Effect of hypoxia on expression of selected proteins involved in regulation of apoptotic activity in striatum of newborn piglets.

The levels of selected neuroregulatory proteins that inhibit or promote apoptotic cell death were measured in the striatum of piglets subjected to precisely controlled 1 h hypoxic insult followed by 0, 2 and 4 h recovery and compared to sham operated animals. The anti-apoptotic proteins: there were increases in Survivin at 0 (157%, P = 0.031) and 4 h (171%, P = 0.033), in Bcl-XL at 0 (138%, P = 0.028) and 4 h (143%, P = 0.007), in VEGF at 4 h (185%, P = 0.019) and Hsp27 at 2 h (144%, P = 0.05) and 4 h (143%, P = 0.05). The pro-apoptotic proteins: caspases-1 and 7 increased at 4 h (135%, P = 0.05) and (129%, P = 0.038), respectively. Bim increased after 4 h (115%, P = 0.028), Apoptosis Inducing Factor after 2 h (127%, P = 0.048) and Calpain after 4 h (143% of control, P = 0.04). Hypoxia causes increase in levels of both anti- and pro-apoptotic proteins. Their relative activity determines the outcome in terms of cell damage and neuronal deficit.

Early regional response of apoptotic activity in newborn piglet brain following hypoxia and ischemia.

Responses of selected neuroregulatory proteins that promote (Caspase 3 and Bax) or inhibit (Bcl-2, high Bcl-2/Bax ratio) apoptotic cell death were measured in the brain of piglets subjected to precisely controlled hypoxic and ischemic insults: 1 h hypoxia (decreasing FiO2 from 21 to 6%) or ischemia (ligation of carotid arteries and hemorrhage), followed by 0, 2 and 4 h recovery with 21% FiO2. Protein expression was measured in cortex, hippocampus and striatum by Western blot. There were no significant differences in expression of Caspase-3 between sham operated, hypoxic and ischemic groups. There were significant regional differences in expression of Bcl-2 and Bax in response to hypoxia and ischemia. The changes in Bcl-2/Bax ratio were similar for hypoxia and ischemia except for striatum at zero time recovery, with ischemia giving lower ratios than hypoxia. The Bcl-2/Bax ratio was also lower for the striatum than for the other regions of the brain, suggesting this region is the more susceptible to apoptotic injury.

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 degrees 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.

Effect of gestational age and hypoxia on activity of ribonucleic acid polymerase in fetal guinea pig brain.

