Oxygen-hemoglobulin dissociation curves: effect of inherited enzyme defects of the red cell.

The blood of a patient with a deficiency of hexokinase in the red cells and a decreased concentration of 2, 3-diphosphoglycerate in the red cells showed an increased affinity for oxygen, whereas a patient with a deficiency of pyruvate kinase and an elevated concentration of 2, 3-diphosphoglycerate in the red cells had blood with a decreased affinity for oxygen. Defects in red cell glycolysis may alter the oxygen affinity of blood by virtue of their effect on 2, 3-diphosphoglycerate concentrations in red cells.

Oxygen affinity in red cells: changes induced in vivo by propranolol.

Propranolol, a blocking agent for the beta adrenergic receptor, produces a redistribution of 2,3-diphosphoglycerate in the red cell. At concentrations of 3.3 x10(-5)M, 2,3-diphosphoglycerate in the red cell membrane becomes unbound in vitro. The administration of propranolol to hunmns produces similar changes and results in a decrease in the affinity of hemoglobin for oxygen.

The effects of deoxygenation of adult and fetal hemoglobin on the synthesis of red cell 2,3-diphosphoglycerate and its in vivo consequences.

Patients over 1 month of age with arterial oxygen pressures of less than 60 mm Hg were found to have elevated red cell 2,3-diphosphoglycerate (2,3-DPG) levels and blood with a decreased affinity for oxygen. The increase in 2,3-DPG was proportional to the degree of hypoxemia. In patients under 1 month of age this relationship was not observed. Red cells from adults, but not newborns, showed rapid increases in 2,3-DPG when incubated under nitrogen. Adult, but not fetal, deoxyhemoglobin was shown to facilitate in vitro synthesis of 2,3-DPG by binding this organic phosphate and relieving the product inhibition of 2,3-DPG mutase. Throughout a wide range change in oxygen affinity as measured by the P(50) is linear with respect to the 2,3-DPG concentration; a change of 430 mmumoles of 2,3-DPG/ml of red blood corpuscles (RBC) resulting in a change of the P(50) of 1 mm Hg. It appears that the 2,3-DPG of the adult's red cells responds rapidly to metabolic and environmental influences and in turn effects metabolism and the cellular environment. Many of these effects are not shared by the red cells of the newborn.

Nitric oxide-mediated mechanism of neuronal nitric oxide synthase and inducible nitric oxide synthase expression during hypoxia in the cerebral cortex of newborn piglets.

Previously, we have shown that hypoxia results in increased generation of nitric oxide free radicals in the cerebral cortex of newborn piglets that may be due to up-regulation of nitric oxide synthases, neuronal nitric oxide synthase and inducible nitric oxide synthase. The present study tests the hypothesis that hypoxia results in increased expression of neuronal nitric oxide synthase and inducible nitric oxide synthase in the cerebral cortex of newborn piglets and that the increased expression is nitric oxide-mediated. Newborn piglets, 2-4 days old, were divided to normoxic (n=4), hypoxic (n=4) and hypoxic-treated with 7-nitro-indazole-sodium salt, a selective neuronal nitric oxide synthase inhibitor (hypoxic-7-nitro-indazole-sodium salt, n=6, 1 mg/kg, 60 min prior to hypoxia). Piglets were anesthetized, ventilated and exposed to an FiO2 of 0.21 or 0.07 for 60 min. Cerebral tissue hypoxia was documented biochemically by determining ATP and phosphocreatine. The expression of neuronal nitric oxide synthase and inducible nitric oxide synthase was determined by Western blot using specific antibodies for neuronal nitric oxide synthase and inducible nitric oxide synthase. Protein bands were detected by enhanced chemiluminescence, analyzed by imaging densitometry and the protein band density expressed as absorbance (OD x mm(2)). The density of neuronal nitric oxide synthase in the normoxic, hypoxic and hypoxic-7-nitro-indazole-sodium salt groups was: 41.56+/-4.27 in normoxic, 61.82+/-3.57 in hypoxic (P<0.05) and 47.80+/-1.56 in hypoxic-7-nitro-indazole-sodium salt groups (P=NS vs normoxic), respectively. Similarly, the density of inducible nitric oxide synthase in the normoxic, hypoxic and hypoxic-7-nitro-indazole-sodium salt groups was: 105.21+/-9.09, 157.71+/-13.33 (P<0.05 vx normoxic), 117.84+/-10.32 (p=NS vx normoxic), respectively. The data show that hypoxia results in increased expression of neuronal nitric oxide synthase and inducible nitric oxide synthase proteins in the cerebral cortex of newborn piglets and that the hypoxia-induced increased expression is prevented by the administration of 7-nitro-indazole-sodium salt. Furthermore, the neuronal nitric oxide synthase inhibition prevented the inducible nitric oxide synthase expression for a period of 7 days after hypoxia. Since administration of 7-nitro-indazole-sodium salt prevents nitric oxide generation by inhibiting neuronal nitric oxide synthase, we conclude that the hypoxia-induced increased expression of neuronal nitric oxide synthase and inducible nitric oxide synthase is mediated by neuronal nitric oxide synthase derived nitric oxide. We speculate that during hypoxia nitric oxide-mediated up-regulation of nitric oxide synthases will continue the perpetual cycle of nitric oxide generation-->NOS up-regulation-->nitric oxide generation resulting in hypoxic neuronal death.

