ to 0.05–0.07 for 1 h. Cerebral tissue hypoxia was documented by decrease of ATP and phosphocreatine levels in both the hypoxic and 7-NINA pretreated hypoxic groups (P < 0.01). An increase in the number of nNOS immunoreactive neurons was observed in the frontal and parietal cortex of the hypoxic as compared to the normoxic groups (P < 0.05) which was attenuated by pretreatment with 7-NINA (P < 0.05 versus hypoxic). 7-NINA affected neither the cerebral energy metabolism nor the cellular distribution of nNOS in the cerebral cortex of normoxic animals. We conclude that nNOS expression in cortical neurons of hypoxic newborn piglets is NO-mediated. We speculate that nNOS inhibition by 7-NINA will protect against hypoxia-induced NO-mediated neuronal death.     

Effect of Allopurinol on Hypoxia-Induced Modification of the NMDA Receptor in Newborn Piglets

The present study tests the hypothesis that pretreatment with allopurinol, a xanthine oxidase inhibitor, will prevent modification of the NMDA receptor during cerebral hypoxia in newborn piglets. Eighteen newborn piglets were studied. Six normoxic control animals were compared to six untreated hypoxic and six allopurinol (20 mg/kg i.v.) pretreated hypoxic piglets. Cerebral hypoxia was induced by lowering the FiO₂ to 0.05–0.07 for 1 hour and tissue hypoxia was confirmed biochemically by the measurement of ATP and phosphocreatine. Brain cell membrane Na⁺,K⁺-ATPase activity was determined to assess membrane function. Na⁺,K⁺-ATPase activity was decreased from control in both the untreated and treated hypoxic animals (46.0 ± 1.0 vs 37.9 ± 2.5 and 37.3 ± 1.4 mol Pi/mg protein/hr, respectively, p < 0.05). [³H]MK-801 binding was determined as an index of NMDA receptor modification. The receptor density (Bmax) in the untreated hypoxic group was decreased compared to normoxic control (1.09 ± 0.17 vs 0.68 ± 0.22 pmol/mg protein, p < 0.01). The dissociation constant (Kd) was also decreased in the untreated group (10.0 ± 2.0 vs 4.9 ± 1.4 nM, p < 0.01), indicating an increase in receptor affinity. However, in the allopurinol treated hypoxic group, the Bmax (1.27 ± 0.09 pmol/mg protein) was similar to normoxic control and the Kd (8.1 ± 1.2 nM, p < 0.05) was significantly higher than in the untreated hypoxic group. The data show that the administration of allopurinol prior to hypoxia prevents hypoxia-induced modification of the NMDA receptor-ion channel binding characteristics, despite neuronal membrane dysfunction. By preventing NMDA receptor-ion channel modification, allopurinol may produce a neuromodulatory effect during hypoxia and attenuate NMDA receptor mediated excitotoxicity.     

NMDA receptor modification in the fetal guinea pig brain during hypoxia

The effect of maternal hypoxia on the modification of the fetal brain cell membrane N-methyl-d-aspartate (NMDA) receptor and its modulatory sites was investigated. Experiments were conducted in pregnant guinea pigs of 60 days of gestation. Guinea pig fetuses were exposed to maternai hypoxia (FiO₂=7%) for 60 minutes. Tissue hypoxia in the fetal brain was documented biochemically by decreased levels of ATP and phosphocreatine (91.3% and 88.6% lower than normoxia, respectively). MK-801 binding characteristics (Bmax = number of receptors, Kd = affinity of receptor) were used as an index of NMDA receptor modification. P2 membrane fraction was prepared from the cortex of normoxic and hypoxic fetal brain and washed thoroughly before carrying out the binding assay. In hypoxic brains, Bmax decreased from the normoxic control level 0.79±0.03 pmol/mg protein to 0.58±0.03 pmol/mg protein (P<0.005) and Kd value decreased (increased affinity) from 8.54±0.27 nM to 4.01±0.23 nM (P<0.005) respectively. The MK-801 binding in the absence of added glutamate and glycine in hypoxic brain was 100% higher as compared to controls, indicating an increased sensitivity of the NMDA receptor to activation. The spermine dependent maximum activation of the NMDA receptor increased to 44% in the hypoxic animals as compared to 25% in controls. The Mg²⁺ response of the NMDA receptor was not affected by hypoxia. The increased affinity and increased basal activation (tone) of the NMDA receptor during hypoxia, as well as its increased activation by spermine, would hyperstimulate the NMDA receptor-ion channel complex function which could increase the susceptibility of the fetal brain to hypoxia. The results of this study indicate that hypoxia causes differential and selective modification of specific sites (recognition, co-activator, and modulatory) of the NMDA receptor ion channel complex. The hypoxia-induced modification of the NMDA receptor modulatory sites appears to be the potential mechanism of neuroexcitotoxicity.     

