Neuronal Nuclei Of Newborn Piglets Research Articles

Effect of hypoxia on nuclear high affinity Ca2+ ‐ATPase activity and nuclear Ca2+ ‐influx in the cerebral cortex of newborn piglets (888.2)

Nuclear calcium signals control a number of critical nuclear functions including regulation of transcription factors, cell cycle regulation, gene transcription and DNA replication. Previous studies have shown that during hypoxia, there is increase in intracellular Ca2+‐and in nuclear high affinity Ca2+‐ATPase activity in the cerebral cortex of newborn piglets.We test the hypothesis that hypoxia results in increased activity of high affinity Ca2+‐ATPase and increased concentration of Ca2+‐influx in cortical neuronal nuclei of newborn piglets, and this hypoxia‐induced increase is Src kinase mediated.Piglets were divided into normoxic (Nx, n=4), hypoxic (Hx, n=4), and hypoxic pretreated with Src kinase inhibitor (Hx+Srci, n=4). Hypoxia was induced by decreasing FiO2 to 0.07 for 1 hr. Src kinase inhibitor (PP2, 1.0 mg/kg i.v.) was administered 30 min prior to hypoxia. Hypoxia was documented by levels of ATP and phosphocreatine (PCr). Nuclear 45Ca2+ concentration and activity of high affinity Ca2+‐ATPase was determined at 37°C for 30 min.Nuclear 45Ca2+ ‐influx (pmoles/mg protein) was 3.95±0.57 in Nx , 13.36±1.56 in Hx (p<0.05) and 9.61±2.76 in the Hx+Srci group. Ca2+‐ATPase activity increased following hypoxia: from 244.66±73.55 nmol Pi/mg protein/h in the normoxic group to 426.33±94.99 nmol Pi/mg protein/h in the hypoxic group (p<0.05 vs. normoxic group). In the Hx+Srci group, the high affinity Ca2+‐ATPase activity increased to 354.25±62.88 nmol Pi/mg protein/h (p<0.05 vs. hypoxic group). The data show that administration of PP2, a selective inhibitor of Src kinase, prevents the hypoxia‐induced increase in nuclear 45Ca2+ ‐influx concentration and Ca2+‐ATPase activity.We conclude that the hypoxia‐induced increase in nuclear membrane high affinity Ca2+‐ATPase activity is Src kinase‐mediated activation of the high affinity Ca2+‐ATPase, a mechanism of ATP‐dependent Ca2+uptake may lead to increase in intranuclear Ca2+during hypoxia.Grant Funding Source: NIH HD200337

Effect of Hyperoxia on Cortical Neuronal Nuclear Function and Programmed Cell Death Mechanisms

There is growing concern over detrimental neurologic effects to human newborns caused by increased inspired oxygen concentrations. We hypothesize that hyperoxia (FiO(2)>0.95) results in increased high-affinity Ca(2+)-ATPase activity, Ca(2+)-influx, and proapoptotic protein expression in cortical neuronal nuclei of newborn piglets. Neuronal cerebral energy metabolism was documented by determining ATP and phosphocreatine levels. Neuronal nuclear conjugated dienes and fluorescent compounds were measured as indices of lipid peroxidation. High-affinity Ca(2+)-ATPase activity and ATP-dependent Ca(2+)-influx were determined to document neuronal nuclear membrane function. Hyperoxia resulted in increases in lipid peroxidation, high-affinity Ca(2+)-ATPase activity, ATP-dependent Ca(2+)-influx, and Bax/Bcl-2 ratio in the cortical neuronal nuclei of newborn piglets. We conclude that hyperoxia results in modification of neuronal nuclear membrane function leading to increased nuclear Ca(2+)-influx, and propose that hyperoxia-induced increases in intranuclear Ca(2+) activates the Ca(2+)/calmodulin-dependent protein kinase pathway, triggering increased CREB protein-mediated apoptotic protein expression in hyperoxic neurons.

