Brain Research, 566 (1991) 342-343
342
Elsevier
BRES 24950
Brain glutamine synthetase activity and hyperoxia in neonatal rats N i n a F e l i c e S c h o r 1"2'3, M a m d o u h a
Ahdab-Barmada 4 and Edwin Nemoto 5
Departments of i Pediatrics, :Neurology, ~Pharmacology, 4Pathology and SAnaesthesiology and Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213 (U.S.A.) (Accepted 27 August 1991)
Key words: Hyperoxia: Oxygen free radical; Glutamine synthetase; Glutamate
We have previously shown that exposure to 100% oxygen for 2 h results in a two-fold decrease in the brain glutamine synthetase activity of neonatal rats. The present study examines whether this decrement in enzyme activity leads to a global accumulation of glutamate, an excitotoxin which is a substrate for this enzyme. Despite a demonstrable decrement in whole brain glutamine synthetase activity, whole brain glutamate content is unaltered in animals exposed to 100% oxygen for 2 h. Furthermore, despite a persistent two-fold decrement in glutamine synthetase activity in animals exposed to 100% oxygen for 6 h, there remained no significant difference in glutamate content or in the glutamatelglutamine ratio between these animals and animals similarly exposed to room air. These results imply that the observed decrease in glutamine synthetase activity does not globally influence the glutamate content of the brain.
Neuronal karyorrhexis has been demonstrated in the brains of human 2 and rat t neonates exposed to acute hyperoxia. We have previously shown that the activity of glutamine synthetase, an enzyme responsible for the conversion of glutamate into its non-toxic metabolite, glutamine, is decreased two-fold in the brains of neonatal rats exposed to 100% oxygen for 2 h 3'4. We hypothesized that the neurotoxicity of hyperoxia results from the inhibition of glutamine synthetase and resultant accumulation of glutamate. We tested this hypothesis by examining whole brain glutamine synthetase activity and glutamate levels in newborn rats exposed to 100% oxygen for 2 or 6 h. Timed pregnant female Wistar albino rats were obtained from Hilltop Laboratories (Scottdale, PA) 2-7 days before the expected day of delivery. All animals had free access to food and water up to the time of the study. All studies were commenced within 24 h of delivery. For the 2 h study, the rats were isolated from their mothers, placed in one of two 5.3 ! glass jars, and exposed to humidified room air (n - 7) or humidified 100% oxygen (n = 6) under normobaric conditions, as we have previously described t.4. For 6 h exposure studies, the newborn rats were maintained in contact with their mothers with food and water available ad libitum. The cage tops were covered by clear plastic bags and sealed with duct tape. Gas lines were introduced into the cages and either room air (n = 15) or 100% oxygen (n = 13), respectively, was introduced into the cages at 2
l/min. In all cases, the oxygen concentration of the gases to which the animals were exposed was measured with an oxygen monitor (Teledyne Analytical Instruments, Pasadena, CA). Gas outlet vents maintained normobadc conditions throughout the experiment. After 2 or 6 h, the rats were removed from the jars or cages and immediately decapitated so that the heads fell directly into liquid nitrogen. The frozen brains were removed from the calvaria and divided in two by mid-saggital section at-15 to-20 °C in a modified cryostat microtome chamber and stored at -80 °C until biochemical analysis. Each brain half was weighed and immediately homogenized at 4 °C in 2 ml of 0.15 M potassium chloride containing 5 mM/~-mercaptoethanol and I mM EDTA. The homogenate was centrifuged at 16,000 g for 30 rain, and 50 #1 of the supernatant was used to assay for whole brain glutamine synthetase activity by the hydroxylamine method of Meister ~, In preparation for the determination of glutamate content, a 10/d aliquot of the supernatant was deproteinized with 100/~! of 35% sulfosalicylic acid at 4 °C. This treated sample was centrifuged, and the supernatant was assayed for glutamate by standard techniques using a Beckman Model 6300 amino acid analyzer (University of Pittsburgh Protein Sequence Facility, Pittsburgh, PA). Statistical analysis of the data involved comparison of the means for room air- and oxygen-treated animals by Student's t-test. A P value maximally 0.05 was considered to be statistically significant. As in our previously reported study 4, glutamine syn-
Correspondence: N.E Schor, Division of Child Neurology, Children's Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213, U.S.A. Fax: (I) {412) 692-5723.
