Eric Lombardini

Modification of the Glutamate Binding Site of the NMDA Receptor During Graded Hypoxia in the Cerebral Cortex of Newborn Piglets. |[dagger]| 183

We have previously shown that during hypoxia the glutamate binding site of the N-methyl-D-aspartate (NMDA) receptor is altered in newborn piglet brain. The present study tests the hypothesis that hypoxia-induced alteration of the glutamate site of the NMDA receptor is progressive and correlates with the decrease in cerebral energy metabolism induced by hypoxia. Studies were performed in 8 ventilated piglets, 4 normoxic and 4 hypoxic. Varying degrees of hypoxia were achieved by exposure to decreased oxygen at different concentrations and confirmed by brain tissue ATP and phosphocreatine (PCr) levels. 3[H]glutamate binding was performed in P2 membrane fractions at concentrations of 25-1000 nM. Specific NMDA-displaceable3[H]-glutamate binding was determined with 100μM NMDA. Non-specific binding was determined with 1mM unlabelled glutamate. Bmax (receptor number) and Kd (dissociation constant) were calculated from Scatchard plots. ATP(μmol/g brain), PCr (μmol/g brain) and Bmax (fmol/mg protein) were as follows: (5.86, 2.77, 402), (4.77, 2.96, 388), (4.50, 2.05, 364), (4.03, 2.01, 332), (3.37, 0.96, 276), (2.49, 0.24, 253), (1.96, 0.77, 322), and (0.24, 0.46, 174). Bmax decreased in a linear relationship as both ATP (r=0.92) and PCr (r=0.83) decreased, but Kd did not. The data show that the number of glutamate binding sites decreased in a linear relationship as oxidative phosphorylation decreased during hypoxia. A decline in oxidative phosphorylation may lead to a reduction in glutamate binding sites by several mechanisms. Since phosphorylation regulates NMDA receptor distribution in neuronal membranes, we speculate that a decrease in oxidative phosphorylation as seen during hypoxia may lead to a loss in number of glutamate binding sites through changing the location and density of NMDA receptors in brain cell membranes. Alternatively, we speculate that dephosphorylation of the receptor may cause an allosteric or conformational change in the receptor preventing glutamate binding and may lead to lipid peroxidation and cell death.

The Effect of Reoxygenation with 21% and 100% Oxygen Following Hypercapnic Hypoxia on the Glutamate Site of the NMDA Receptor in the Brains of Newborn Piglets. |[dagger]| 904

The Effect of Reoxygenation with 21% and 100% Oxygen Following Hypercapnic Hypoxia on the Glutamate Site of the NMDA Receptor in the Brains of Newborn Piglets. † 904

Spermine Dependent Activation of the N-Methyl-D-Aspartate (NMDA) Receptor in the Cerebral Cortex of Newborn Piglets. |[dagger]| 1771

Previous studies have shown that maximal spermine-dependent activation of the NMDA receptor-ion channel complex is increased during hypoxia. The aim of the present study is to investigate the mechanism by which spermine activates the NMDA receptor. 3H-MK-801 binding was used to assess receptor affinity (Kd) and density (Bmax). Twelve piglets (<7 days old) were exposed for 60 min to either FiO2 0.21 (normoxia, n=7) or an FiO2 0.05-0.07 (PaO2≤ 20 mmHg, hypoxia, n=7). Brain tissue hypoxia was documented biochemically by a decrease in ATP and phosphocreatine. P2 membrane fractions were prepared from cerebral cortices and 3H-MK-801 binding was performed at 32°C in 200 μl of medium containing either buffer or 10 μM spermine plus 100 μM glutamate, 100 μM glycine, 10 mM HEPES/1 mM EDTA buffer (pH 7.0), increasing concentrations of 3H-MK-801(2.5 to 50 nM), and 75 μg protein. Saturation curves and Scatchard plots were constructed to determine the Bmax and Kd. Bmax was not significantly different among the 4 groups (1.5±0.3 pmol/mg protein in normoxia to 1.5±0.3 pmol/mg protein in normoxia+spermine, and from 1.3±0.5 pmol/mg protein in hypoxia to 1.3±0.5 pmol/mg protein in hypoxia+spermine). Kd (mean±SD) decreased from 5.4±0.7 nM in normoxia to 3.6±1.9 nM in normoxia+spermine, and from 6.4±0.7 nM in hypoxia to 3.1±1.2 nM in hypoxia+spermine (p<0.001). The results demonstrate that spermine increases the affinity of the NMDA receptor ion channel for MK-801 in both normoxia and hypoxia without changing the number of binding sites. We speculate that the spermine dependent activation of the NMDA receptor ion-channel occurs by alteration of the ion-channels affinity through the glutamate recognition site and/or acts as a polycationic molecule to influence the micro environment of the channel. (Funded by MOD 6-FY94-0135, NIH #HD-20337)