Effect of post-hypoxic reoxygenation on DNA fragmentation in cortical neuronal nuclei of newborn piglets

Previous studies have shown an increased fragmentation of genomic DNA following hypoxia in cortical neuronal nuclei of newborn piglets. The present study tests the hypothesis that DNA fragmentation following hypoxia persists during reoxygenation in cortical neuronal nuclei of newborn piglets. To test this hypothesis, DNA fragmentation was assessed in 36 newborn piglets divided into six groups: normoxic (Nx), hypoxic (Hx) and hypoxic/reoxygenated for 6, 12, 24 h and 7 days. The Hx groups were exposed to 7% oxygen for 1 h followed by reoxygenation to room air for 6, 12, 24 h and 7 days. Cerebral tissue hypoxia was confirmed biochemically by ATP and phosphocreatine (PCr) levels. Nuclei were isolated and purified using discontinuous sucrose gradient. DNA was isolated by phenol/chloroform/isoamyl-alcohol extraction method. ATP/PCr (μmol/g brain) were 4.11 ± 0.15/3.67 ± 0.30 for Nx, 1.31 ± 0.68/0.74 ± 0.30 for Hx, 3.81 ± 0.11/3.24 ± 0.14 for 6 h reoxygenation, 4.21 ± 0.12/3.27 ± 0.09 for 12 h reoxygenation and 4.63 ± 0.09/3.75 ± 0.27 for 24 h reoxygenation and 4.31 ± 0.12/3.70 ± 0.21 for 7 days reoxygenation. There was a significant difference in the ATP and PCr values between Nx and Hx groups ( p < 0.05) and between Hx and hypoxic reoxygenated groups ( p < 0.05). DNA fragments (OD/mm 2) increased from 1776 ± 267 in the Nx group to 3211 ± 285 in the Hx group ( p < 0.05). In the reoxygenation groups, DNA fragments (OD/mm 2) decreased to 2018 ± 249 after 6 h ( p < 0.05 versus Hx) but increased to 3408 ± 206, 2782 ± 406 and 3256 ± 302 after 12 24 h and 7 days, respectively. The data show a decrease in DNA fragmentation in the early phase (6 h) of reoxygenation but is comparable to acute hypoxia during the later phases (12, 24 h and 7 days) of reoxygenation. We propose that the biphasic pattern of DNA fragmentation during reoxygenation occurs by an initial oxidative DNA injury followed by an enzymatic cleavage of DNA by endonucleases activation.

Nuclear Ca++-influx, Ca++/Calmodulin-Dependent Protein Kinase IV Activity and CREB Protein Phosphorylation during Post-Hypoxic Reoxygenation in Neuronal Nuclei of Newborn Piglets: The Role of Nitric Oxide

The present study tests the hypothesis that post-hypoxic reoxygenation results in an nitric oxide (NO)-mediated increase in nuclear Ca(++)-influx, increased calmodulin kinase (CaM kinase) IV activity, and increased Ser(133) phosphorylation of cyclic AMP response element binding (CREB) protein in neuronal nuclei of the cerebral cortex of newborn piglets. Piglets were divided into normoxic (Nx), hypoxic (Hx, FiO(2) = 0.07 for 1 h), hypoxic with 6 h reoxygenation (Hx + reox), and Hx + reox injected with 7-nitroindazole sodium salt (7-NINA), a nNOS inhibitor, immediately after hypoxia (Hx + 7-NINA). Cerebral tissue hypoxia was documented by ATP and phosphocreatine (PCr) levels. Nuclear Ca(++)-influx was determined using (45)Ca(++) and CaM kinase IV activity determined by (33)P-incorporation into syntide-2. Ser(133) phosphorylation of CREB protein was determined by Western blot analysis using a specific anti-phosphorylated Ser(133)-CREB protein antibody. ATP and PCr values in Hx, Hx + reox, and Hx + 7-NINA were significantly different from Nx (P < 0.05 versus Nx). Ca(++)-influx (pmoles/mg protein/min) was 3.79 +/- 0.91 in Nx; 11.81 +/- 2.54 in Hx (P < 0.05 versus Nx), 16.55 +/- 3.55 in Hx + reox (P < 0.05 versus Nx), and 12.40 +/- 2.93 in Hx + 7-NINA (P = NS versus Hx). CaM kinase IV activity (pmoles/mg protein/min) was 1,220 +/- 76 in Nx, 2,403 +/- 254 in Hx (P < 0.05 versus Nx), 1,971 +/- 147 in Hx + reox (P < 0.05 versus Hx), and 1,939 +/- 125 Hx + 7-NINA (P < 0.05 versus Hx). Ser(133) phosphorylated CREB protein expression (OD x mm(2)) was 87 +/- 2 in Nx, 203 +/- 24 in Hx (P < 0.05 versus Nx), 186 +/- 23 in Hx + reox (P < 0.05 Nx, P = NS versus Hx), and 128 +/- 10 in Hx + 7-NINA (P < 0.05 versus Hx and Hx + reox). The results show that post-Hx administration of 7-NINA prevents the increased nuclear Ca(++)-influx and CREB protein phosphorylation at Ser(133) during reox. We conclude that post-Hx increase in nuclear Ca(++)-influx leading to increased phosphorylation of CREB protein is mediated by NO derived from nNOS. However, hypoxia-induced increase in CaM Kinase IV activity decreased during the post-Hx reox. We propose that hypoxia-induced increase in CaM Kinase IV activity leads to increased phosphorylation of CREB protein and transcription of proapoptotic genes during post-Hx reox resulting in Hx neuronal death.