343 TABLE I Glutamine synthetase activity and glutamate content of the brains of neonatal rats exposed to room air or 100% oxygen for 2 h
Neonatal rat pups were isolated from their mothers and exposed to room air (n = 7) or 100% oxygen (n = 7) for 2 h, or maintained with their mothers and exposed to room air (n = 15) or 100% oxygen (n = 13) for 6 h. Glutamine synthetase activity was determined on whole brain by the method of Meister3. Glutamate content was determined by amino acid analysis. Values are expressed as the mean ± S.E.M. The values for glutamine synthetase activity differ with a P maximally 0.02 (Student's t test) for the 2 h exposure, and with a P maximally 0.05 for the 6 h exposure.
Glutamine synthetase at 2 h (U/g wet tissue weight) Glutamate at 2 h ~mol/g wet tissue weight) Glutamine synthetase at 6 h (Ulg wet tissue weight) Glutamate at 6 h (,umol/g wet tissue weight) Glutamine at 6 h (~mol/g wet tissue weight)
Room air
100% oxygen
1.15 - 0.19
0.66 - 0.19
1.46 +- 0.11
1.46 - 0.16
0.68 ± 0.15
0.28 ± 0.15
0.82 ± 0.05
0,85 ± 0.05
0.76 ± 0.05
0.88 ± 0.08
thetase levels were two-fold lower in the brains of animals isolated from their mothers and treated with 100% oxygen for 2 h, ~han in animals similarly isolated and treated with room air for the same length of time. Despite the reduction in glutamine synthetase activity, there was no difference between control and hyperoxic animals with respect to the glutamate content of their brains (see Table I). This was true whether the values were normalized to wet tissue weight (shown) or to glycine content (data not shown), making it unlikely that this
1 Ahdab-Barmada, M., Moossy, J., Nemoto, E.M. and Lin, M.R., Hyperoxia produces neuronal necrosis in the rat, ./. Neuropath. Exp. Neurol., 45 (1986) 233-246.
2 Ahdab-Barmada, M., Moossy, J. and Painter, M.J., Pontosubicular necrosis and hyperoxemia, Pediatrics, 66 (1980) 840-847.
effect is due solely to edema formation in hyperoxic animals. Preliminary studies (Ahdab-Barmada, Nemoto, and Schor, unpublished results) indicated that isolation of neonatal animals from their mothers for more than 2 h resulted in severe dehydration. Even over a 2 h period, brain giycine concentration, used here as a marker for hydration status, progressively increased up to two-fold in control animals exposed to room air. For this reason, animals studied for 6 h were maintained in the presence of the mother. This restored the brain glycine concentration to its native value, approximately two-fold lower than that seen in isolated animals. This discrepancy is therefore assumed to be dilutional in nature. In agreement with this is our finding that control glutamine synthetase and glutamate levels are approximately two-fold higher in the isolated control animals (room air, 2 h, Table I) than in control animals maintained in the presence of the mother (room air, 6 h, Table I). As was the case at 2 h, animals exposed to 100% oxygen for 6 h exhibited a two-fold reduction in glutamine synthetase activity relative to animals similarly exposed to room air. Despite this, there was no difference in the contents of glutamate or glutamine between the two groups. The present study shows that the decrement in glutamine synthetase activity seen after a 2 or 6 h exposure to 100% oxygen is not accompanied by an alteration in whole brain glutamate content or in its global conversion to glutamine. These studies do not preclude local changes (e.g. in the pons and subiculum - areas particularly affected by hyperoxic insults 1'2) in the concentration of glutamate, changes in the uptake of glutamate by astrocytes as a result of exposure to 100°/'o oxygen, or a role for glutamate in hyperoxic injury which is independent of the inactivation of glutamine synthetase. The authors wish to thank Yvette M. Walker-Cox and John Melick for expert technical assistance during this study. Supported by a Biomedical Research Studies Grant from the National Institutes of Health to Children's Hospital of Pittsburgh.
3 Meister, A., Giutamine synthetase from mammalian tissues, Meth. Enzymoi., 113 (1985) 185-199. 4 Schor, N.F., Inactivation of mammalian brain glutamine synthetase by oxygen radicals, Brain Research, 456 (1987) 17-21.