Cyclooxygenase-Mediated Generation of Free Radicals During Hypoxia in the Cerebral Cortex of Newborn Piglets

Previous studies have demonstrated that free radicals are formed under hypoxic conditions in newborn piglet brain. To test the hypothesis that the cyclooxygenase pathway serves as a source of free radical generation during hypoxia studies were performed on 24 piglets divided into four groups. Six saline (group 3) and six indomethacin treated (group 4) were exposed to hypoxia (FiO2 0.05-0.07) for 60 min. Cerebral hypoxia was documented biochemically by determination of ATP and phosphocreatine. Fluorescent compounds and conjugated dienes were determined as indices of lipid peroxidation. Free radical formation was determined by using n-tert butyl phenyl nitrone (PBN) as a spin trap agent and measuring spin adduct formation in duplicate using a Varian E-109 spectrometer. Groups 1 and 2 (normoxic) showed no spin adduct formation. Group 3 showed a significant increase in spin adduct formation compared to normoxia (372+/-125 vs. 63+/-15, P<0.001). Hypoxic animals pretreated with indomethacin had a spin adduct level of 197+/-132 and were similar to normoxic animals. ATP/PCr levels were the same in groups 3 and 4 denoting the same degree of cerebral hypoxia in all hypoxic animals. Conjugated dienes increased significantly during hypoxia as compared to normoxia (0.142+/-0.017 vs. 0.0+/-0.0) and were decreased insignificantly with indomethacin treatment. Fluorescent compounds were not significantly different among the four groups. Na+,K+-ATPase activity decreased during hypoxia but was not preserved in hypoxic animals pretreated with indomethacin. These data provide direct evidence of the presence of free radicals during hypoxia and the contribution of cyclooxygenase metabolism to their formation.     

NMDA Receptor-Mediated Calcium Influx in Cerebral Cortical Synaptosomes of the Hypoxic Guinea Pig Fetus

Calcium influx via the NMDA receptor has been proposed as a mechanism of hypoxia-induced neuronal injury. The present study tests the hypothesis that the increase of [Ca²⁺]_i observed under hypoxic conditions is the result of an NMDA-mediated Ca²⁺ influx. Changes of [Ca²⁺]_i, measured fluorometrically with Fura-2, were followed after activation of the NMDA receptor with NMDA and glutamate, in the presence of glycine, in cortical synaptosomes prepared from six normoxic and six hypoxic guinea pig fetuses. [Ca²⁺]_i was significantly higher in hypoxic vs normoxic synaptosomes, at baseline and in the presence of glycine as well as following activation of the NMDA receptor. Increase in [Ca²⁺]_i was not observed in a Ca²⁺ free medium and was significantly decreased by MK-801 and thapsigargin. These results demonstrate that hypoxia-induced modifications of the NMDA receptor ion-channel results in increased [Ca²⁺]_i in hypoxic vs normoxic synaptosomes. This increased accumulation may be due to an initial influx of Ca²⁺ via the altered NMDA receptor with subsequent release of Ca²⁺ from intracellular stores. Increase in intracellular calcium may initiate several pathways of free radical generation including cyclooxygenase, lipoxygenase, xanthine oxidase and nitric oxide synthase, and lead to membrane lipid peroxidation resulting in neuronal cell damage.     