Effect of nitric oxide synthase inhibition during post-hypoxic reoxygenation on Bax and Bcl-2 protein expression and DNA fragmentation in neuronal nuclei of newborn piglets

Previous studies have shown that cerebral tissue hypoxia results in increased generation of oxygen-free radicals including nitric oxide (NO), expression of the proapoptotic protein Bax and fragmentation of nuclear DNA. The present study tests the hypothesis that post-hypoxic reoxygenation for 6 h following hypoxia (FiO 2 = 0.06 for 1 h) results in continued hypoxia-induced, NO-mediated expression of the Bax protein and nuclear DNA fragmentation in the cerebral cortex of newborn piglets. Piglets were divided into normoxic (Nx), hypoxic (Hx, FiO 2 = 0.06 for 1 h), hypoxic with 6 h reoxygenation (Hx + reox) and hypoxic with 6 h reoxygenation injected with 7-nitroindazole sodium salt (7-NINA), a selective nNOS inhibitor, immediately after hypoxia (Hx + 7-NINA). Cerebral tissue hypoxia was documented by levels of ATP and phosphocreatine (PCr). Bax and Bcl-2 were analyzed by Western blot and DNA fragmentation was determined by agarose gel electrophoresis. ATP and PCr values in Hx, Hx + reox and Hx + 7-NINA were significantly different from Nx ( P < 0.05 vs. Nx). Bax protein (OD × mm 2) was 128.9 ± 38.7 in Nx; 223.6 ± 45.8 in Hx ( P < 0.05 vs. Nx); 340.5 ± 73.2 in Hx + reox ( P < 0.05 vs. Nx, Hx and Hx + 7-NINA); and 202.2 ± 34.8 in Hx + 7-NINA ( P = NS vs. Hx). Bcl-2 protein (OD × mm 2) was 14.9 ± 2.7 in Nx, 12.4 ± 2.1 in Hx, ( P < 0.05 vs. Nx), 15.7 ± 3.8 in Hx + reox, ( P < 0.05 vs. Hx) and 13.1 ± 2.2 in Hx + 7-NINA ( P = NS among groups). Nuclear DNA fragmentation (OD × mm 2) was 147 ± 15 in Nx; 797 ± 84 in Hx ( P < 0.05 vs. Nx); 1134 ± 127 in Hx + reox ( P < 0.05 vs. Nx, Hx and Hx + 7-NINA); and 778 ± 146 in Hx + 7-NINA ( P = NS vs. Hx, P < 0.05 vs. Hx + reox). The results show that post-hypoxic reoxygenation results in increased expression of Bax protein without affecting Bcl-2 protein and increased fragmentation of nuclear DNA, which are prevented by 7-NINA. We conclude that during post-hypoxic reoxygenation the increase in Bax protein expression and fragmentation of nuclear DNA are mediated by NO derived from nNOS. We propose that in addition to NO-mediated nuclear DNA damage, the hypoxia-induced increased ratio of Bax/Bcl-2 protein will lead to caspase-activated cascade of hypoxic neuronal death during post-hypoxic reoxygenation.