OBJECTIVE: The aim of this study was to determine the effect of gestational age and hypoxia on the activity of ribonucleic acid polymerase in fetal guinea pig brain. STUDY DESIGN: Fetal cerebral cortical neuronal nuclei were isolated at 40, 50, and 60 days (term) of gestation to determine the effect of gestational age on the activity of ribonucleic acid polymerase I, II, and III. Pregnant guinea pigs at 60 days' gestation were randomly assigned to a normoxic or hypoxic group to determine the effect of hypoxia on ribonucleic acid polymerase activity. The fetal neuronal nuclei were pooled from 6 pregnant animals in each group. In the normoxic group the pregnant guinea pigs were exposed to room air before delivery. In the hypoxic group delivery occurred after the pregnant guinea pig had been exposed to 7% oxygen for 60 minutes. The fetuses were delivered by cesarean, and the fetal cerebral cortical neuronal nuclei were isolated immediately. Ribonucleic acid polymerase activity was determined with nuclei suspended in a buffer containing adenosine triphosphate, guanosine triphosphate, cytidine triphosphate, and tritiated uridine triphosphate. Dactinomycin (actinomycin D) and polydeoxyadenylic-thymidylic acid were used to determine the activity of bound and free ribonucleic acid polymerase. alpha-Amanitin was used to determine the activity of ribonucleic acid polymerase II. RESULTS: The activity of total (bound and free) ribonucleic acid polymerase I and III increased from 85.4 +/- 9.4 fmol of tritiated uridine triphosphate incorporated per milligram of protein per hour at 40 days' gestation to 233.3 +/- 82.1 fmol at 50 days and to 343.4 +/- 231.6 fmol at 60 days (P =.02). Total ribonucleic acid polymerase II activity increased from 19.9 +/- 6.0 fmol of tritiated uridine triphosphate incorporated per milligram of protein per hour at 40 days to 123.8 +/- 53.0 fmol at 50 days and to 200.9 +/- 77.8 fmol at 60 days (P <.01). In the term fetal guinea pig brain the activity of bound ribonucleic acid polymerase I and III decreased from 116.8 +/- 107.2 fmol of tritiated uridine triphosphate incorporated per milligram of protein per hour under normoxic conditions to 92.8 +/- 76.0 fmol in hypoxic fetal brain, a decrease of 20.5%. Free ribonucleic acid polymerase I and III activity decreased from 199.2 +/- 115.2 fmol of tritiated uridine triphosphate incorporated per milligram of protein per hour in normoxic fetal brain to 132.0 +/- 66.4 fmol in hypoxic fetal brain, a decrease of 33.8%. Free ribonucleic acid polymerase II activity decreased from 62.4 +/- 70.4 fmol of tritiated uridine triphosphate incorporated per milligram of protein per hour in normoxic fetuses to 13.6 +/- 9.6 fmol in hypoxic fetal brain, a decrease of 78.2%. In contrast, however, in term fetal guinea pig brain, bound ribonucleic acid polymerase II activity increased from 8.0 +/- 10.4 fmol of tritiated uridine triphosphate incorporated per milligram of protein per hour under normoxic conditions to 35.2 +/- 8.8 fmol in hypoxic fetal brain, an increase of 340% (P <.01). CONCLUSION: The activity of ribonucleic acid polymerases I, II, and III increases throughout the latter half of gestation, from 40 to 60 days, in the fetal guinea pig brain. Hypoxia in utero is associated with a decrease in ribonucleic acid polymerase I and III activity. Although hypoxia is associated with a decrease in free ribonucleic acid polymerase II activity, we observed a marked increase in bound ribonucleic acid polymerase II activity, which may represent a hypoxia-induced alteration of gene expression.

On the unidirectionality of arginine uptake in the yeast Saccharomyces cerevisiae.

The reversibility of arginine accumulation was followed in exponentially growing cells of Saccharomyces cerevisiae and in the same cells transferred to non-growing energized conditions. Under non-growing conditions the accumulated arginine is retained in the cells while in exponentially growing cells the accumulated radioactivity is released after the addition of high external concentrations of arginine. There are indications that the process is saturable. The accumulated arginine is not exchanged for other related amino acids (L-citrulline, L-histidine). Only L-lysine (a low-affinity substrate of the specific arginine permease) provokes partial radioactivity efflux from the cells. The switch of the arginine-related radioactive label efflux to its complete retention in the cells after changing the growth conditions occurs within a few minutes and is tentatively attributed to two concomitantly occurring events: (1) the actual presence of radioactive arginine (not its metabolite(s)) in the cell and (2) a modification of the specific arginine permease. The specific exchange of arginine described in the present study contrasts with the currently widely accepted opinion of unidirectionality of amino acid fluxes in yeast. The reasons why this phenomenon has not been observed before are discussed.

The in vivo effect of bilirubin on the N-methyl-D-aspartate receptor/ion channel complex in the brains of newborn piglets.