Nitration as a Mechanism of Na+, K+-ATPase Modification During Hypoxia in the Cerebral Cortex of the Guinea Pig Fetus

Previous studies have shown that hypoxia induces nitric oxide synthase-mediated generation of nitric oxide free radicals leading to peroxynitrite production. The present study tests the hypothesis that hypoxia results in NO-mediated modification of Na⁺, K⁺-ATPase in the fetal brain. Studies were conducted in guinea pig fetuses of 58-days gestation. The mothers were exposed to FiO₂ of 0.07% for 1 hour. Brain tissue hypoxia in the fetus was confirmed biochemically by decreased ATP and phosphocreatine levels. P₂ membrane fractions were prepared from normoxic and hypoxic fetuses and divided into untreated and treated groups. The membranes were treated with 0.5 mM peroxynitrite at pH 7.6. The Na⁺, K⁺-ATPase activity was determined at 37°C for five minutes in a medium containing 100 mM NaCl, 20 mM KCl, 6.0 mM MgCl₂, 50 mM Tris HCl buffer pH 7.4, 3.0 mM ATP with or without 10 mM ouabain. Ouabain sensitive activity was referred to as Na⁺, K⁺-ATPase activity. Following peroxynitrite exposure, the activity of Na⁺, K⁺-ATPase in guinea pig brain was reduced by 36% in normoxic membranes and further 29% in hypoxic membranes. Enzyme kinetics was determined at varying concentrations of ATP (0.5 mM-2.0 mM). The results indicate that peroxynitrite treatment alters the affinity of the active site of Na⁺, K⁺-ATPase for ATP and decreases the Vmax by 35% in hypoxic membranes. When compared to untreated normoxic membranes Vmax decreases by 35.6% in treated normoxic membranes and further to 52% in treated hypoxic membranes. The data show that peroxynitrite treatment induces modification of Na⁺, K⁺-ATPase. The results demonstrate that peroxynitrite decreased activity of Na⁺, K⁺-ATPase enzyme by altering the active sites as well as the microenvironment of the enzyme. We propose that nitric oxide synthase-mediated formation of peroxynitrite during hypoxia is a potential mechanism of hypoxia-induced decrease in Na⁺, K⁺-ATPase activity.     

Lipid Free Radical Generation and Brain Cell Membrane Alteration Following Nitric Oxide Synthase Inhibition During Cerebral Hypoxia in the Newborn Piglet

Abstract: Nitric oxide (NO) is reported to cause neuronal damage through various mechanisms. The present study tests the hypothesis that NO synthase inhibition by N ^ω-nitro- l -arginine (NNLA) will result in decreased oxygen-derived free radical production leading to the preservation of cell membrane structure and function during cerebral hypoxia. Ten newborn piglets were pretreated with NNLA (40 mg/kg); five were subjected to hypoxia, whereas the other five were maintained with normoxia. An additional 10 piglets without NNLA treatment underwent the same conditions. Hypoxia was induced with a lowered FiO₂ and documented biochemically by decreased cerebral ATP and phosphocreatine levels. Free radicals were detected by using electron spin resonance spectroscopy with a spin trapping technique. Results demonstrated that free radicals, corresponding to alkoxyl radicals, were induced by hypoxia but were inhibited by pretreatment with NNLA before inducing hypoxia. NNLA also inhibited hypoxia-induced generation of conjugated dienes, products of lipid peroxidation. Na⁺,K⁺-ATPase activity, an index of cellular membrane function, decreased following hypoxia but was preserved by pretreatment with NNLA. These data demonstrate that during hypoxia NO generates free radicals via peroxynitrite production, presumably causing lipid peroxidation and membrane dysfunction. These results suggest that NO is a potentially limiting factor in the peroxynitrite-mediated lipid peroxidation resulting in membrane injury.     