Effects of Magnesium Sulfate Administration During Hypoxia on Ca2+ Influx and IP3 Receptor Modification in Cerebral Cortical Neuronal Nuclei of Newborn Piglets

Magnesium is a non-competitive antagonist of the NMDA receptor. Hypoxic insults to the brain are associated with a significant increase in the intranuclear Ca(2+) due to altered nuclear membrane Ca(2+) influx mechanisms including hypoxia-induced modifications of nuclear membrane IP(3) receptors. In this study we have examined the effects of magnesium sulfate administration to newborn piglets subjected to normoxia and severe hypoxia. The animals were randomly divided into normoxic (n=4), hypoxic (n=4) and magnesium sulfate treated hypoxic (n=4) groups. Hypoxia was confirmed biochemically by measuring ATP and phosphocreatine (PCr) levels in the brain tissue. Intranuclear Ca(2+) influx was assessed by measuring (45)Ca(2+) uptake. Results show a significant (P<0.05) decrease in ATP and PCr levels in hypoxic group in comparison with normoxia. On the other hand magnesium-treated hypoxic group showed a significantly (P<0.05) higher ATP and PCr in comparison with the hypoxic group. Intranuclear Ca(2+) was significantly (P<0.05) higher in the hypoxic group in comparison with both normoxic and magnesium-treated hypoxic groups. In addition results show that magnesium prevented hypoxia-induced modification of the IP(3) receptor. Magnesium treatment significantly reduced the hypoxia-induced increase in the number of receptors (reduced B(max) --P<0.05-treated hypoxia vs. hypoxia and normoxia), and reversed the receptor affinity (reduced dissociation coefficient-K(d)--P<0.05-treated hypoxia vs. normoxia). The results demonstrate that the administration of magnesium sulfate prior to hypoxia prevents the hypoxia-induced increase in intranuclear Ca(2+) and IP(3) receptor modifications. We conclude that Mg(2+ )administration prevents hypoxia-induced modification of neuronal nuclear membrane function that leads to intranuclear Ca(2+)-dependent transcription of apoptotic proteins leading to hypoxic neuronal programmed cell death.

Hypercapnia-Induced Modifications of Neuronal Function in the Cerebral Cortex of Newborn Piglets

There is significant controversy over the effects of hypercapnia on the human newborn brain. Previous studies have shown that 1 h of an arterial CO2 pressure (Paco2) of 80 mm Hg alters brain cell membrane Na+K+-ATPase enzyme activity in the cerebral cortex of newborn piglets. The present study tests the hypothesis that hypercapnia (either a Paco2 of 65 or 80 mm Hg) results in decreased energy metabolism and alters neuronal nuclear enzyme activity and protein expression, specifically Ca++/calmodulin-dependent kinase (CaMK) IV activity, phosphorylation of cAMP response element binding protein (CREB), and expression of apoptotic proteins in cortical neuronal nuclei of newborn piglets. Studies were performed in 20 anesthetized normoxic piglets ventilated at either a Paco2 of 65 mm Hg, 80 mm Hg, or 40 mm Hg for 6 h. Energy metabolism was documented by ATP and phosphocreatine (PCr) levels. Results show ATP and PCr levels were significantly lower in the hypercapnic groups than the normocapnic. CaMK IV activity, phosphorylated CREB density, and Bax protein expression were all significantly higher in the hypercapnic groups than the normocapnic group. Bcl-2 protein was similar in all three groups, making the ratio of Bax/Bcl-2 significantly higher in the hypercapnic groups than in the normocapnic group. We conclude that hypercapnia alters neuronal energy metabolism, increases phosphorylation of transcription factors, and increases the expression of apoptotic proteins in the cerebral cortex of newborn piglets and therefore may be deleterious to the newborn brain.

Inositol tetrakisphosphate (IP4)- and inositol triphosphate (IP3)-dependent Ca2+ influx in cortical neuronal nuclei of newborn piglets following graded hypoxia.