Hypoxia-induced Bax and Bcl-2 protein expression, caspase-9 activation, DNA fragmentation, and lipid peroxidation in mitochondria of the cerebral cortex of newborn piglets: the role of nitric oxide.

The present study tests the hypothesis that cerebral hypoxia results in increased ratio of Bax/Bcl-2, activation of caspase-9, lipid peroxidation, and DNA fragmentation in mitochondria of the cerebral cortex of newborn piglets and that the inhibition of nitric oxide synthase by N-nitro-L-arginine during hypoxia will prevent the events leading to mitochondrial DNA fragmentation. To test this hypothesis, six piglets, 3-5 days old, were divided into three groups: normoxic (n=5), hypoxic (n=5), and hypoxic-nitric oxide synthase (n=4). Hypoxic animals were exposed to a FiO2 of 0.6 for 60 min. Nitric oxide synthase (40 mg/kg) was infused over 60 min prior to hypoxia. Tissue hypoxia was confirmed by measuring levels of ATP and phosphocreatine. Cerebral cortical tissue mitochondria were isolated and purified using a discontinuous ficoll gradient. Mitochondrial Bax and Bcl-2 proteins were determined by Western blot. Caspase-9 activity in mitochondria was determined spectro-fluorometrically using fluorogenic substrate for caspase-9. Fluorescent compounds, an index of mitochondrial membrane lipid peroxidation, were determined spectrofluorometrically. Mitochondrial DNA was isolated and separated by electrophoresis on 1% agarose gel and stained with ethidium bromide. ATP levels (micromol/g brain) were 4.52+/-0.34 in normoxic, 1.18+/-0.29 in hypoxic (P<0.05) and 1.00+/-0.26 in hypoxic-nitric oxide synthase animals (P<0.05 vs. normoxic). Phosphocreatine levels (micromol/g brain) were 3.61+/-0.33 in normoxic, 0.70+/-0.20 in hypoxic (P<0.05 vs. normoxic) and 0.57+/-0.14 in hypoxic-nitric oxide synthase animals (P<0.05 vs. normoxic, P=NS vs. hypoxic). Bax density in mitochondrial membranes was 160+/-28 in normoxic and 324+/-65 in hypoxic (P<0.001 vs. normoxic). Bcl-2 density mitochondria was 96+/-18 in normoxic and 98+/-20 in hypoxic (P=NS vs. normoxic). Mitochondrial caspase-9 activity (nmol/mg protein/h) was 1.32+/-0.23 in normoxic and 2.25+/-0.24 in hypoxic (P<0.01 vs. normoxic). Levels of fluorescent compounds (microg of quinine sulfate/g protein) were 12.48+/-4.13 in normoxic and 37.92+/-7.62 in hypoxic (P=0.003 vs. normoxic). Densities (ODxmm2) of low molecular weight DNA fragments were 143+/-38 in normoxic, 365+/-152 in hypoxic, (P<0.05 vs. normoxic) and 163+/-25 in hypoxic-nitric oxide synthase animals (P<0.05 vs. hypoxic, P=NS vs. normoxic). The data demonstrate that hypoxia results in increased mitochondrial proapoptotic protein Bax, increased mitochondrial caspase-9 activity, increased mitochondrial lipid peroxidation, and increased fragmentation of DNA in mitochondria of the cerebral cortex of newborn piglets. The administration of a nitric oxide synthase inhibitor, nitric oxide synthase, prior to hypoxia prevented fragmentation of mitochondrial DNA, indicating that the hypoxia-induced mitochondrial DNA fragmentation is NO-mediated. We propose that NO free radicals generated during hypoxia lead to NO-mediated altered expression of Bax leading to increased ratio of pro-apoptotic/anti-apoptotic protein resulting in modification of mitochondrial membrane, and subsequently Ca2+-influx and fragmentation of mitochondrial DNA.

Effect of dexamethasone treatment on maturational changes in the NMDA receptor in sheep brain.