Hypoxia-induced caspase-3 activation and DNA fragmentation in cortical neurons of newborn piglets: role of nitric oxide

Hypoxia results in generation of nitric oxide (NO) free radicals, activation of caspase-3, and genomic DNA fragmentation. The present study tests the hypothesis that hypoxia-induced caspase-3 activation and DNA fragmentation are nitric oxide mediated. Studies were conducted in newborn piglets, divided into normoxic (n = 5), hypoxic (n = 5), and hypoxic-7-NINA (n = 6). Hypoxic-7-NINA group received the neuronal nitric oxide synthase inhibitor, 7-Nitroindazole (7-NINA). Caspase-3 activity was determined spectrofluorometrically using enzyme-specific substrates. Sections from the neocortex were stained with an antiserum recognizing active caspase-3. Purified DNA was separated by gel electrophoresis. Administration of 7-NINA resulted in decreased immunoreactivity of caspase-3 (mean LI: 20.2%) as compared to the untreated hypoxia group (mean LI: 57.5%) (P < 0.05). 7-NINA attenuated caspase-3 enzymatic activity as well in comparison to the untreated hypoxia group (P < 0.05). Furthermore, multiple low molecular weight bands corresponding to DNA fragments were present in the hypoxic but not in the normoxic or hypoxic-7-NINA groups. Inhibition of nNOS abates the hypoxia-induced increase in active caspase-3 immunoreactivity, as well as enzymatic activity in cortical neurons, and DNA fragmentation in brain homogenates. We conclude that the coordinate increase of capase-3 activity and fragmentation of nuclear DNA in the hypoxic newborn piglet brain are NO mediated.     

Hypoxic Response of Synaptosomal Proteins in Term Guinea Pig Fetuses

Early events in the hypoxia-induced response trigger tyrosine phosphorylation cascades involving a large number of enzymes and substrates. The resolving power of advanced two-dimensional gel electrophoresis, followed by immunoblotting with specific antibodies to phosphotyrosine, has been used to analyze hypoxia-induced modifications in guinea pig brain synaptosomes. These procedures, in conjunction with computer-aided image analysis, are useful in the differential display of gene products, providing comparison at the level of posttranslationally modified products. Studies were performed in cerebral cortical synaptosomes from three normoxic and three hypoxic newborn guinea pigs. To filter off background noise consisting of nonreproducible migrating protein spots, only reproducible features of electrophoretic patterns were considered. Immunoreactivity patterns obtained with anti-phosphotyrosine antibodies proved to be different in normoxic and hypoxic synaptosomes: of a total of 130 immunoreactive spots, 49 were tyrosine-phosphorylated in hypoxic synaptosomes only and 20 in the normoxic ones only. Our data suggest that hypoxia extensively remodels the signaling pathway by switching off tyrosine phosphorylation of some cellular components (i.e., alpha-internexin) and switching on tyrosine phosphorylation of some other proteins (i.e., heat shock cognate 70, aconitase, 2',3'-cyclic nucleotide 3'-phosphodiesterase, and pyruvate kinase).     

N-acetylcysteine inhibits hypoxic pulmonary hypertension most effectively in the initial phase of chronic hypoxia