Previous studies have shown that hypoxia results in a modification of the binding characteristics of the neuronal nuclear membrane inositol tetrakisphosphate (IP4) and inositol triphosphate (IP3) receptors. The present study tests the hypothesis that hypoxia-induced modification of the IP4 and IP3 receptors results in increased IP4 and IP3 dependent Ca2+ influx in neuronal nuclei as a function of the degree of cerebral tissue hypoxia in newborn piglets. Studies were performed in piglets, 3-5 days old, divided into normoxic (N = 5) and hypoxic (N = 6) groups. The hypoxic group was exposed to decreased FiO2 ranging from 0.15 to 0.05 for 1 h. Brain tissue hypoxia was documented biochemically by determining ATP and phosphocreatine (PCr) levels. Neuronal nuclei were isolated and 45Ca2+ influx was determined in a medium containing 50 mM Tris buffer (pH 7.4), neuronal nuclei (150 microg protein), 1 microM 45Ca2+, with or without 10 microM IP4 or IP3. In normoxic and hypoxic groups, ATP levels were 4.27 +/- 0.80 and 1.40 +/- 0.69 micromoles/g brain, respectively, P < .001 (ranging from 4.78 to 0.82). PCr levels were 3.40 +/- 0.99 and 0.91 +/- 0.57 micromoles/g brain, respectively, P < .001 (raning from 4.07 to 0.60). During hypoxia, IP4-dependent intranuclear 45Ca2+ influx increased from 3.39 +/- 0.64 in normoxic nuclei to 13.30 +/- 2.18 pM/mg protein in hypoxic nuclei (P < .01). There was an inverse correlation between the 45Ca2+ influx in neuronal nuclei and the levels of cerebral tissue ATP (r = 0.83) and PCr (r = 0.85). Similarly, IP3-dependent intranuclear 45Ca2+ influx increased from 2.26 +/- 0.38 pmoles/mg protein in normoxic nuclei to 11.12 +/- 1.65 pmoles/mg protein in hypoxic nuclei and showed an inverse correlation between 45Ca2+ influx in neuronal nuclei and the levels of cerebral tissue ATP (r = 0.86) and PCr (r = 0.71). The data demonstrate that there is an IP4- as well as IP3-dependent increase in nuclear Ca2+ influx with increasing cerebral tissue hypoxia, suggesting a hypoxia-induced modification of the nuclear membrane IP4 and IP3 receptors. We propose that there is a specific level of tissue hypoxia that results in a critical increase of intranuclear Ca2+ that leads to altered transcription of apoptotic genes and activation of nuclear endonucleases resulting in hypoxia-induced programmed neuronal death.

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 FiO 2 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×mm 2. 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.

Nitric oxide-mediated activation of extracellular signal-regulated kinase (erk) and c-jun n-terminal kinase (jnk) during hypoxia in cerebral cortical nuclei of newborn piglets

Previous studies have shown that mitogen-activated protein kinases, such as extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK), mediate signal transduction from cell surface receptors to the nucleus and phosphorylate anti-apoptotic proteins thereby regulating programmed cell death. The present study tests the hypotheses that hypoxia activates ERK and JNK in neuronal nuclei of newborn piglets and the hypoxia-induced activation of ERK and JNK is mediated by nitric oxide (NO). Activated ERK and JNK were assessed by determining phosphorylated ERK and JNK using immunoblotting in six normoxic (Nx) and 10 hypoxic (Hx) and five N-nitro- l-arginine (a NOS inhibitor, 40 mg/kg,) -pretreated hypoxic ( N-nitro- l-arginine [NN lA]-Hx) 3–5 day old piglets. 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). Cortical neuronal nuclei were isolated and the nuclear protein was analyzed for activated ERK and JNK using anti-phosphorylated ERK and JNK antibodies. Protein bands were detected using enhanced chemiluminescence method and analyzed by imaging densitometry. Protein density was expressed as absorbance OD×mm 2. ATP levels were 4.57±0.45 μmoles/g brain in the Nx group, 1.29±0.23 μmoles/g brain in the Hx group ( P<0.05 vs Nx) and 1.50±0.14 μmoles/g brain in the NN lA-Hx group ( P<0.05 vs Nx). PCr levels were 3.77±0.36 μmoles/g brain in the Nx group, 0.77±0.13 μmoles/g brain in the hypoxic group ( P<0.05) and 1.02±0.24 in the NN lA-Hx group ( P<0.05 vs Nx). Density of phosphorylated ERK protein was 170.5±53.7 OD×mm 2 in the Nx group as compared with 419.6±63.9 OD×mm 2 in the hypoxic group ( P<0.001 vs Nx) and 270.0±28.7 in the NN lA-Hx group ( P<0.002 vs Hx). Density of phosphorylated JNK protein was 172.8±42.8 OD×mm 2 in the normoxic group as compared with 364.6±60.1 OD×mm 2 in the Hx group ( P<0.002) and 254.8±24.8 in the NN lA-Hx group ( P<0.002 vs Hx). The data demonstrate increased phosphorylation of ERK and JNK during hypoxia indicating that hypoxia results in activation of ERK and JNK in neuronal nuclei of newborn piglets. The administration of NN lA, a NOS inhibitor, prevented the hypoxia-induced phosphorylation of ERK and JNK indicating that the hypoxia-induced activation of ERK and JNK in the cerebral cortical nuclei of newborn piglets is NO-mediated

