Karen I. Fritz

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 FiO2 (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 (micromol/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 (Axmm(2)). The expression of Bax protein (Axmm(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.

Nitric oxide-mediated expression of Bax protein and DNA fragmentation during hypoxia in neuronal nuclei from newborn piglets

The present study tests the hypothesis that nitric oxide mediates the hypoxia-induced increase in expression of Bax and in DNA fragmentation in the cerebral cortex of newborn piglets, and that administration of N-nitro-L-arginine (NNLA), a nitric oxide synthase inhibitor, will prevent a change in hypoxia-induced expression of apoptotic genes and DNA damage. Piglets were assigned to normoxic, hypoxic, or NNLA-pretreated hypoxic groups. Cerebral tissue hypoxia was documented biochemically by measuring ATP and phosphocreatine (PCr) levels. Cerebral cortical neuronal nuclei were isolated and nuclear proteins were separated electrophoretically and probed with specific antibodies against Bcl-2 or Bax proteins. Neuronal nuclear DNA from normoxic, hypoxic, and NNLA-pretreated hypoxic animals was isolated, separated by electrophoresis on 1% agarose gel and stained with ethidium bromide. Cerebral hypoxia resulted in an increase in nuclear membrane Bax protein levels from 121.33+/-47.7 optical density (OD)xmm(2) in normoxic to 273.67+/-67.3 ODxmm(2) in hypoxic group (P<0.05 vs. normoxic), but levels in NNLA-pretreated hypoxic group were 155.78+/-48.3 ODxmm(2) (P<0.05 vs. hypoxic, P=NS vs. normoxic). Similarly, cerebral hypoxia resulted in the density of DNA fragments increasing from 1530.3+/-309.8 OD/mm(2) in the normoxic group to 5383.3+/-775 OD/mm(2) in the hypoxic group (P<0.05), while levels in NNLA-pretreated hypoxic group were 3574.0+/-952 OD/mm(2) (P<0.05 compared to hypoxic and normoxic groups). The data show that NNLA-pretreatment prevents the hypoxia-induced increase in Bax expression and DNA fragmentation demonstrating that the hypoxia-induced Bax gene expression and the DNA fragmentation are NO-mediated.

Nitric oxide-mediated Ca2+/calmodulin-dependent protein kinase IV activity during hypoxia in neuronal nuclei from newborn piglets.

The present study tested the hypothesis that hypoxia results in increased Ca(2+)/calmodulin-dependent protein kinase IV (CaM kinase IV) activity and that inhibition of nitric oxide (NO) synthase by N-nitro-L-arginine (NNLA) prevents the hypoxia- induced increase in neuronal nuclear CaM kinase IV activity in newborn piglets. CaM kinase IV activity was determined in normoxic (Nx), hypoxic (Hx), and NNLA-pretreated Hx piglets. Cerebral hypoxia was confirmed biochemically. There was a significant difference between CaM kinase IV activity (pmoles/mg protein/min) in Nx (285.22+/-86.12), Hx (494.77+/-99.79, P<0.05 vs. Nx), and NNLA-pretreated Hx (249.55+/-53.85)(P=NS vs. Nx, P<0.05 vs. Hx) animals. The results demonstrate that the cerebral tissue hypoxia results in an increase in neuronal nuclear CaM kinase IV activity, and the hypoxia-induced increase in CaM kinase IV activity is NO-mediated.

The effect of hypocapnia (PaCO2 27 mmHg) on CaM kinase IV activity, Bax/Bcl-2 protein expression and DNA fragmentation in the cerebral cortex of newborn piglets

The present study tests the hypothesis that a PaCO(2) of 27 mmHg for 1 hr results in increased neuronal nuclear Ca(++)/calmodulin-dependent protein kinase IV (CaM kinase IV) activity, pro-apoptotic protein expression and DNA fragmentation in the cerebral cortex of newborn piglets. Hypocapnic (HC) and normocapnic newborn piglets were studied. Tissue levels of ATP and phosphocreatine (PCr) were lower in the HC group. CaM kinase IV activity and Bax protein density were higher in the HC group. Bcl-2 protein density was the same in both groups, resulting in an increased ratio of Bax/Bcl-2 in the HC group. Density of nuclear DNA fragments was greater in the HC group and varied inversely with ATP and PCr levels. We conclude that hypocapnia (PaCO(2) 27 mmHg) results in increased expression of pro-apoptotic proteins and fragmentation of nuclear DNA in newborn piglets.

Effect of graded hypoxia on the high-affinity CPP binding site of the NMDA receptor in the cerebral cortex of newborn piglets