Exposure to chronic hypoxia results in hypoxic pulmonary hypertension (HPH). In rats HPH develops during the first two weeks of exposure to hypoxia, then it stabilizes and does not increase in severity. We hypothesize that free radical injury to pulmonary vascular wall is an important mechanism in the early days of the hypoxic exposure. Thus antioxidant treatment just before and at the beginning of hypoxia should be more effective in reducing HPH than antioxidant therapy of developed pulmonary hypertension. We studied adult male rats exposed for 4 weeks to isobaric hypoxia (F(iO2) = 0.1) and treated with the antioxidant, N-acetylcysteine (NAC, 20 g/l in drinking water). NAC was given "early" (7 days before and the first 7 days of hypoxia) or "late" (last two weeks of hypoxic exposure). These experimental groups were compared with normoxic controls and untreated hypoxic rats (3-4 weeks hypoxia). All animals kept in hypoxia had significantly higher mean pulmonary arterial blood pressure (PAP) than normoxic animals. PAP was significantly lower in hypoxic animals with early (27.1 +/- 0.9 mmHg) than late NAC treatment (30.5 +/- 1.0 mmHg, P < 0.05; hypoxic without NAC 32.6 +/- 1.2 mmHg, normoxic controls 14.9 +/- 0.7 mmHg). Early but not late NAC treatment inhibited hypoxia-induced increase in right ventricle weight and muscularization of distal pulmonary arteries assessed by quantitative histology. We conclude that release of free oxygen radicals in early phases of exposure to hypoxia induces injury to pulmonary vessels that contributes to their structural remodeling and development of HPH.     

Chronic hypoxia increases MCA contractile response to U-46619 by reducing NO production and/or activity.

Chronic hypoxia alters contractile sensitivity of isolated arteries to alpha-adrenergic stimulation and other agonists. However, most studies have been performed in thoracic aortas or other large vessels making little contribution to vascular resistance in their respective circulations. To determine the effect of chronic hypoxia on the vasoconstrictor response in a small, resistance-sized vessel, we studied second and third generation middle cerebral arteries (MCA; approximately 75-microm internal diameter before mounting). MCA were isolated from normoxic (inspired oxygen = 125 Torr) and hypoxic (8 wk at 3,960 m; inspired oxygen = 90 Torr) guinea pigs, and their vasoconstrictor responses were determined to the thromboxane mimetic U-46619 by using dual-pipette video microscopy. Arteries from hypoxic animals had greater contractile sensitivity to U-46619 compared with those of the normoxic animals (-log EC50 = 7.86 +/- 0.11 vs. 7.62 +/- 0.06, respectively, P < 0.05). Addition of the nitric oxide (NO) inhibitor nitro-L-arginine (200 microM) to the vessel bath eliminated the differences in contractile sensitivity between the MCA from the normoxic and chronically hypoxic groups. Supplementation with L-arginine in the drinking water sufficient to raise plasma L-arginine levels 41% reduced MCA contractile sensitivity to U-46619 in the normoxic group (-log EC50 = 7.22 +/- 0.31, P < 0.05 compared with the nonsupplemented normoxic group) but not in the chronically hypoxic group. These results show that chronic hypoxia increases the sensitivity of the MCA to the vasoconstrictor U-46619, likely because of a reduction in NO production and/or activity.     

Nitric Oxide–Mediated Modification of the Glycine Binding Site of the NMDA Receptor During Hypoxia in the Cerebral Cortex of the Newborn Piglet

This study tested the hypothesis that cerebral hypoxia results in nitric oxide (NO)-mediated modification of the glycine-binding site of the N-methyl-D-aspartate (NMDA) receptor. Glycine binding characteristics were determined in normoxic, hypoxic, and hypoxic with 7-nitroindazole (7-NINA)-pretreated newborn piglets. The role of nitration was evaluated by determining binding characteristics in non-nitrated and in-vitro nitrated membranes. Bmax and Kd values were 30% higher in the hypoxic group than the normoxic and 7-NINA pretreated hypoxic groups. Kd values in the in-vitro normoxic nitrated membranes were similar to the non-nitrated hypoxic group. Bmax values in the in-vitro) normoxic nitrated membrane samples were 16% lower than in the non-nitrated hypoxic group. We conclude cerebral hypoxia causes modification of the glycine-binding site of the NMDA receptor and this modification of the glycine-binding site may be NO mediated. We propose that NO-mediated modification of the glycine-binding site of the NMDA receptor regulates calcium influx through its ion-channel.     

