Karen I Fritz

Effect of cerebral hypoxia on NMDA receptor binding characteristics after treatment with 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) in newborn piglets.

Previous studies have shown that hypoxia modifies the NMDA receptor/ion channel complex in cortical brain cell membranes of newborn piglets. The present study tests the hypothesis that blockade of the glutamate recognition site of the NMDA receptor with the competitive antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) prevents modification of the receptor during hypoxia. Twenty seven anesthetized, ventilated newborn piglets were randomized into four groups: 7 normoxic (Nx), 6 CPP-treated normoxic (CPP-Nx), 8 hypoxic (Hx) and 6 CPP-treated hypoxic (CPP-Hx). Treatment groups received CPP 2 mg/kg i.v. The CPP-Hx group received CPP 30 min prior to hypoxia, which was induced by lowering the FiO2 to 5-7% for 1 h. Physiologic data showed no change in heart rate, blood pressure, arterial blood gas values, glucose or lactate following CPP administration. During hypoxia there was a significant decrease in PaO2, pH and an increase in lactate compared to baseline values. The CPP-Hx group had significantly higher lactate levels than the Hx group during hypoxia. P2 membrane fractions were prepared and thoroughly washed. Characteristics of the NMDA receptor ion channel were determined by [3H]MK-801 binding assays and characteristics of the glutamate recognition site by specific NMDA-displaceable [3H]glutamate binding assays. Brain tissue ATP and PCr levels confirmed tissue hypoxia, and were not preserved by CPP administration. [3H]MK-801 binding assays revealed that CPP treatment attenuated the hypoxia-induced decrease in the number of receptors (Bmax) and receptor binding affinity (Kd) during hypoxia. CPP treatment also decreased receptor affinity (increased Kd) for [3H]MK-801 binding during normoxia and hypoxia. Assays of [3H]glutamate binding revealed that hypoxia decreased both the Bmax and the Kd of the NMDA receptor for [3H]glutamate and both were preserved by CPP treatment prior to hypoxia. CPP had no effect on [3H]glutamate Bmax or Kd during normoxia. We conclude that hypoxia decreases the Bmax and Kd of the NMDA receptor glutamate recognition site for [3H]glutamate and the ion channel site for [3H]MK-801 in newborn piglets. These changes are prevented by CPP administration prior to hypoxia. The different effects of CPP binding during normoxia and hypoxia suggest a use-dependent mechanism for CPP binding during hypoxia, possibly through an hypoxia-induced alteration of the high-affinity binding site for CPP. During both normoxia and hypoxia CPP binding appeared to induce a conformational change in the receptor causing a decrease in binding affinity for [3H]MK-801. CPP administration did not preserve brain tissue ATP or PCr levels during hypoxia and may alter cellular metabolism in addition to its action at the NMDA receptor. However, even with depletion of the energy precursors ATP and PCr, and with higher lactate levels in the CPP-Hx group, CPP was able to maintain NMDA receptor binding characteristics during hypoxia and may decrease excitotoxic cellular damage from hypoxia.

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.

Effect of Moderate Hypocapnic Ventilation on Nuclear DNA Fragmentation and Energy Metabolism in the Cerebral Cortex of Newborn Piglets

Previous studies have shown that severe hypocapnic ventilation [arterial carbon dioxide partial pressure (Paco2) 7–10 mm Hg] in newborn animals results in decreased cerebral blood flow and decreased tissue oxidative metabolism. The present study tests the hypothesis that moderate hypocapnic ventilation (Paco2 20 mm Hg) will result in decreased cerebral oxidative metabolism and nuclear DNA fragmentation in the cerebral cortex of normoxemic newborn piglets. Studies were performed in 10 anesthetized newborn piglets. The animals were ventilated for 1 h to achieve a Paco2 of 20 mm Hg in the hypocapnic (H) group (n = 5) and a Paco2 of 40 mm Hg in the normocapnic, control (C) group (n = 5). Tissue oxidative metabolism, reflecting tissue oxygenation, was documented biochemically by measuring tissue ATP and phosphocreatine (PCr) levels. Cerebral cortical nuclei were purified, nuclear DNA was isolated, and DNA content was determined. DNA samples were separated, stained, and compared with a standard DNA ladder. Tissue PCr levels were significantly lower in the H group than the C group (2.32 ± 0.66 versus 3.73 ± 0.32 μmol/g brain, p < 0.05), but ATP levels were preserved. Unlike C samples, H samples displayed a smear pattern of small molecular weight fragments between 100 and 12,000 bp. The density of DNA fragments was eight times higher in the H group than the C group, and DNA fragmentation varied inversely with levels of PCr (r = 0.93). These data demonstrate that moderate hypocapnia of 1 h duration results in decreased oxidative metabolism that is associated with DNA fragmentation in the cerebral cortex of newborn piglets. We speculate that hypocapnia-induced hypoxia results in increased intranuclear Ca2+ flux, which causes protease and endonuclease activation, DNA fragmentation, and periventricular leukomalacia in newborn infants.