NMDA Receptor Characteristics in the Brain of Fetal Guinea Pigs Following In Utero Hypoxia and Recovery. † 201

Previous studies have shown that in utero hypoxia results in brain cell membrane dysfunction and alters N-methyl-D-aspartate (NMDA) receptor characteristics in fetal guinea pigs. The present study tests the hypothesis that modification of brain cell membrane function during in utero hypoxia persists following recovery. Six pregnant guinea pigs at term were exposed to 7% oxygen for 1 hour. Brain tissue obtained from fetal guinea pigs immediately after in utero hypoxia was compared to that of fetuses exposed to hypoxia but allowed to recover in utero for 24 hours. ATP and phosphocreatine (PCr) levels were determined biochemically as indices of brain tissue energy metabolism. ATP and PCr levels were significantly higher in the recovery group compared to the hypoxic group(5.22±0.95 vs. 1.5±0.02 and 3.73±0.28 vs. 0.66±0.17 μ mol/g brain, respectively, p<0.01). Brain cell membrane Na+,K+-ATPase activity was measured as an index of brain cell membrane function. The recovery group demonstrated a significantly higher activity of Na+,K+-ATPase compared to the hypoxic group(35.1±4.0 vs. 25.2±7.0 μ moles Pi/mg protein/hr, p<0.05).3[H]MK-801 binding (2.5-50 nM) in the presence of glutamate and glycine was performed as an index of NMDA receptor modification. Receptor number(Bmax) in the recovery group was similar to the hypoxic group(1.28±0.04 vs 1.25±0.18 pmol/mg protein). In addition receptor dissociation constant (Kd) was similar in the two groups (5.61±0.30 vs 4.94±0.84 nM). This preliminary data show that following recovery from in utero hypoxia, brain cell energy metabolism and brain cell membrane function improve, but that hypoxia-induced alterations in NMDA receptor characteristics persist. We speculate that alterations in the NMDA receptor following in utero hypoxia may contribute to delayed brain cell injury despite recovery of cellular energy metabolism and cell membrane function.

Peroxynitrite-Induced Modification of the NMDA Receptor in the Cerebral Cortex of the Term Guinea Pig Fetus. |[bull]| 342

Peroxynitrite-Induced Modification of the NMDA Receptor in the Cerebral Cortex of the Term Guinea Pig Fetus ♦ 1911

Inhibition of the Glutamate Dependent Activation of the Nmda Receptor By Adenosine in Cortical Tissue of the Newborn Piglet. 17