The objective of the present study was to examine the effect of antenatal or postnatal treatment with corticosteroids on the NMDA receptor, one of the mediators of both normal brain development and hypoxic-ischemic injury, by determining the characteristics of the receptor MK-801 binding site in untreated and corticosteroid-treated fetal and newborn lambs. (3)H-MK-801 binding was performed in cerebral cortical cell membranes from fetal sheep at 88, 120, and 136 d gestation (term = 150 d), and from 5-d-old lambs and adult ewes. Animals were randomized to receive dexamethasone [fetuses: 6 mg, i.m. every 12 hr for four doses to mother; lambs: 0.01 mg/kg (low dose) or 0.25 mg/kg (high dose) every 12 hr for four doses] or placebo. During development, B(max) (apparent number of receptors) increased, reaching a maximum in 5-d-old lambs (p < 0.05) and decreasing in the adult brain. K(d) (dissociation constant) did not change, suggesting that receptor affinity was not altered during maturation. Dexamethasone treatment had no effect on MK-801 binding in the fetus or adult, but in lambs was associated with a significant decrease in B(max) from 2.17 +/- 0.18 pmol/mg protein in placebo-treated animals to 1.65 +/- 0.8 and 1.62 +/- 0.07 pmol/mg protein in low-dose and high-dose animals, respectively. Affinity for (3)H-MK-801 decreased 20% after dexamethasone treatment in lambs only (p < 0.05). Thus, dexamethasone treatment appears to modify the NMDA receptor only during a specific period of brain development.

Hypoxia modifies nuclear calcium uptake pathways in the cerebral cortex of the guinea-pig fetus.

Nuclear Ca2+ signals are thought to play a critical role in the initiation and progression of programmed cell death. The present study tests the hypothesis that hypoxia alters nuclear Ca2+ transport pathways and leads to an increase in nuclear Ca(2+)-influx in cerebral cortical neuronal nuclei. To test this hypothesis the effect of tissue hypoxia on high affinity Ca(2+)-ATPase activity and the binding characteristics of inositol 1,4,5-triphosphate (IP3) and inositol 1,3,4,5-tetrakisphosphate (IP4) receptors were studied in neuronal nuclei from the cerebral cortex of guinea-pig fetuses. Results show increased high-affinity Ca(2+)-ATPase activity (nmol/mg protein/h) in the hypoxic group 969.7+/-79 as compared with 602.4+/-90.9 in the normoxic group, P<0.05. The number of IP3 receptors (Bmax, fmol/mg protein) increased from 61+/-21 in the normoxic group to 164+/-49 in the hypoxic group, P<0.05. K(d) values did not change following hypoxia. In contrast, IP4 receptor Bmax (fmol/mg protein) and K(d) (nM) values increased from 360+/-32 in the normoxic group to 626+/-136 in the hypoxic group (P<0.001) and, from 26+/-1 in the normoxic group to 61+/-9 in the hypoxic group (P<0.001), respectively. 45Ca(2+)-influx (pmol/mg protein) significantly increased from 6.3+/-1.9 in the normoxic group to 10.9+/-1.1 the hypoxic group (P<0.001). The data show that hypoxia modifies nuclear Ca2+ transport pathways and results in increased nuclear Ca(2+)-influx. We speculate that hypoxia increases nuclear Ca2+ uptake from the cytoplasm to the nucleoplasm, resulting in increased transcription of proapoptotic genes and subsequent activation of programmed cell death pathways.

Acute hypoxia-induced alterations of calbindin-D28k immunoreactivity in cerebellar Purkinje cells of the guinea pig fetus at term.

Purkinje cells (PCs) are vulnerable to hypoxic/ischemic insults and rich in calcium and calcium-buffering/sequestering systems, including calcium-binding proteins (CaBPs). Calbindin-D28k is an EF-hand CaBP, which is highly expressed in PCs where it acts primarily as a cellular Ca++ buffer. Elevation of [Ca++] in the cytosol and nuclei of PCs is pivotal in hypoxic/ischemic cell death. We hypothesize that hypoxia results in decreased concentration, or availability of calbindin-D28k in PCs, thereby decreasing their buffering capacity and resulting in increase of intracellular and intranuclear [Ca++]. Cerebellar tissues from normoxic fetuses were compared to fetuses obtained from term pregnant guinea pigs exposed to hypoxia [7% FiO2] for 60 min. The pregnant guinea pigs were either killed upon delivery immediately following hypoxia (Hx0h) or were subsequently allowed to recover for 24 h (Hx24h) or 72 h (Hx72h). Fetal brain hypoxia was documented biochemically by a decrease in brain tissue levels of ATP and phosphocreatine. Compared to normoxic fetuses, there is a predominantly somatodendritic loss or decrease of calbindin-D28k immunohistochemical staining in PCs of Hx0h (p < 0.005), Hx24h (p < 0.05), and Hx72h (p < 0.005) fetuses. Hypoxia-induced alterations of calbindin-D28k immunoreactivity are qualitatively similar at all time points and include a distinctive intranuclear localization in subpopulations of PCs. A similar trend is demonstrated by immunoblotting. Subpopulations of TUNEL+/calbindin-D28k- PCs lacking morphologic features of apoptosis or necrosis are demonstrated in Hx24h and Hx72h fetuses. The present study demonstrates an abrogating effect of perinatal hypoxia on calbindin-D28k immunoreactivity in cerebellar PCs. The perturbation of this Ca++ buffer protein in hypoxia-induced neuronal injury may herald delayed cell death or degeneration.