Hypoxia-induced modification of the inositol triphosphate receptor in neuronal nuclei of newborn piglets: role of nitric oxide.

Previous studies have shown that hypoxia results in increased Ca2+ influx in neuronal nuclei and generation of nitric oxide (NO) free radicals in the cerebral cortical tissue of newborn piglets. The present study tests the hypothesis that hypoxia results in modification of the inositol triphosphate (IP3) receptor characteristics in neuronal nuclei and that the hypoxia-induced modification of the IP3 receptor is NO mediated. Studies were performed in piglets, 3-5 days old, divided into normoxic (n = 5), hypoxic (n = 5), and NO synthase (NOS) inhibitor N-nitro-L-arginine (NNLA)-treated hypoxic (n = 5) groups. The NNLA-treated hypoxic group received an infusion of NNLA (40 mg/kg) over 1 hr prior to hypoxic exposure. The hypoxia was induced by lowering the FiO2 to 0.05-0.07 for 1 hr. Brain tissue hypoxia was documented biochemically by determining ATP and phosphocreatine (PCr) levels. Neuronal nuclei were isolated from the cerebral cortical tissue, and IP3 receptor binding was performed in a medium containing 50 mM HEPES (pH 8.0), 2 mM EDTA, 3H-IP3 (7.5-100 nM), and 100 microg nuclear protein. Nonspecific binding was determined in the presence of 10 microM unlabelled IP3. The IP3 receptor characteristics Bmax (number of receptor sites) and Kd (dissociation constant) were determined. In normoxic, hypoxic, and NNLA-hypoxic groups, ATP levels were 4.46 +/- 0.35, 1.52 +/- 0.10 (P <.05 vs. normoxic), and 1.96 +/- 0.33 micromoles/g brain, respectively (P <.05 vs. normoxic). PCr levels were 3.75 +/- 0.35, 0.87 +/- 0.09 (P <.05 vs. normoxic), and 1.31 +/- 0.10 micromoles/g brain, respectively (P <.05 vs. normoxic). IP3 receptor binding characteristics in normoxic nuclear membranes showed that the Bmax value was 150.0 +/- 14.1 pmoles/mg protein compared with 239.3 +/- 13.6 pmoles/mg protein in the hypoxic group (P <.05). In the NNLA-treated hypoxic group, the Bmax value was 159.0 +/- 42.6 pmoles/mg protein (P <.05 vs. hypoxic a, P = NS vs. normoxic). Similarly, the Kd was 25.2 +/- 0.28 nM in the normoxic group, 44.6 +/- 5.4 nM in the hypoxic group (P <.05), and 28.1 +/- 6.4 nM in the NNLA-treated hypoxic group. (P <.05 vs. hypoxic and P = NS vs. normoxic). The results show that hypoxia results in increased Bmax and Kd values for the IP3 receptor. Furthermore, the data demonstrate that administration of NNLA prior to hypoxia prevents the hypoxia-induced modification of the IP3 receptor in neuronal nuclei of newborn piglets. Because NNLA inhibits NOS and prevents generation of NO, we conclude that the mechanism of hypoxia-induced modification of the neuronal nuclear membrane IP3 receptor is NO mediated. We propose that NO-mediated modification of the IP3 receptor during hypoxia may lead to increased intranuclear Ca2+, resulting in altered transcription of apoptotic genes and activation of cascades of hypoxia-induced programmed neuronal death.