Bilirubin neurotoxicity can be mediated by numerous mechanisms due to its increased permeability in neuronal membranes. The present study tests the hypothesis that a prolonged bilirubin infusion modifies the N-methyl-D-aspartate (NMDA) receptor/ ion channel complex in the cerebral cortex of newborn piglets. Studies were performed in seven control and six bilirubin-exposed piglets, 2-4 d of age. Piglets in the bilirubin group received a 35 mg/kg bolus of bilirubin followed by a 4-h infusion (25 mg/kg/h) of a buffer solution containing 0.1 N NaOH, 5% human albumin, and 0.055 Na2HPO4 with 3 mg/mL bilirubin. The final mean bilirubin concentration in the bilirubin group was 495.9 +/- 85.5 mumol/L (29.0 +/- 5.0 mg/dL). The control group received a bilirubin-free buffer solution. Sulfisoxazole was administered to animals in both groups. P2 membrane fractions were prepared from the cerebral cortex. [3H]MK-801 binding assays were performed to study NMDA receptor modification. The Bmax in the control and bilirubin groups were 1.20 +/- 0.10 (mean +/- SD) and 1.32 +/- 0.14 pmol/mg protein, respectively. The value for Kd in the control brains was 6.97 +/- 0.80 nM compared with 4.80 +/- 0.28 nM in the bilirubin-exposed brains (p < 0.001). [3H]Glutamate binding studies did not show a significant difference in the Bmax and Kd for the NMDA-specific glutamate site in the two groups. The results show that in vivo exposure to bilirubin increases the affinity of the receptor (decreased Kd) for [3H]MK-801, indicating that bilirubin modifies the function of the NMDA receptor/ion channel complex in the brain of the newborn piglet. We speculate that the affinity of bilirubin for neuronal membranes leads to bilirubin-mediated neurotoxicity, resulting in either short- or long-term disruption of neuronal function.

Modification of the glycine (co-activator) binding site of the N-methyl-D-aspartate receptor in the guinea pig fetus brain during development following hypoxia.

The present study was designed to investigate the mechanism of NMDA receptor activation as a function of brain maturation by studying the development of the glycine binding site of the NMDA receptor and its modification by in-utero hypoxia in the guinea pig fetus brain during gestation. Measurements of Bmax (number of functional receptors) and Kd (apparent receptor affinity) of glycine binding sites of the NMDA receptor were performed in eleven (45 days, n = 5; 60 days, n = 6) synaptosomal membranes from normoxic (control) fetuses and ten (45 days, n = 4; 60 days, n = 6) synaptosomal membranes constituted the hypoxic (experimental) group. In the experimental group, fetuses were exposed to maternal hypoxia (FiO2 0.07) for 1 h. Synaptosomal membranes were prepared and strychnine-insensitive specific [3H]glycine binding was determined During development, the number of glycine binding sites increased (Bmax:392 +/- 30 vs. 583 +/- 30 fmol/mg protein at 45 and 60 days respectively, P < 0.05) where as the affinity remained unchanged (Kd: 190 +/- 9 vs. 211 +/- 30 nM at 45 and 60 days respectively). Following hypoxia, glycine binding sites increased at 45 days (Bmax:392 +/- 30 vs. 561 +/- 96 fmol/mg protein, P < 0.005) but decreased at 60 days (Bmax:583 +/- 85 vs. 411 +/- 65 fmol/mg protein, P < 0.005) with change in Kd only at 60 days (Kd:211 +/- 30 vs. 149 +/- 52 nM, P < 0.05). The data show that there are alterations in the characteristics of the glycine binding site during development and following hypoxia. We conclude that developmental changes in the glycine binding site might modulate NMDA receptor activation as a function of brain maturation. Furthermore, hypoxia-induced modification of the glycine binding site might be a potential mechanism of neurotoxicity and might increase susceptibility of the fetal brain to excitotoxicity at term.

Brain cell membrane Na+,K(+)-ATPase activity following severe hypoxic injury in the newborn piglet.

This study tests the hypothesis that severe brain hypoxia causes decreased Na+,K(+)-ATPase activity, resulting in permanent alterations in the neuronal cell membranes. Seventeen anesthetized piglets (normoxic control (NC), no recovery after hypoxia (Group 1), 6 h normoxic recovery (Group 2), and 48 h normoxic recovery (Group 3)) were studied. Hypoxia was induced by lowering the FiO2 to maintain PCr/Pi ratio at 25% of baseline for 1 h as monitored by 31P-NMR spectroscopy. PCr/Pi returned to 57% of baseline by 6 h and was normal by 48 h. At termination, cortical tissue Na+,K(+)-ATPase activity was determined. Na+,K(+)-ATPase activity was measured in cortical membrane preparations by determining the rate of ATP hydrolysis. NC membranes had Na+,K(+)-ATPase activity of 58.3 +/- 1.3 microM Pi/mg protein/h (mean +/- S.E.M.). Na+,K(+)-ATPase activity was reduced in Groups 1, 2, and 3 (45.8 +/- 1.3, 47.4 +/- 3.6, 48.7 +/- 2.9 microM Pi/mg protein/h) (P < 0.05 compared to NC). There was no difference in enzyme activity among Groups 1, 2, or 3. The data show that in spite of recovery of neuronal oxidative phosphorylation (PCr/Pi) by 48 h, there is a permanent decrease in Na+,K(+)-ATPase activity in cells that have undergone severe hypoxic injury. The persistent decrease in Na+,K(+)-ATPase activity indicates ongoing cell injury following severe cerebral hypoxia, and that recovery of oxidative phosphorylation as indicated by PCr/Pi values cannot be used as an index of recovery of cell function.