The effect of hematocrit and systolic blood pressure on cerebral blood flow in newborn infants.

The effects of hematocrit and systolic blood pressure on cerebral blood flow were measured in 15 stable, low birth weight babies. CBF was measured with a modification of the xenon-133 (133Xe) clearance technique, which uses an intravenous bolus of 133Xe, an external chest detector to estimate arterial 133Xe concentration, eight external cranial detectors to measure cephalic 133Xe clearance curves, and a two-compartmental analysis of the cephalic 133Xe clearance curves to estimate CBF. There was a significant inverse correlation between hematocrit and CBF, presumably due to alterations in arterial oxygen content and blood viscosity. Newborn CBF varied independently of systolic blood pressure between 60 and 84 mm Hg, suggesting an intact cerebrovascular autoregulatory mechanism. These results indicate that at least two of the factors that affect newborn animal CBF are operational in human newborns and may have important clinical implications.

Noninvasive method of estimating human newborn regional cerebral blood flow.

A noninvasive method of estimating regional cerebral blood flow (rCBF) in premature and full-term babies has been developed. Based on a modification of the xenon-133 inhalation rCBF technique, this method uses eight extracranial NaI scintillation detectors and an i.v. bolus injection of xenon-133 (approximately 0.5 mCi/kg). Arterial xenon concentration was estimated with an external chest detector. Cerebral blood flow was measured in 15 healthy, neurologically normal premature infants. Using Obrist's method of two-compartment analysis, normal values were calculated for flow in both compartments, relative weight and fractional flow in the first compartment (gray matter), initial slope of gray matter blood flow, mean cerebral blood flow, and initial slope index of mean cerebral blood flow. The application of this technique to newborns, its relative advantages, and its potential uses are discussed.

Alterations in oxygen transport following WR-2721.

The toxicity of radiation and alkylating agent chemotherapy is in part related to free radical formation in DNA and associated proteins, which is enhanced by oxygen and other electron affinic compounds. During the Phase I clinical trials of WR-2721 and alkylating agent chemotherapy, venous blood samples were obtained prior to and immediately following the WR-2721 infusion. We have observed a marked increase in the oxygen saturation of venous blood following WR-2721 (430-910 mg/m2 at a rate of 15 mg/m2/minute). The mean venous PO2 (PvO2) rose from a baseline of 34.0 +/- 13.54 torr to a mean value of 54.60 +/- 12.47 torr (p less than 0.001). The percent venous oxygen saturation increased from 54.52 +/- 21.38% to 82.73 +/- 7.66%. The highest values generally occurred within the first 2 hours after the completion of the WR-2721 infusion. No significant differences were found between the pre and post WR-2721 values for either the venous pH, CO2, or bicarbonate levels. Despite increased PvO2, the patients' heart rates, blood pressures, or respiratory rates did not change pre and post WR-2721. No apparent differences were noted in the effects of WR-2721 or PvO2 when given at doses of 740 or 910 mg/m2. However, when WR-2721 (418-1100 mg/m2) was administered over 15 minutes, the PvO2 increased in only 8/13 courses. We postulated that the marked increase in venous blood oxygen concentration was secondary to one of the following mechanisms: 1) Increased affinity of hemoglobin for oxygen, 2) microcirculatory shunting, or 3) decreased tissue requirements for oxygen. In 12 patients oxygen-hemoglobin dissociation curves and intracellular red cell pH's were obtained before and after 740-910 mg/m2 of WR-2721. Despite the significant increase in oxygenation of venous blood after WR-2721, there were no changes in either oxygen-hemoglobin dissociation or intracellular red cell pH to account for this increase. The oxygen consumption of peripheral blood granulocytes was measured prior to and following 15 courses of WR-2721. A marked decrease in granulocyte oxygen consumption was observed in 6/8 patients who had a significant increase in PvO2 and only 1/7 patients who had either a decrease or no change in PvO2 after WR-2721. Our preliminary investigations suggest that the increased venous blood oxygen content may be secondary to a reduction in normal tissue oxygen consumption. WR-2721 may afford protection by both decreasing oxygen consumption and delivery to normal tissues and increasing intracellular sulfhydryls.