Hypoxia-induced Ca 2+-influx in cerebral cortical neuronal nuclei of newborn piglets

The present study was designed to investigate the effect of hypoxia on nuclear calcium-influx in the cerebral cortex of newborn piglets. Anesthetized and ventilated newborn piglets divided into normoxic ( n=4) and hypoxic groups with varying degrees of tissue hypoxia ( n=10) were studied. Nuclear Ca 2+-influx was determined using 45Ca 2+ and plotted against ATP and phosphocreatine levels. The plots were analyzed by non-linear regression (exponential) analysis that showed a curvilinear relationship ( r=0.92 for ATP and r=0.88 for phosphocreatine). These data suggest a threshold at which there is a sudden increase in the nuclear calcium-influx that then continues to increase with further decrease in the ATP and phosphocreatine levels. The results demonstrate an increase in the nuclear Ca 2+-influx during hypoxia in newborn piglets and that this increase correlates in a curvilinear fashion with the increase in the degree of cerebral tissue hypoxia. We propose that the hypoxia-induced increase in intranuclear Ca 2+ is due to altered nuclear membrane Ca 2+-influx mechanisms and will lead to Ca 2+-mediated alteration of apoptotic gene expression as well as Ca 2+-dependent activation of endonucleases that result in DNA fragmentation and subsequent programmed neuronal cell death.

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)×mm 2. ATP levels (μmol/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 (μmol/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 (OD×mm 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.

Expression of Bax and Bcl-2 proteins during hypoxia in cerebral cortical neuronal nuclei of newborn piglets: effect of administration of magnesium sulfate

This study tests the hypothesis that administration of magnesium sulfate, an antagonist of the NMDA receptor ion-channel, will prevent the hypoxia-induced alteration in the expression and the ratio of Bax and Bcl-2 proteins in cerebral cortical neuronal nuclear membranes. Anesthetized, ventilated and instrumented newborn piglets were divided into three groups: normoxic controls (Nx), untreated hypoxic (Hx), and magnesium sulfate-treated hypoxic (Mg-Hx) groups. Cerebral hypoxia was induced by lowering the FiO 2 (0.05–0.07) for 1 h and the cerebral cortex was harvested immediately for isolation of neuronal nuclei and hypoxia was confirmed biochemically by a decrease in the tissue levels of ATP and phosphocreatine (PCr). Brain tissue PCr (μmol/g brain) was 2.74±0.77 (Nx), 0.38±0.09 (Hx, P<0.05 vs. Nx) and 0.69±0.60 (Mg-Hx, P<0.05 vs. Nx). The density of immunoblotted proteins was expressed as absorbance ( A×mm 2). The expression of Bax protein ( A×mm 2) was 222±31 (Nx), 279±32 (Hx), and 148±44 (Mg-Hx, P<0.05 vs. Hx). Bcl-2 protein expression was 77±1.0 (Nx), 37±5.0 (Hx) and 46±15 (Mg-Hx, P<0.05 vs. Nx). The ratio of Bax to Bcl-2 proteins increased more than twofold during hypoxia as compared to normoxia (7:1 Hx vs. 3:1 Nx). However, in the magnesium sulfate-treated group the Bax:Bcl-2 ratio was similar to normoxic controls. The data demonstrate that magnesium sulfate treatment prevents both the hypoxia-induced increase in Bax protein expression and the alteration of Bax:Bcl-2 protein ratios. We suggest that magnesium sulfate treatment before and during hypoxia may decrease hypoxia-induced programmed cell death by maintaining the normal ratio of Bax to Bcl-2 proteins.

Effect of graded hypoxia on high-affinity Ca2+-ATPase activity in cortical neuronal nuclei of newborn piglets.