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 phosphocreatine. [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 degrees C for 30 min in a 500 microliters medium containing 50 mM Tris-HCl buffer, 0.1 mM EDTA (pH 7.4), 300 micrograms 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 receptor-mediated excitotoxic mechanisms. We propose that hypoxia-induced modification of the high affinity kainate receptor in the fetus is a potential mechanism of neuroexcitotoxicity.

Brain cell membrane modification following hypercapnia and recovery in newborn piglets.

The effect of hypercapnia on brain cell membrane structure and function was studied in anesthetized newborn piglets. Lipid peroxidation products (conjugated dienes and fluorescent compounds), Na+,K(+)-ATPase activity and enzyme affinity to ATP (substrate), K+ and Na+ ions (activators), and strophanthidin (inhibitor) were measured in three groups of animals: controls, those exposed to 90 minutes of PaCO2 > 80 mmHg (hypercapnia) and those exposed the same way, following restoration of normal PaCO2 (recovery). Enzyme activity was unchanged by hypercapnia, but enzyme affinity was altered as indicated by an increase in ATP affinity. Affinities to Na+, K+, and strophanthidin were unchanged. Restoration of normal PaCO2 resulted in an increase in conjugated dienes. The data demonstrate that hypercapnia followed by restoration of normal PaCO2 in healthy term newborn piglets is associated with mild modification of brain cell membrane Na+,K(+)-ATPase, possibly due to lipid peroxidation.

The suppressive activity of T-lymphocytes and serum factors in burned patients.

This study was performed to investigate the cell-mediated immune response in burned patients with no septic episodes. The results show that burned patients with percentage body burn higher than 20 had an impaired lymphocyte reactivity to phytohaemagglutinin and conconavalin A. This hyporesponsiveness appeared on day 3-4 and in all cases reached its maximum on day 7-8 post burn, while recovery occurred between day 11 and 29 depending on the severity of the injury. The serum from immunodepressed patients was able to inhibit the response to phytohaemagglutinin and conconavalin A of normal lymphocytes. This immunosuppressive activity was present very early after injury (on day 1-2) and before the onset of lymphocyte hyporesponsiveness to mitogens and was no longer detectable on day 7-8 post burn, when patient lymphocytes showed the greatest hyporesponsiveness to mitogens. This late depression was due to T suppressor cells.

Antigenic characterization of urea-extractable crude burn toxin.

Crude burn toxin was obtained from human skin scalded for 1 min at 90 degrees C in vitro. The toxin solubilized with 8 M urea was administered to rabbits to obtain antiserum. The antigenic analysis carried out by immunodiffusion and immunoelectrophoresis demonstrated that urea-extractable toxin solution differed from its analogue obtained from untreated skin in antigenic properties and presence of high-molecular weight protein fractions. Presence of a tissue antigen of low grade electrophoretic mobility in the preparation of burn toxin was shown. This antigen was present, probably also in small amounts in the preparation of native skin which was analogous to the tested skin.

Isolation and immunochemical properties of burn toxin.

Human skin and the skin of some laboratory animals was minced, scalded and homogenized. The preparation from scalded human skin (the "crude burn toxin") revealed to be highly toxic to mice after i.v. administration. It was found by immunodiffusion and immunoelectrophoresis, that the urea and buthanol extracts of the crude "toxin" contain tissue antigens which were absent in a control preparation from a native (non scalded) skin.