Effect of hypoxia on the expression and activity of mitogen-activated protein (MAP) kinase-phosphatase-1 (MKP-1) and MKP-3 in neuronal nuclei of newborn piglets: the role of nitric oxide.

Mitogen-activated protein kinase-1 (MAPK-1) and MAPK-3 regulate survival and programmed cell death of neurons under stress conditions. The activity of MAPK-1 and MAPK-3 is regulated by dual specificity phosphatases: MKP-1 and MKP-3. In previous studies, we have shown that cerebral hypoxia results in increased activation of MAPK-1 and MAPK-3. Furthermore, we have shown that the hypoxia-induced activation of MAPK is nitric oxide (NO)-mediated. The present study tested the hypothesis that hypoxia results in altered expression and activity of MKP-1 and MKP-3 in neuronal nuclei and the administration of 7-nitro-indazole (7-NINA; 1 mg/kg, 60 min prior to hypoxia), a selective nNOS inhibitor, will prevent the hypoxia-induced alteration in the expression and activity of MKP-1 and MKP-3. To test this hypothesis expression and activity of MKP-1 and MKP-3 were determined in neuronal nuclei of normoxic (Nx; n=5), hypoxic (Hx; n=5) and 7-NINA-pretreated-hypoxic (7-NINA-Hx; n=5). Hypoxia was achieved by exposing the animals to an FiO2 of 0.07 for 60 min. Cerebral tissue hypoxia was documented biochemically by determining ATP and phosphocreatine levels. Neuronal nuclei were isolated using discontinuous sucrose gradient centrifugation and purified. Nuclear proteins were analyzed by Western blot using specific antibodies for MKP-1 and MKP-3 (Santa Cruz, CA, USA). The protein band density was determined by imaging densitometry and expressed as OD x mm2. The density of MKP-1 was 61.57+/-5.68, 155.86+/-44.02 and 69.88+/-25.54 in the Nx, Hx and 7-NINA-Hx groups, respectively (P<0.05, ANOVA). Similarly, the density of MKP-3 was 66.46+/-5.88, 172.04+/-33.10 and 116.88+/-14.66 in the Nx, Hx and 7-NINA-Hx groups, respectively (P<0.05, ANOVA). The data show an increased expression of MKP-1 and MKP-3 during hypoxia in neuronal nuclei of newborn piglets and the administration of 7-NINA, an nNOS inhibitor, prevented the hypoxia-induced increased expression of MKP-1 and MKP-3. The activity of MKP-1 (pmol/min) was 176.17+/-16.95 in Nx, 97.56+/-10.64 in Hx and 130+/-14.42 in the 7-NINA-Hx groups, respectively (P<0.05, ANOVA). Similarly the activity of MKP-3 was 104.11+/-12.17 in Nx, 36.29+/-16.88 in Hx and 77.89+/-20.18 in the 7-NINA groups, respectively (P<0.05, ANOVA). The results demonstrate that cerebral hypoxia results in increased expression of MKP-1 and MKP-3 expression that was prevented by the administration of 7-NINA. In contrast, hypoxia resulted in decreased activity of MKP-1 and MKP-3 that was prevented by the administration of a nNOS inhibitor. We conclude that hypoxia-induced decrease in MKP-1 and MKP-3 activity is not due to altered expression but due to NO-mediated modification of the cysteine residue at the active site of these dual specificity phosphatases, a mechanism of their inactivation that leads to activation of MAP kinases.

Phosphorylation of cAMP response element binding (CREB) protein during hypoxia in cerebral cortex of newborn piglets and the effect of nitric oxide synthase inhibition.