Hypothesis: Adenosine will competitively inhibit the glutamate site and decrease the glutamate dependent activation of the NMDA receptor ion-channel. Methods: Studies were performed in brain cell membranes from 6 normoxic (Nx) and 6 hypoxic (Hx) newborn piglets. 3H-MK-801 binding was performed in the presence of 0 to 10μM glutamate and 100μM glycine as an index of NMDA receptor activation. Assays were performed in the presence and absence of 100μM adenosine. Results: Maximum binding(mean ± sd) was 238 ± 25nM/mg protein in untreated Nx membranes as compared with 155 ± 23 nM/mg protein in the treated Nx group, a 35% decrease of NMDA receptor activation. Ka (concentration of glutamate needed for 50% of maximum activation) was 0.22μM glutamate in untreated, Nx tissue vs 0.31 μM glutamate in treated, Nx tissue, an increase of 40%. In Hx tissue, untreated samples showed maximum binding of 216 ± 34 nM/mg protein as compared with 124 ± 33 nM/mg protein (42% inhibition of activation). Hx Ka values were 0.32 μM glutamate in untreated samples and 0.36 μM glutamate in treated samples, an increase of 11%.Conclusions: Adenosine inhibits the NMDA receptor ion-channel activation, probably by interacting at the glutamate site of the receptor. The altered response in the hypoxic tissue indicates a modification of the glutamate site on the receptor during hypoxia. The NMDA receptor ion-channel mediated effect of adenosine appears to be a new mechanism of its action, resulting in inhibitory neurotransmission by counteracting the excitatory pathway.

Effect of Dephosphorylation on the Glutamate Site of the NMDA Receptor in the Cerebral Cortex of the Newborn Piglet. † 1716

Effect of Dephosphorylation on the Glutamate Site of the NMDA Receptor in the Cerebral Cortex of the Newborn Piglet. † 1716

MODIFICATION OF THE NMDA RECEPTOR GLUTAMATE BINDING SITE DURING ACUTE HYPOGLYCEMIA IN NEWBORN PIGLETS. |[dagger]| 1360

We have previously shown that acute hypoglycemia alters both the kainate-type glutamate receptor and the MK-801 binding site of the N-methyl-D-aspartate(NMDA)-type glutamate receptor in newborn piglet brain. The present study tests the hypothesis that acute hypoglycemia modifies the glutamate binding site of the NMDA receptor, potentially increasing excitotoxicity. Studies were performed in 3 control (C) and 5 hypoglycemic (H) newborn piglets. Hypoglycemia (blood glucose concentration 1 mmol/L) was induced with 150 U/kg IV bolus of regular insulin and maintained for 2 hr with continuous insulin infusion. Brain cell membranes were prepared from cerebral cortex and saturation binding assays were performed using 150 μg of membrane protein and 3H-glutamate at concentrations of 25-1000 nM. NMDA-dependent binding was determined in the presence of 1 mM NMDA. Bmax(number of receptors) and Kd (dissociation constant) were calculated from Scatchard plots. Bmax (mean ± SD) in hypoglycemic animals was significantly increased from the control value of 746 ± 42 to 992± 64 fmol/mg protein (p < 0.05). Kd was 296 ± 64 nM in H, not significantly different from the value of 251 ± 4 nM in C. Thus acute hypoglycemia increased the apparent number of glutamate binding sites of the NMDA receptor but did not alter receptor affinity for glutamate. The increased number of glutamate sites may be the mechanism for the increase in activation of MK-801 binding by glutamate during hypoglycemia which we reported previously. The results of the present study support the speculation that hypoglycemia enhances the sensitivity of cerebral cortex to excitotoxicity under conditions which increase extracellular glutamate concentration in the newborn. (Supported by NIH #HD-20337, UCP #R-503-93)

EFFECT OF LIPID PEROXIDATION ON THE NMDA RECEPTOR CPP BINDING SITE DURING CEREBRAL HYPOXIA IN NEWBORN PIGLETS. |[utrif]| 2229