342
Elsevier
BRES 24950
Brain glutamine synthetase activity and hyperoxia in neonatal rats N i n a F e l i c e S c h o r 1"2'3, M a m d o u h a
Ahdab-Barmada 4 and Edwin Nemoto 5
Departments of i Pediatrics, :Neurology, ~Pharmacology, 4Pathology and SAnaesthesiology and Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213 (U.S.A.) (Accepted 27 August 1991)
Key words: Hyperoxia: Oxygen free radical; Glutamine synthetase; Glutamate
We have previously shown that exposure to 100% oxygen for 2 h results in a two-fold decrease in the brain glutamine synthetase activity of neonatal rats. The present study examines whether this decrement in enzyme activity leads to a global accumulation of glutamate, an excitotoxin which is a substrate for this enzyme. Despite a demonstrable decrement in whole brain glutamine synthetase activity, whole brain glutamate content is unaltered in animals exposed to 100% oxygen for 2 h. Furthermore, despite a persistent two-fold decrement in glutamine synthetase activity in animals exposed to 100% oxygen for 6 h, there remained no significant difference in glutamate content or in the glutamatelglutamine ratio between these animals and animals similarly exposed to room air. These results imply that the observed decrease in glutamine synthetase activity does not globally influence the glutamate content of the brain.
Neuronal karyorrhexis has been demonstrated in the brains of human 2 and rat t neonates exposed to acute hyperoxia. We have previously shown that the activity of glutamine synthetase, an enzyme responsible for the conversion of glutamate into its non-toxic metabolite, glutamine, is decreased two-fold in the brains of neonatal rats exposed to 100% oxygen for 2 h 3'4. We hypothesized that the neurotoxicity of hyperoxia results from the inhibition of glutamine synthetase and resultant accumulation of glutamate. We tested this hypothesis by examining whole brain glutamine synthetase activity and glutamate levels in newborn rats exposed to 100% oxygen for 2 or 6 h. Timed pregnant female Wistar albino rats were obtained from Hilltop Laboratories (Scottdale, PA) 2-7 days before the expected day of delivery. All animals had free access to food and water up to the time of the study. All studies were commenced within 24 h of delivery. For the 2 h study, the rats were isolated from their mothers, placed in one of two 5.3 ! glass jars, and exposed to humidified room air (n - 7) or humidified 100% oxygen (n = 6) under normobaric conditions, as we have previously described t.4. For 6 h exposure studies, the newborn rats were maintained in contact with their mothers with food and water available ad libitum. The cage tops were covered by clear plastic bags and sealed with duct tape. Gas lines were introduced into the cages and either room air (n = 15) or 100% oxygen (n = 13), respectively, was introduced into the cages at 2
l/min. In all cases, the oxygen concentration of the gases to which the animals were exposed was measured with an oxygen monitor (Teledyne Analytical Instruments, Pasadena, CA). Gas outlet vents maintained normobadc conditions throughout the experiment. After 2 or 6 h, the rats were removed from the jars or cages and immediately decapitated so that the heads fell directly into liquid nitrogen. The frozen brains were removed from the calvaria and divided in two by mid-saggital section at-15 to-20 °C in a modified cryostat microtome chamber and stored at -80 °C until biochemical analysis. Each brain half was weighed and immediately homogenized at 4 °C in 2 ml of 0.15 M potassium chloride containing 5 mM/~-mercaptoethanol and I mM EDTA. The homogenate was centrifuged at 16,000 g for 30 rain, and 50 #1 of the supernatant was used to assay for whole brain glutamine synthetase activity by the hydroxylamine method of Meister ~, In preparation for the determination of glutamate content, a 10/d aliquot of the supernatant was deproteinized with 100/~! of 35% sulfosalicylic acid at 4 °C. This treated sample was centrifuged, and the supernatant was assayed for glutamate by standard techniques using a Beckman Model 6300 amino acid analyzer (University of Pittsburgh Protein Sequence Facility, Pittsburgh, PA). Statistical analysis of the data involved comparison of the means for room air- and oxygen-treated animals by Student's t-test. A P value maximally 0.05 was considered to be statistically significant. As in our previously reported study 4, glutamine syn-
Correspondence: N.E Schor, Division of Child Neurology, Children's Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213, U.S.A. Fax: (I) {412) 692-5723.