Previous studies have shown that hypoxia results in increased phosphorylation of CREB protein that mediates gene expression including that of the pro-apoptotic gene bax. We also have shown that hypoxia-induced expression of Bax protein is prevented by blocking nitric oxide synthase (NOS). The present study tests the hypothesis that inhibition of NOS by N-nitro-L-arginine (NNLA) will prevent the hypoxia-induced increased phosphorylation of CREB protein in neuronal nuclei of newborn piglets. To test this hypothesis, phosphorylation of CREB protein was assessed by immunoblotting neuronal nuclear proteins from five normoxic (Nx), 10 hypoxic (Hx) and five Hx-NNLA-treated 3-5-day-old piglets. NNLA (40 mg/kg) or saline was infused over 60 min prior to induction of hypoxia. Hypoxia was achieved by reducing the FiO(2) (0.15 to 0.05) for 60 min and documented biochemically by ATP and phosphocreatine (PCr) levels. Neuronal nuclei were isolated using discontinuous sucrose gradient centrifugation and purified. Nuclear proteins were separated on 12% sodium dodecylsulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, reacted with anti-phosphorylated CREB protein antibody and conjugated with horseradish peroxidase antibody. Protein bands were detected using the enhanced chemiluminescence method and quantitated by imaging densitometry. Protein density was expressed as absorbance (OD)xmm(2). ATP levels (micromol/g brain) were 4.3+/-0.6 in the Nx group, 1.3+/-0.5 in the Hx group (P<0.001) and 1.1+/-0.2 in the Hx-NNLA group (P<0.001 vs. Nx and Hx). Similarly, PCr levels (micromol/g brain) were 3.8+/-0.6 in the Nx group, 0.7+/-0.2 in the Hx group (P<0.001) and 0.6+/-0.1 in the Hx-NNLA group (P<0.001 vs. Nx and Hx). Density of phosphorylated CREB protein (ODxmm(2)) was 134.2+/-52.4 in the Nx group compared to 746.0+/-76.8 in the Hx group (P<0.05) and 491.1+/-40.9 in the Hx-NNLA group (P<0.05 Hx). The data show that NOS inhibition attenuates the hypoxia-induced increase in CREB protein phosphorylation in the cerebral cortex of newborn piglets.

Hypoxia-induced modification of poly (ADP-ribose) polymerase and dna polymerase beta activity in cerebral cortical nuclei of newborn piglets: role of nitric oxide.

Previous studies have shown that poly (ADP-ribose) polymerase (PARP) and DNA polymerase beta, nuclear enzymes, are associated with cell replication and DNA repair. The present study tests the hypothesis that hypoxia results in increased PARP and DNA polymerase activity in cerebral cortical neuronal nuclei to repair the hypoxia-induced damage to genomic DNA. Studies were conducted in 13 anesthetized and ventilated newborn piglets (age 3-5 days) divided into normoxic (n=5) and hypoxic (n=8) groups. Hypoxia was induced by decreasing inspired oxygen from 21% to 7% for 60 min. Cerebral tissue hypoxia was documented biochemically by determining the tissue levels of ATP and phosphocreatine (PCr). Following isolation of the cortical neuronal nuclei, the activity of PARP and DNA polymerase beta was determined. During hypoxia, the tissue ATP level decreased by 73% from 4.12+/-0.67 micromol/g brain to 1.12+/-0.34 micromol/g brain, and PCr decreased by 78% from 4.14+/-0.68-0.90+/-0.20 micromol/g brain. In hypoxic neuronal nuclei, PARP activity significantly increased from 5.88+/-0.51 pmol NAD/mg protein/h in normoxic nuclei to 10.04+/-2.02 (P=0.001). PARP activity inversely correlated with tissue ATP (r=0.78) and PCr levels (r=0.81). Administration of N-nitro-L-arginine prior to hypoxia decreased the hypoxia-induced increase in PARP activity by 67%. Endogenous DNA polymerase beta activity increased from 0.96+/-0.13 in normoxic nuclei to 1.39+/-0.18 nmol/mg protein/h in hypoxic nuclei (P<0.005). DNA polymerase beta activity in the presence of exogenous template increased from 1.54+/-0.14 in the normoxic to 2.42+/-0.26 nmol/mg protein/h in the hypoxic group (P<0.005). DNA polymerase beta activity in the presence or absence of template inversely correlated with the tissue ATP (r=0.95 and 0.84, respectively) and PCr levels (r=0.93 and 0.77, respectively). These results demonstrate that the activity of PARP and DNA polymerase beta enzymes increase with the increase in degree of cerebral tissue hypoxia. Furthermore, the results demonstrate a direct correlation between the PARP and the DNA polymerase beta activity. We conclude that tissue hypoxia results in increased PARP and DNA polymerase beta activities indicating activation of DNA repair mechanisms that may result in potential neuronal recovery following hypoxia and the hypoxia-induced increase in PARP activity is NO-mediated.

Effect of propentofylline on free radical generation during cerebral hypoxia in the newborn piglet.