Effects of Magnesium Sulfate Administration During Hypoxia on CaM Kinase IV and Protein Tyrosine Kinase Activities in the Cerebral Cortex of Newborn Piglets

The present study tested the hypothesis that magnesium sulfate administration prior to hypoxia prevents hypoxia-induced increase in Ca²⁺/Calmodulin-dependent-kinase (CaM Kinase) IV and Protein Tyrosine Kinase (PTK ) activities. Animals were randomly divided into normoxic (Nx), hypoxic (Hx) and magnesium-pretreated hypoxic (Mg²⁺-Hx) groups. Cerebral hypoxia was confirmed biochemically by measuring ATP and phosphocreatine (PCr) levels. CaM Kinase IV and PTK activities were determined in Nx, Hx and Mg²⁺-Hx newborn piglets. There was a significant difference between CaM kinase IV activity (pmoles/mg protein/min) in Nx (270 ± 49), Mg²⁺-Hx (317 ± 82) and Hx (574 ± 41, P < 0.05 vs. Nx and Mg²⁺-Hx) groups. Similarly, there was a significant difference between Protein Tyrosine Kinase activity (pmoles/mg protein/h) in normoxic (378 ± 68), Mg²⁺-Hx (455 ± 67) and Hx (922 ± 66, P < 0.05 vs. Nx and Mg²⁺-Hx ) groups. We conclude that magnesium sulfate administration prior to hypoxia prevents hypoxia-induced increase in CaM Kinase IV and Protein Tyrosine Kinase activities. We propose that by blocking the NMDA receptor ion-channel mediated Ca²⁺-flux, magnesium sulfate administration inhibits the Ca²⁺/calmodulin-dependent activation of CaMKIV and prevents the generation of nitric oxide free radicals and the subsequent increase in PTK activity. As a result, phosphorylation of CREB and Bcl-2 family of proteins is prevented leading to prevention of programmed cell death.     

Differential Expression of Apoptotic Proteins Following Hypoxia-induced CREB Phosphorylation in the Cerebral Cortex of Newborn Piglets

The present study investigates the correlation between the hypoxia-induced phosphorylation of cyclic AMP response element binding protein and the expression of apoptotic proteins (proapoptotic proteins Bax and Bad and antiapoptotic proteins Bcl-2 and Bcl-xl) during hypoxia in the cerebral cortex of newborn piglets. Piglets were divided into normoxic (Nx) and hypoxic (Hx, FiO₂ = 0.06 for 1 h) groups. Cerebral tissue hypoxia was documented by ATP and phosphocreatine (PCr) levels. Ser¹³³ phosphorylation of cyclic AMP response element binding (CREB) protein was determined by Western blot analysis using a specific anti-phosphorylated Ser¹³³-CREB protein antibody. The expression of apoptotic proteins was determined by using specific anti-Bax, anti-Bad, anti-Bcl-2 and anti-Bcl-xl antibodies. ATP and PCr values (μmoles/g brain) in Hx were significantly different from Nx (ATP: 4.40 ± 0.39 in Nx vs. 1.19 ± 0.44 in Hx, P < 0.05 vs. Nx; PCr: 3.60 ± 0.40 in Nx vs. 0.70 ± 0.31 in Hx, P < 0.05 vs. Nx). Ser¹³³ phosphorylated CREB protein (OD × mm²) was 74.55 ± 4.75 in Nx and 127.13 ± 19.36 in Hx (P < 0.05 vs. Nx). The expression of proapoptotic proteins Bax and Bad increased and strongly correlated with the increase in CREB protein phosphorylation (correlation coefficient r = 0.82 and r = 0.85, respectively). The expression of antiapoptotic proteins Bcl-2 and Bcl-xl did not show correlation with CREB protein phosphorylation. We conclude that cerebral hypoxia results in differential regulation of CREB protein-mediated expression of proapoptotic and antiapoptotic proteins in the cerebral cortex of newborn piglets. We propose that the increased expression of proapoptotic vs antiapoptotic genes will lead to an increased potential for apoptotic programmed cell death in the Hx newborn brain.     