343 TABLE I Glutamine synthetase activity and glutamate content of the brains of neonatal rats exposed to room air or 100% oxygen for 2 h
Neonatal rat pups were isolated from their mothers and exposed to room air (n = 7) or 100% oxygen (n = 7) for 2 h, or maintained with their mothers and exposed to room air (n = 15) or 100% oxygen (n = 13) for 6 h. Glutamine synthetase activity was determined on whole brain by the method of Meister3. Glutamate content was determined by amino acid analysis. Values are expressed as the mean ± S.E.M. The values for glutamine synthetase activity differ with a P maximally 0.02 (Student's t test) for the 2 h exposure, and with a P maximally 0.05 for the 6 h exposure.
Glutamine synthetase at 2 h (U/g wet tissue weight) Glutamate at 2 h ~mol/g wet tissue weight) Glutamine synthetase at 6 h (Ulg wet tissue weight) Glutamate at 6 h (,umol/g wet tissue weight) Glutamine at 6 h (~mol/g wet tissue weight)
Room air
100% oxygen
1.15 - 0.19
0.66 - 0.19
1.46 +- 0.11
1.46 - 0.16
0.68 ± 0.15
0.28 ± 0.15
0.82 ± 0.05
0,85 ± 0.05
0.76 ± 0.05
0.88 ± 0.08
thetase levels were two-fold lower in the brains of animals isolated from their mothers and treated with 100% oxygen for 2 h, ~han in animals similarly isolated and treated with room air for the same length of time. Despite the reduction in glutamine synthetase activity, there was no difference between control and hyperoxic animals with respect to the glutamate content of their brains (see Table I). This was true whether the values were normalized to wet tissue weight (shown) or to glycine content (data not shown), making it unlikely that this
1 Ahdab-Barmada, M., Moossy, J., Nemoto, E.M. and Lin, M.R., Hyperoxia produces neuronal necrosis in the rat, ./. Neuropath. Exp. Neurol., 45 (1986) 233-246.
2 Ahdab-Barmada, M., Moossy, J. and Painter, M.J., Pontosubicular necrosis and hyperoxemia, Pediatrics, 66 (1980) 840-847.
effect is due solely to edema formation in hyperoxic animals. Preliminary studies (Ahdab-Barmada, Nemoto, and Schor, unpublished results) indicated that isolation of neonatal animals from their mothers for more than 2 h resulted in severe dehydration. Even over a 2 h period, brain giycine concentration, used here as a marker for hydration status, progressively increased up to two-fold in control animals exposed to room air. For this reason, animals studied for 6 h were maintained in the presence of the mother. This restored the brain glycine concentration to its native value, approximately two-fold lower than that seen in isolated animals. This discrepancy is therefore assumed to be dilutional in nature. In agreement with this is our finding that control glutamine synthetase and glutamate levels are approximately two-fold higher in the isolated control animals (room air, 2 h, Table I) than in control animals maintained in the presence of the mother (room air, 6 h, Table I). As was the case at 2 h, animals exposed to 100% oxygen for 6 h exhibited a two-fold reduction in glutamine synthetase activity relative to animals similarly exposed to room air. Despite this, there was no difference in the contents of glutamate or glutamine between the two groups. The present study shows that the decrement in glutamine synthetase activity seen after a 2 or 6 h exposure to 100% oxygen is not accompanied by an alteration in whole brain glutamate content or in its global conversion to glutamine. These studies do not preclude local changes (e.g. in the pons and subiculum - areas particularly affected by hyperoxic insults 1'2) in the concentration of glutamate, changes in the uptake of glutamate by astrocytes as a result of exposure to 100°/'o oxygen, or a role for glutamate in hyperoxic injury which is independent of the inactivation of glutamine synthetase. The authors wish to thank Yvette M. Walker-Cox and John Melick for expert technical assistance during this study. Supported by a Biomedical Research Studies Grant from the National Institutes of Health to Children's Hospital of Pittsburgh.
3 Meister, A., Giutamine synthetase from mammalian tissues, Meth. Enzymoi., 113 (1985) 185-199. 4 Schor, N.F., Inactivation of mammalian brain glutamine synthetase by oxygen radicals, Brain Research, 456 (1987) 17-21.