The present study tests the hypothesis that propentofylline, an adenosine re-uptake inhibitor, will reduce free radical generation during cerebral hypoxia. Ten newborn piglets were pretreated with propentofylline (10 mg/kg), five of which were subjected to hypoxia, while the other five were maintained at normoxia. Five untreated control piglets underwent the same conditions. Hypoxia was induced through a decrease in FiO2 to 0.11 and documented biochemically by a decrease in ATP and phosphocreatine levels. Free radical formation in the cortex was detected directly using electron spin resonance spectroscopy with a spin trap technique. Results demonstrate that free radicals, corresponding to the alkoxyl radical, increased significantly following hypoxia, and that this increase was inhibited by pretreatment with propentofylline. Conjugated dienes, a lipid peroxidation product, also increased following hypoxia and were subsequently inhibited by propentofylline. The administration of propentofylline also significantly limited the hypoxia-induced decrease in tissue levels of ATP and phosphocreatine. These data demonstrate that pretreatment with propentofylline decreased free radical generation and lipid peroxidation as well as preserved high energy phosphates during cerebral hypoxia.

Anti-oxidant enzymes in the brain of newborn piglets during ischemia followed by reperfusion.

The activity of anti-oxidant enzymes in the brains of newborn piglets were studied under the condition of ischemic hypoxia followed by reperfusion. The activity of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase, was determined in the brain tissue of control animals and animals exposed to 60 min of hypoxia followed by 30 min of normoxia. The results showed that the activities of these enzymes were not significantly affected by hypoxia and subsequent reperfusion, suggesting that under these conditions the anti-oxidant system is not a target for, nor is its inhibition a cause of, cellular damage. It is proposed that the anti-oxidant enzyme system in the brain is non-responsive to and may not play a role during hypoxia/ischemia and subsequent reperfusion.

Mg2+-dependent modification of the N-methyl-D-aspartate receptor following graded hypoxia in the cerebral cortex of newborn piglets.

The present study tests the hypothesis that Mg2+ modification of N-methyl-D-aspartate receptor ion channel opening is altered during hypoxia and correlates with the progressive decrease in cerebral energy metabolism induced by hypoxia. Studies were performed in five normoxic and nine hypoxic ventilated piglets. In the hypoxic group, varying degrees of cerebral energy metabolism were achieved by administration of different fractions of inspired oxygen (FiO2) (5-9%) for varying durations of time and were documented by cortical tissue phosphocreatine levels. [3H]Dizocilpine maleate binding was performed with increasing concentrations of MgSO4 from 0.01 to 15 mM in cortical P2 membrane fractions. Mg2+ percentage activation and Mg2+ 50% inhibitory concentrations (IC50) were determined. The mean +/- S.D. phosphocreatine value was 3.0 +/- 1.3 micromol/g brain in the normoxic group and 1.4 +/- 1.0 micromol/g brain in the hypoxic group (P < 0.01). Low concentrations of Mg2+ (0.01-1 mM) increased [3H]dizocilpine maleate binding in the normoxic group (to 137 +/- 26% of baseline), significantly greater than in the hypoxic group (109 +/- 13%, P < 0.05). Receptor activation correlated with brain tissue levels of phosphocreatine, with percentage maximal activation decreasing linearly as phosphocreatine levels decreased (r=0.7). Higher levels of Mg2+ (1.5-15 mM) caused inhibition of [3H]dizocilpine maleate binding, with IC50 levels significantly higher in the normoxic group (3.2 +/- 1.1 mM) than in the hypoxic group (1.9 +/- 0.4 mM). Mg2+ IC50 values decreased in a linear fashion as phosphocreatine values decreased (r=0.9). The data demonstrate that, as brain cell energy metabolism decreases during hypoxia, maximal receptor activation by low levels of Mg2+ decreases and receptor inhibition by high levels of Mg2+ increases in a linear fashion. We speculate that, during hypoxia, dephosphorylation of the ion channel of the N-methyl-D-aspartate receptor increases Mg2+ blockade of the receptor by increasing Mg2+ accessibility to its binding site and that receptor modification may be initiated by subtle decreases in cortical oxygenation in the newborn brain.

Effect of hypoxia on protein phosphatase 2A activity, subcellular distribution and expression in cerebral cortex of newborn piglets.