Previous studies have shown that the N-methyl-D-aspartate (NMDA) receptor is modified during hypoxia in the cerebral cortex of newborn piglets. The present study tests the hypothesis that the NMDA receptor 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) high-affinity binding site is modified during hypoxia and that the degree of modification correlates with the progressive decrease in cerebral cellular energy metabolism and increase in lipid peroxidation induced by hypoxia. Studies were conducted in twelve anesthetized, ventilated newborn piglets, five normoxic and seven hypoxic which were exposed to decreased fraction of inspired oxygen (FiO2) to achieve varying phosphocreatine (PCr) levels. 3[H]-CPP binding was performed with CPP concentrations ranging from 0.5 to 1500 nM at 23 degrees C for 40 min in P2 membrane fractions. Brain tissue PCr levels were determined biochemically. Conjugated dienes (CDs) were measured as an index of lipid peroxidation. In the normoxic group, B(max) (receptor number) for the CPP binding site was 329+/-93 fmol/mg protein and Kd (dissociation constant) 137+/-44 nM, the mean PCr value was 2.5+/-0.4 micromol/g brain and the CD level was 0.0 nmol/g brain. As tissue hypoxia worsened, there was a gradual decline in tissue PCr as well as receptor B(max) and K(d) values, and there was an increase in conjugated dienes. Both the receptor B(max) (r=0.90) and Kd (r=0.72) decreased in a linear relationship as PCr decreased. As the levels of CDs increased both the receptor B(max) (r=0.88) and Kd (r=0.68) decreased in a linear fashion. The data show that there is not a critical hypoxic threshold for modification of the CPP binding site of the NMDA receptor, but that modification is coupled to a gradual decrease in brain cell energy metabolism and increase in lipid peroxidation. We speculate that hypoxia-induced modification of the NMDA receptor is mediated not only by changes in the receptor recognition site but also by an alteration of brain cell membrane structure secondary to conjugated diene formation.

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

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

NMDA Receptor Modification during Graded Hypoxia in the Cerebral Cortex of Newborn Piglets

Previous studies have shown that the cerebral N-methyl-D-aspartate (NMDA) receptor is altered during hypoxia in newborn piglets. The present study tests whether modification of the glutamate and ion channel sites of the NMDA receptor correlates with the progressive decrease in cerebral energy metabolism induced by hypoxia. Degrees of cerebral hypoxia were attained by exposure of ventilated piglets to decreased oxygen at different concentrations and confirmed by tissue phosphocreatine levels. During graded hypoxia, the number of glutamate sites decreased, the affinity of the ion channel site increased, the inhibition by Zn2+ increased, the activation by glutamate increased, and the activation by glycine decreased. Therefore, modification of the NMDA receptor correlates with the energy state of the tissue. Alterations in receptor phosphorylation may gradually modify the NMDA receptor and may be initiated by subtle decreases in tissue oxygenation in the newborn brain.

Effects of Severe Hypocapnia on Expression of Bax and Bcl-2 Proteins, DNA Fragmentation, and Membrane Peroxidation Products in Cerebral Cortical Mitochondria of Newborn Piglets

Background: Hypocapnia occurs in the newborn infant inadvertently or as a therapeutic modality and may result in neuronal and mitochondrial alterations in the newborn brain. Since mitochondria regulate apoptosis, these alterations may initiate a cascade of reactions that lead to apoptotic cell death. Objectives: This study tests the hypothesis that hypocapnia results in increased expression of the pro-apoptotic protein Bax, fragmentation of DNA and membrane lipid peroxidation in cerebral cortical mitochondria (mt) of newborn piglets. Methods: Studies were performed in three groups of anesthetized normoxic newborn piglets: hypocapnic (H, n = 5), ventilated at a PaCO2 of 11–15 mm Hg; normocapnic (N, n = 5), ventilated at a PaCO2 of 40 mm Hg; and corrected normocapnic (CN, n = 4), ventilated as H with CO2 added to maintain normocapnia. Tissue ATP and phosphocreatine levels were determined. Mitochondrial membrane proteins were separated, transblotted and probed with antibodies to Bax and Bcl-2. Bands were detected by enhanced chemiluminescence and analyzed by imaging densitometry. mtDNA was isolated. Cell and mitochondrial membrane lipid peroxidation products were measured spectrofluorometrically. Results: ATP and PCr concentrations were similar in the 3 groups. The ratio of Bax/Bcl-2 increased significantly in H compared to N and CN. mtDNA fragmentation was also significantly greater in H compared to N or CN. Membrane lipid peroxidation was higher in H than in N or CN; and in CN compared to N. Conclusions: The data demonstrate that severe hypocapnia results in increased Bax expression, DNA fragmentation, and membrane lipid peroxidation in mitochondria of cerebral cortical neurons of newborn piglets, and may result in apoptotic cell death.

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.

The effect of moderate hypocapnic ventilation on nuclear Ca2+-ATPase activity, nuclear Ca2+ flux, and Ca2+/calmodulin kinase IV activity in the cerebral cortex of newborn piglets.

Previous studies have shown that hypocapnia results in fragmentation of nuclear DNA in the cerebral cortex of newborn piglets. We tested the hypothesis that hypocapnia results in decreased ATP and phosphocreatine (PCr) levels and increased nuclear high-affinity Ca++-ATPase activity, intranuclear Ca++ flux, and CaM kinase IV activity in neuronal nuclei of piglets. Three groups of piglets were ventilated as either hypocapnic (a PaCO2 of 20 mm Hg), normocapnic (a PaCO2 of 40 mm Hg), or corrected hypocapnic (ventilated as hypocapnic but with CO2 added to maintain normocapnia) for 1 h. Tissue ATP levels were lower in the hypocapnic than in the normocapnic group. PCr levels were lower and 45Ca++-influx, Ca++-ATPase activity and CaM kinase IV activity were higher in hypocapnic than in normocapnic or corrected hypocapnic piglets. We conclude that hypocapnia alters nuclear membrane Ca++ flux mechanisms and may alter neuronal phosphorylation mechanisms in the cerebral cortex of piglets.