Kainate receptor modification in the fetal guinea pig brain during hypoxia

The present study tests the hypothesis that hypoxia alters the high-affinity kainate receptors in fetal guinea pig brain. Experiments were conducted in normoxic and hypoxic guinea pig fetus at preterm (45 days of gestation) and term (60 days of gestation). Hypoxia in the guinea pig fetus was induced by exposure to maternal hypoxia (FiO₂=7%) for 60 min. Brain tissue hypoxia in the fetus was documented biochemically by decreased levels of ATP and phosphorreatine. [³H]-Kainate binding characteristics (Bmax=number of receptors, Kd=dissociation constant) were used as indices of kainate receptor modification. P₂ membrane fractions were prepared from the cortex of normoxic and hypoxic fetuses and were washed six times prior to performing the binding assays. [³H]kainate binding was performed at 0°C for 30 min in a 500 l medium containing 50 mM Tris-HCl buffer, 0.1 mM EDTA (pH 7.4), 300 g protein and varying concentrations of radiolabelled kainate ranging from 1 to 200 nM. Non-specific binding was determined in the presence of 1.0 mM glutamate. During brain development from 45 to 60 days gestation, Bmax value increased from 330±16 to 417±10 fmoles/mg protein; however, the Kd was unchanged (8.2±0.4 vs 8.8±0.5 nM, respectively). During hypoxia at 60 days, the Kd value significantly increased as compared to normoxic control (15.5±0.7 vs 8.8±0.5 nM, respectively), whereas the Bmax was not affected (435±12 vs 417±10 fmol/mg protein, respectively). At 45 days, hypoxia also increased the Kd (11.9±0.6 vs 8.2±0.4 nM) without affecting the Bmax (290±15 vs 330±16 fmol/mg protein, respectively). The results show that the number of kainate receptors increase during gestation without change in affinity and demonstrate that hypoxia modifies the high-affinity kainate receptor sites at both ages; however the effect is much stronger at 60 days (term). The decreased affinity of the site could decrease the kainate receptor-mediated fast kinetics of desensitization and provide a longer period for increased Na⁺-influx, leading to increased accumulation of intracellular Ca²⁺ by reversal of the Na⁺–Ca²⁺ exchange mechanism. In addition, Kd values for kainate-type glutamate receptor sites are 30–40 fold lower (i.e. higher affinity) than those for NMDA-displaceable glutamate sites. The higher affinity suggests that the activation of the kainate-type glutamate receptor during hypoxia could precede initiation of NMDA receptormediated excitotoxic mechanisms. We propose that hypoxia-induced modification of the high affinity kainate receptor in the fetus is a potential mechanism of neuroexcitotoxicity.     

Hypoxia-induced pulmonary endothelin-1 expression is unaltered by nitric oxide.

Nitric oxide (NO) attenuates hypoxia-induced endothelin (ET)-1 expression in cultured umbilical vein endothelial cells. We hypothesized that NO similarly attenuates hypoxia-induced increases in ET-1 expression in the lungs of intact animals and reasoned that potentially reduced ET-1 levels may contribute to the protective effects of NO against the development of pulmonary hypertension during chronic hypoxia. As expected, hypoxic exposure (24 h, 10% O(2)) increased rat lung ET-1 peptide and prepro-ET-1 mRNA levels. Contrary to our hypothesis, inhaled NO (iNO) did not attenuate hypoxia-induced increases in pulmonary ET-1 peptide or prepro-ET-1 mRNA levels. Because of this surprising finding, we also examined the effects of NO on hypoxia-induced increases in ET peptide levels in cultured cell experiments. Consistent with the results of iNO experiments, administration of the NO donor S-nitroso-N-acetyl-penicillamine to cultured bovine pulmonary endothelial cells did not attenuate increases in ET peptide levels resulting from hypoxic (24 h, 3% O(2)) exposure. In additional experiments, we examined the effects of NO on the activity of a cloned ET-1 promoter fragment containing a functional hypoxia inducible factor-1 binding site in reporter gene experiments. Whereas moderate hypoxia (24 h, 3% O(2)) had no effect on ET-1 promoter activity, activity was increased by severe hypoxic (24 h, 0.5% O(2)) exposure. ET-1 promoter activity after S-nitroso-N-acetyl-penicillamine administration during severe hypoxia was greater than that in normoxic controls, although activity was reduced compared with that in hypoxic controls. These findings suggest that hypoxia-induced pulmonary ET-1 expression is unaffected by NO.     