Previous studies have shown that nuclear calcium signals control a variety of nuclear functions including gene transcription, DNA synthesis, DNA repair and nuclear envelope breakdown. The present study tested the hypothesis that the activity of the neuronal nuclear high affinity Ca2+-ATPase increases as a function of decreased energy metabolism in the cerebral cortex. Studies were performed in 11 ventilated newborn piglets, age 3-5 days, divided into normoxic (Nx, n = 4) and hypoxic (Hx, n = 7) groups. The animals were exposed to a single FiO2 in the range from 0.21 to 0.05 for one hr. Cerebral tissue hypoxia was confirmed biochemically by determining brain tissue ATP and phosphocreatine levels. Neuronal nuclei were isolated and the high-affinity Ca2+-ATPase activity determined. During graded hypoxia, cerebral tissue ATP decreased from 4.80 +/- 0.58 (normoxic) to 1.03 +/- 0.38 (ranging from 0.61-1.63) micromol/g brain (p < 0.05) and PCr decreased from 3.94 +/- 0.75 (normoxic) to 0.99 +/- 0.27 (ranging from 0.50 to 1.31) micromol/g brain (p < 0.05). The total high affinity Ca2+-ATPase activity in the hypoxic nuclei increased and ranged from 541 to 662 nmol/mg protein/hr, compared to activity in normoxic group of 327 to 446 nmol/mg protein/hr. During graded hypoxia, the level of nuclear high affinity Ca2+-ATPase activity correlated inversely with ATP (r = 0.91) and PCr levels (r = 0.82), with activity increasing as tissue high energy phosphates decreased. The results demonstrate that the decrease in cerebral energy metabolism during hypoxia is linearly correlated with an increase in activity of high affinity Ca2+-ATPase in cerebral cortical nuclei from immature brain. We propose that increased nuclear membrane high affinity Ca2+-ATPase activity, leading to increased nuclear Ca2+, will result in altered expression of apoptotic genes that could initiate programmed neuronal death.

Effect of nitric oxide synthase inhibition on high affinity Ca 2+-ATPase during hypoxia in cerebral cortical neuronal nuclei of newborn piglets

Previous studies have shown that during hypoxia, neuronal nuclear high affinity Ca 2+-ATPase activity is increased in the cerebral cortex of newborn piglets. The present study tests the hypothesis that pretreatment with N-nitro- l-arginine (NNLA) will prevent the hypoxia-induced increase in high affinity Ca 2+-ATPase activity in cortical neuronal nuclear membrane of newborn piglets. We also tested the hypothesis that nitration is a mechanism of elevation of the high affinity Ca 2+-ATPase activity during hypoxia. Studies were performed in five normoxic, five hypoxic, and six NNLA-pretreated (40 mg/kg) hypoxic newborn piglets. Cerebral cortical neuronal nuclei were isolated and the high affinity Ca 2+-ATPase activity was determined. Further, normoxic samples were aliquoted into two sub-groups for in vitro nitration with 0.5 mM peroxynitrite and subsequent determination of the high affinity Ca 2+-ATPase activity. The activity increased from 309±40 nmol Pi/mg protein/h in the normoxic group to 520±108 nmol Pi/mg protein/h in the hypoxic group ( P<0.05). In the NNLA-pretreated group, the activity was 442±53 nmol Pi/mg protein/h ( P<0.05), which is 25% lower than in the hypoxic group. In the nitrated group the enzyme activity increased to 554±59 nmol Pi/mg protein/h ( P<0.05). Thus peroxynitrite-induced nitration in vitro increased the high affinity Ca 2+-ATPase activity and NNLA administration in vivo partially prevented the hypoxia-induced increase in neuronal nuclear high affinity Ca 2+-ATPase activity. We conclude that the hypoxia-induced increase in nuclear membrane high affinity Ca 2+-ATPase activity is NO-mediated and that nitration of the enzyme is a mechanism of its modification.

Hypoxia-Induced Modification of Poly (ADP-Ribose) Polymerase in Cerebral Cortical Neuronal Nuclei of Newborn Piglets

Hypoxia-Induced Modification of Poly (ADP-Ribose) Polymerase in Cerebral Cortical Neuronal Nuclei of Newborn Piglets