Previous studies have shown that hypoxia induces membrane lipid peroxidation and alters the N-methyl-D-aspartate (NMDA) receptor in the cerebral cortex of newborn piglets. The present study tests the hypothesis that hypoxia-induced modification of the 3-(2-carboxypiperazin-4-yl)-1-phosphonic acid (CPP) binding site of the NMDA receptor is correlated with hypoxia-induced lipid peroxidation in brain cell membranes. Studies were performed in 18 anesthetized, ventilated piglets divided into 2 groups - 8 normoxic and 10 exposed to decreased FiO2 at different concentrations and durations to achieve various degrees of hypoxia. P2 membrane fractions were prepared and 3H-CPP binding was performed at concentrations ranging from 2.5 to 1500 nM at 23°C for 40 mins. Conjugated dienes (CDs) were measured as an index of lipid peroxidation.[ATP] and [phosphocreatine] (PCr) were measured biochemically to document tissue hypoxia. In the normoxic group the [CD] was 0.0 nmol/g brain. Bmax(receptor number) 343±80 fmole/mg protein and Kd (dissociation constant) 130±46 nM. The animals exposed to decreased FiO2 concentrations demonstrated [CDs] (nmol/g brain), Bmax (fmole/mg protein) and Kd (nM) concentrations as follows: (3.5, 142, 80), (25, 193, 118), (49, 195, 80), (56, 261, 119), (70, 326, 112), (98, 97, 47), (112, 61, 65), (117, 166, 156), (123, 59, 28) and (202, 50, 58). The results show that as the [CDs] increase both the receptor Bmax (r=0.7) and Kd (r=0.6) decrease in a linear fashion. The data demonstrate that during hypoxia progressive NMDA receptor modification occurs as lipid peroxidation increases. We speculate that the hypoxia-induced progressive modification of the NMDA recognition site may be due either to an alteration of brain cell membrane structure secondary to conjugated diene formation or to a direct modification of the receptor by hypoxia-induced free radicals. (Funded by NIH-HD-20337, MOD #6-FY94-0135, UCPR 506-93)

EFFECT OF 100|[percnt]| OXYGEN AND 21|[percnt]| OXYGEN DURING RESUSCITATION ON CEREBRAL CELLULAR FUNCTION FOLLOWING NEONATAL ASPHYXIA. |[dagger]| 1279

Previous studies have shown that hypoxia and severe repeated asphyxia modify the N-methyl-D-aspartate (NMDA) receptor, decrease Na+,K+-ATPase activity, and increase lipid peroxidation and free radical production. The present study investigates the effect of hyperoxic and normoxic reventilation following asphyxia in piglets on NMDA receptor activation, lipid peroxidation and free radical generation. Asphyxia was induced in 8 anesthetized and paralyzed piglets by discontinuing mechanical ventilation for 6-12 min until the heart rate was 120mmHg and PO2<5mmHg). After asphyxia, animals in Grp 1 (n=4) and Grp 2 (n=4) were reventilated with 21% oxygen and 100% oxygen for 20 min. The cerebral cortices were removed for analysis.3[H]MK-801 binding, an index of NMDA receptor activation, showed the Bmax (number of receptors) in Grp 1 and Grp 2 to be 1.32±0.13 and 1.57±0.34 pmol/mg protein (mean±SD). Kd for the same groups were 4.45±1.45 and 3.79±0.96 nM. Bmax and Kd in controls were 1.23±0.06 pmol/mg protein and 5.8±0.5 nM. Na+,K+-ATPase activity in Grps 1 and 2 were 23.5±0.9 and 24.4±3.9 μmol Pi/mg protein/hr, as compared to control(49.9±0.5, p<0.05). Lipid peroxidation products, conjugated dienes(0.07 vs. 0.05 μmol/g brain) and fluorescent compounds (0.78 vs. 0.54 μg quinine sulfate/g brain) were similar. Electron spin resonance spectroscopy performed after trapping spin adducts in α-phenyl-N-tert-butylnitrone for the detection of alkoxyl radical showed similar spin adduct signal height intensity (10.39 vs. 13.68 mm/g tissue). The results show that NMDA receptor activation, lipid peroxidation. Na+,K+-ATPase activity and alkoxyl radical generation were similar following reventilation with 100% and 21% oxygen. These data did not reveal immediate cerebral cellular dysfunction with either normoxic or hyperoxic reventilation following asphyxia. Since previous data have shown a 48 h delay in the appearance of neuronal membrane injury, the long term effect of reventilation with oxygen on neuronal membrane function remains unknown. (Funded by NIH HD-20337, MOD-6FY940135, UCP#R-506-93).