Spermine dependent activation of the N-methyl-d-aspartate receptor and the effect of nitric oxide synthase inhibition during hypoxia in the cerebral cortex of newborn piglets

This study tests the hypothesis that brain tissue hypoxia results in modification of spermine-dependent activation of the cerebral N-methyl-D-aspartate (NMDA) receptor ion-channel in newborn piglet brains and that pretreatment with N(omega)-nitro-L-arginine (NNLA), an inhibitor of nitric oxide synthase, will reduce the hypoxia-induced modification of the spermine-dependent activation of the receptor. Piglets were assigned to one of four groups; normoxia or hypoxia with or without NNLA. The infusion of NNLA or vehicle lasted for 60 min while the animals were ventilated under either hypoxic or normoxic conditions. Cerebral tissue hypoxia was confirmed by measuring ATP and phosphocreatine (PCr) levels. P2 membranes were isolated and 3H-MK-801 binding was measured in the presence of spermine. Steady state 3H-MK-801 binding in the presence of spermine, showed an increase in receptor affinity in both normoxic (47% of control) and hypoxic (42% of control) animals without change in receptor density. During hypoxia, the spermine-dependent increase in the maximal response of the 3H-MK-801 binding correlated inversely with the ATP concentrations. NNLA pretreatment prior to hypoxia, resulted in a decrease in the slope of the regression line describing the relationship between cellular energy state (ATP) and percent change in maximal response to spermine compared with vehicle treated animals indicating attenuation of the response to hypoxia. We conclude that the spermine-dependent modification of the affinity of the NMDA receptor ion-channel as assessed by 3H-MK-801 binding is similar in hypoxic and normoxic cortical tissue. NNLA administration reduces the hypoxia-induced spermine-dependent activation of the receptor indicating that nitric oxide mediates modification of the spermine site activation of the NMDA receptor ion-channel complex.

EFFECT OF GRADED HYPOXIA ON THE NMDA RECEPTOR CPP BINDING SITE IN THE CEREBRAL CORTEX OF NEWBORN PIGLETS. |[utrif]| 267

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)-1-phosphonic acid (CPP) binding site is altered in response to the progressive decrease in cerebral cellular energy metabolism induced by hypoxia. Studies were conducted in 17 anesthetized, ventilated newborn piglets, 8 normoxic and 9 exposed to decreased FiO2 at different concentrations and durations to achieve varying phosphocreatine(PCr) levels. 3H-CPP binding was performed at concentrations ranging from 2.5 to 1500 nM at 23°C for 40 minutes in P2 membrane fractions. Brain tissue ATP and PCr levels were determined biochemically. In the normoxic group the mean PCr value was 2.7 ± 0.3 μmol/g brain, Bmax (receptor number) 343 ± 80 fmoles/mg protein and Kd (dissociation constant) 130 ± 46 nM. For the group exposed to varying degrees of hypoxia, PCr (μmol/g brain), Bmax (fmoles/mg protein) and Kd (nM) were as follows: (0.04, 50, 58), (0.5, 166, 156), (0.8, 61, 65), (0.9, 59, 28), (0.9, 97, 47), (1.5, 142, 80), (2.7, 193, 188), (3.2, 261, 119), and (4.4, 326, 112). In contrast to PCr, tissue ATP levels decreased significantly only when PCr values were less than 1.0 μmol/g brain (4.8 ± 1.0 versus 1.5± 1.9 μmol/g brain, p<0.001). The results demonstrate that both the receptor Bmax (r = 0.9) and Kd (r = 0.83) decrease in a linear relationship as PCr decreases. 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 as reflected by brain tissue PCr levels. We speculate that NMDA receptor modulation may be mediated by changes in phosphorylation of the receptor recognition site and may be initiated by subtle decreases in tissue oxygenation in the newborn brain. (Funded by NIH-HD-20337, MOD #6-FY94-0135, UCPR 506-93)

Mechanism of Modification of the NR2A Subunit of the NMDA Receptor during Graded Hypoxia in the Cerebral Cortex of Newborn Piglets

Mechanism of Modification of the NR2A Subunit of the NMDA Receptor during Graded Hypoxia in the Cerebral Cortex of Newborn Piglets

Hypoxia-Induced Nitration of NMDA Receptor Subunits in the Brain of the Guinea Pig Fetus at Term

Hypoxia-Induced Nitration of NMDA Receptor Subunits in the Brain of the Guinea Pig Fetus at Term

Expression of the NR1, NR2A and NR2B Subunits of the NMDA Receptor During Graded Hypoxia in the Cerebral Cortex of Newborn Piglets |[dagger]| 1866