Protein phosphatase (PP) 2A (PP2A), a major serine/threonine phosphatase highly active in the brain, is known to regulate programmed cell death by different mechanisms including downregulation of Ca++/calmodulin-dependent kinase IV (CaMK IV). Previous studies have shown that CaMK IV activity is increased following cerebral hypoxia. In the present study, we tested the hypothesis that PP2A activity and expression in neuronal nuclei are decreased following hypoxia in newborn piglets. PP and PP2A activities were determined in cerebral subcellular fractions spectrophotometrically using a serine phosphopeptide in the presence or absence of microcystine. The activity of CaMK IV in neuronal nuclei was determined by 33P-incorporation into syntide 2 in the presence or absence of either 1 mM EGTA or 0.8 mM CaCl2 and 1 mM calmodulin. The expressions of PP2A and CaMK IV were measured using Western blot. Following hypoxia, nuclear Ca++-dependent kinase IV activity increased two-fold (P<0.001), whereas PP2A and PP activities significantly decreased (P<0.05) in the neuronal nuclei and membranes but not in the cytosol (P=NS). The distribution of the activity of PP2A was 60% in the cytosol, 35% in membranes and 5% in the neuronal nuclei. The expression of PP2A protein showed a 14% increase and for CaMK IV protein a 100% increase during hypoxia. We propose that due to the decreased activity of PP and PP2A following hypoxia in the neuronal nuclei there is a shift in the balance of the phosphorylation/dephosphorylation system toward increased phosphorylated state thereby increasing activity of the nuclear CaMK IV, modulator of programmed cell death. Since there is only slight increase in the PP2A protein expression, we conclude that the changes observed in the activity of PP2A are due to hypoxia-induced modification of the enzyme itself. We also provide evidence that PP2A is a potential regulator of CaMK IV during hypoxia.

Effect of neuronal nitric oxide synthase inhibition on CA2+/calmodulin kinase kinase and CA2+/calmodulin kinase IV activity during hypoxia in cortical nuclei of newborn piglets.

The present study tests the hypothesis that cerebral tissue hypoxia results in increased Ca(2+)/calmodulin (CaM) kinase kinase activity and that the administration of nitric oxide synthase inhibitors (N-nitro-l-arginine [NNLA], or 7-nitroindazole sodium [7-NINA]) prior to the onset of hypoxia will prevent the hypoxia-induced increase in the enzyme activity. To test this hypothesis, CaM kinase kinase and CaM kinase IV activities were determined in normoxic, hypoxic, NNLA-treated hypoxic, and 7-NINA-treated hypoxic piglets. Hypoxia was induced (FiO(2)=0.05-0.08x1 h) and confirmed biochemically by tissue levels of ATP and phosphocreatine. CaM kinase kinase activity was determined in a medium containing protein kinase and phosphatase inhibitors, calmodulin, and a specifically designed CaM kinase kinase target peptide. CaM kinase IV activity was determined by (33)P-incorporation into syntide-2 in a buffer containing protein kinase and phosphatase inhibitors. Compared with normoxic animals, ATP and phosphocreatine levels were significantly lower in all hypoxic piglets whether or not pretreated with nitric oxide synthase inhibitors. There was a significant difference among CaM kinase kinase activity (pmol/mg protein/min) in normoxic (76.84+/-14.1), hypoxic (138.86+/-18.2, P<0.05 vs normoxia), NNLA-pretreated hypoxic (91.34+/-19.3; P=NS vs normoxia, P<0.05 vs hypoxia) and 7-NINA-pretreated hypoxic animals (100.12+/-23.3; P=NS vs normoxia, P<0.05 vs hypoxia). There was a significant difference among CaM kinase IV activity (pmol/mg protein/min) in normoxia (1270.80+/-126.1), hypoxia (2680.80+/-136.7; P<0.05 vs normoxia), NNLA-pretreated hypoxia (1666.00+/-154.8; P<0.05 vs normoxia, P<0.05 vs hypoxia), and 7-NINA-pretreated hypoxic (1712.9+/-231.5; P=NS vs normoxia, P<0.05 vs hypoxia). We conclude that the hypoxia-induced increase in CaM kinase kinase and CaM kinase IV activity is mediated by neuronal NOS-derived NO.

In vivo 31P nuclear magnetic resonance measurement of chronic changes in cerebral metabolites following neonatal intraventricular hemorrhage.

The purpose of this study was to determine whether cerebral metabolic changes occur after intraventricular hemorrhage in the newborn. Five babies with bilateral grade 3 to 4 intraventricular hemorrhage were compared with 15 preterm infants without intraventricular hemorrhage. Cerebral high-energy phosphorus metabolites and intracellular pH were measured with in vivo 31P nuclear magnetic resonance spectroscopy. Spectra were collected initially within the first 2 weeks of life, and then every other week until discharged from the hospital. The phosphocreatine to inorganic phosphate ratio and the phosphocreatine to adenosine triphosphate ratio were significantly lower in the group with intraventricular hemorrhage, but differences in intracellular pH were not significant. Differences between babies with and without intraventricular hemorrhage varied with postconceptional age: in those with intraventricular hemorrhage, the phosphocreatine to adenosine triphosphate ratio was decreased at all postconceptional ages, and the phosphocreatine to inorganic phosphate ratio was lower in babies with intraventricular hemorrhage and younger than 30 weeks. Results of this study confirm the presence of chronic metabolic changes following intraventricular hemorrhage which may exacerbate neurologic damage after intraventricular hemorrhage in the newborn.