Chronic hypoxia alters the function of NOS nerves in cerebral arteries of near-term fetal and adult sheep.

In addition to adrenergic innervation, cerebral arteries also contain neuronal nitric oxide synthase (nNOS)-expressing nerves that augment adrenergic nerve function. We examined the impact of development and chronic high-altitude hypoxia (3,820 m) on nNOS nerve function in near-term fetal and adult sheep middle cerebral arteries (MCA). Electrical stimulation-evoked release of norepinephrine (NE) was measured with HPLC and electrochemical detection, whereas nitric oxide (NO) release was measured by chemiluminescence. An inhibitor of NO synthase, N(omega)-nitro-l-arginine methyl ester (l-NAME), significantly inhibited stimulation-evoked NE release in MCA from normoxic fetal and adult sheep with no effect in MCA from hypoxic animals. Addition of the NO donor S-nitroso-N-acetyl-dl-penicillamine fully reversed the effect of l-NAME in MCA from normoxic animals with no effect in MCA from hypoxic animals. Electrical stimulation caused a significant increase in NO release in MCA from normoxic animals, an effect that was blocked by the neurotoxin tetrodotoxin, whereas there was no increase in NO release in MCA from hypoxic animals. Relative abundance of nNOS as measured by Western blot analysis was similar in normoxic fetal and adult MCA. However, after hypoxic acclimitization, nNOS levels dramatically declined in both fetal and adult MCA. These data suggest that the function of nNOS nerves declines during chronic high-altitude hypoxia, a functional change that may be related to a decline in nNOS protein levels.     

Sodium-Coupled Neutral Amino Acid Transporter 1 (SNAT1) Modulates L-Citrulline Transport and Nitric Oxide (NO) Signaling in Piglet Pulmonary Arterial Endothelial Cells

Rationale

There is evidence that impairments in nitric oxide (NO) signaling contribute to chronic hypoxia-induced pulmonary hypertension. The L-arginine-NO precursor, L-citrulline, has been shown to ameliorate pulmonary hypertension. Sodium-coupled neutral amino acid transporters (SNATs) are involved in the transport of L-citrulline into pulmonary arterial endothelial cells (PAECs). The functional link between the SNATs, L-citrulline, and NO signaling has not yet been explored.

Objective

We tested the hypothesis that changes in SNAT1 expression and transport function regulate NO production by modulating eNOS coupling in newborn piglet PAECs.

Methods and Results

A silencing RNA (siRNA) technique was used to assess the contribution of SNAT1 to NO production and eNOS coupling (eNOS dimer-to-monomer ratios) in PAECs from newborn piglets cultured under normoxic and hypoxic conditions in the presence and absence of L-citrulline. SNAT1 siRNA reduced basal NO production in normoxic PAECs and prevented L-citrulline-induced elevations in NO production in both normoxic and hypoxic PAECs. SNAT1 siRNA reduced basal eNOS dimer-to-monomer ratios in normoxic PAECs and prevented L-citrulline-induced increases in eNOS dimer-to-monomer ratios in hypoxic PAECs.

Conclusions

SNAT1 mediated L-citrulline transport modulates eNOS coupling and thus regulates NO production in hypoxic PAECs from newborn piglets. Strategies that increase SNAT1-mediated transport and supply of L-citrulline may serve as novel therapeutic approaches to enhance NO production in patients with pulmonary vascular disease.