Previous studies have shown that during hypoxia the N-methyl-D-aspartate(NMDA) receptor ion-channel complex is modified, resulting in activation of the receptor and cellular injury through intracellular Ca++ flux. The degree of NMDA receptor modification correlates with the decrease in cerebral energy metabolism induced by hypoxia. The present study tests the hypothesis that expression of the NR1, NR2A and NR2B subunits of the NMDA receptor increases as cerebral energy metabolism decreases. Studies were performed in 3 normoxic (Nx) and 7 hypoxic (Hx) ventilated piglets. In the Hx group varying degrees of cerebral energy metabolism were achieved by administration of different concentrations of O2 (5-9%) for 20 min to 1 hr, and were documented biochemically by tissue ATP and phosphocreatine (PCr) levels. Immunoprecipation of NR1, NR2A and NR2B NMDA receptor subunits was performed with primary rat, mouse and mouse antibodies respectively, and separated using 8% SDS-PAGE. Subunit expression was quantified by imaging densitometry and expressed as absorbance × mm2. During hypoxia, expression(absorbance × mm2) of the NR1 (4.6±1.5 Hx vs 0.4±0.0 Nx, p <0.01), NR2A (5.0±1.2 Hx vs 1.7±0.0 Nx, p<0.05) and NR2B (3.1±0.6 Hx vs 1.3±0.0 Nx, p<0.05) subunits were increased compared to normoxic controls. The increased expression of the NMDA receptor NR1 subunit correlated inversely with tissue ATP and PCr levels (r=0.62, r=0.66) as did the increase in NR2A (r=0.99, r=0.99) and NR2B (r=0.99, r=0.97) subunit expression, with expression increasing as tissue high energy phosphates decreased during hypoxia. The results demonstrate that as cerebral energy metabolism decreases, expression of the NR1, NR2A and NR2B subunits of the NMDA receptor increases in a linear relationship. We speculate that as cerebral energy metabolism decreases, altered phosphorylation of the NMDA receptor results in increased intracellular Ca++ flux which increases NMDA receptor subunit expression and may increase NMDA receptor recycling during hypoxia.

Phosphorylation of Serine Residues of the NR1 Subunit of the NMDA Receptor During Graded Hypoxia in the Cerebral Cortex of Newborn Piglets |[dagger]| 195

Previous studies have shown that N-methyl-D-aspartate (NMDA) receptor - mediated intracellular Ca++ flux increases with receptor phosphorylation by protein kinase C (PKC) during normoxia and increases with the hypoxia-induced decrease in cerebral energy metabolism. The present study tests the hypothesis that the serine residues of the NMDA receptor NR1 subunit are phosphorylated during hypoxia, and that the degree of phosphorylation correlates with the decrease in cerebral energy metabolism induced by hypoxia. Studies were performed in 3 normoxic (Nx) and 3 hypoxic (Hx) ventilated newborn piglets. In the Hx group varying degrees of cerebral energy metabolism were achieved by administration of different concentrations of O2(5-9%) and documented biochemically by tissue ATP and phosphocreatine (PCr) levels. P2 membrane proteins were immunoprecipitated with antiphosphoserine antibodies and separated by 8% SDS-PAGE. Proteins were transblotted and probed with NR1 antibody. The phosphorylated NR1 subunit protein was visualized with horseradish peroxidase conjugate. Protein bands were analyzed by imaging densitometry and expressed as absorbance/mm2. During hypoxia, absorbance/mm2 of the phosphorylated serine NR1 residues was 1.7±0.7, significantly greater than in the Nx group 0.6±0.1,(p< 0.05). The ATP, PCr and absorbance/mm2 of phosphorylated serine residues were as follows: (5.9, 0.7, 1.4), (5.2, 1.2, 0.5), (4.9, 2.0, 0.7),(4.6, 0.6, 0.8), (4.4, 0.7, 2.4) and (0.0, 0.0, 2.2), respectively, demonstrating an inverse linear correlation between both ATP (r=0.5) and PCr(r=0.7) levels and phosphorylation of NR1 serine residues during graded hypoxia. The data suggest that as cerebral energy metabolism decreases, phosphorylation of serine residues of the NR1 subunit of the NMDA receptor increases, with phosphorylation and potential activation of the NMDA receptor increasing despite a decrease in tissue high energy phosphates. We speculate that increased phosphorylation of the NMDA receptor serine residues may be due to either an increase in PKC phosphorylation or to conformational change in the receptor exposing new serine residues to PKC. Phosphorylation of the NMDA receptor may be a potential mechanism of receptor activation and intracellular Ca++ flux during hypoxia.

Glycine Activation of the NMDA Receptor During Graded Hypoxia in the Cerebral Cortex of Newborn Piglets † 333

Glycine Activation of the NMDA Receptor During Graded Hypoxia in the Cerebral Cortex of Newborn Piglets † 333