528
BIOCHEMICAL SOCIETY TRANSACTIONS
of the label in glutamine in the NIUI-treated animals (Fig. 2). From these two experiments we conclude that the primary action of ammonia on the metabolism of glutamate and glutamine in brain is an inhibition of the rate of degradation of glutamine and not an enhancement of the rate of synthesis. From the data obtained after acute injectionof NH4CI into rats (Hawkins et al., 1973), it has been concluded that there is a rapid increase in the rate of formation of glutamine from glucose; in unpublished experiments we found no change in the rate of incorporation of 8-hydroxybutyrate into brain amino acids after NH4Cl loading over the same time-period. Differences between the results of Hawkins et al. (1973) and those of ourselves could be due to species variation, but the mechanism shown to exist by us could be present in rats under different experimental conditions. It is to be noted that from 11 to 16min after injection of NH4Cl there is a definite increase in the total amino nitrogen, which is indicative of the occurrence of the mechanism postulated by Hawkins et al. (1973). The dose chosen by us did not produce spontaneous convulsions, but after 5min there were very clear signs of excitability and other neurological symptoms, such as an extreme susceptibility to sound. Cheng, S-C. & Mela, P. (1966)J. Neurochem. 13,281-287 Cremer, J. E. & Lucas, H. M. (1971) Brain Res. 35,619-621 Hawkins, R. A., Miller, A. L., Nielson, R. C. & Veech, R. (1973) Biochem.J. 134,1001-1008 Krebs, H. (1935) Biochem.J. 29,1951-1969 O”ea1, R. M. & Koeppe, R. E. (1966)J. Neurochem. 13,835-847 Richter, D. & Dawson, R. M. C. (1948)J. Biol. Chem. 176,1189-1198 Van den Berg, C. J. & Garfinkel, D. (1971) Biochem.J. 123,211-218 Van den Berg, C. J., Krzalic, Lj., Mela, P. & Waelsch, H. (1969) Biochem. J. 113, 281-290 Weil-Malherbe, H. (1950)Physiol. Rev. 30,549-568 Weil-Malherbe, H. (1974) Mol. Cell. Biochem. 4, 31-44
The Formation of Glutamine in Mouse Brain: Effect of Amino-oxyacetic Acid and Ammonia C. J. VAN D E N BERG and D. F. MATHESON Study Group: Inborn Errors and Brain, Department of Biological Psychiatry, Psychiatric University Clinic, Oostersingel59, Groningen, The Netherlands
In the foregoing paper (Matheson &Van den Berg, 1975) we have provided evidence for the hypothesis that the effect of acute ammonia intoxication is initially to inhibit the degradation of the glutamine labelled by acetate. It has been previously postulated from an analysis of data on the incorporation of labelled glucose and acetate into brain amino acids that the degradation of y-aminobutyric acid was coupled with the formation of glutamine(Vanden Berg & Garfinkel, 1971).As amino-oxyacetic acid has been shown to inhibit transaminases, particularly y-aminobutyrate transaminase (EC 2.6.1.19) (Van Gelder, 1966; Rognstad & Katz, 1970), we decided to investigate the effect of amino-oxyacetic acid, withand without ammonia, on theconcentrations and thelabelling by radioactive acetate of glutamate and glutamine in adult mouse brain. In addition we wanted to see if the relative contributions of glutamatea-oxoglutarate transaminase and glutamate dehydrogenase activities to the labelling could be discerned. It has been shown already that amino-oxyacetic acid in oitro decreased the labelling of glutamate and glutamine by various labelled precursors (Haber, 1965; Berl et al., 1970). Amino-oxyacetic acid, administered at a dose of both 20 and 80mgIkg because y-aminobutyrate transaminase is fully inhibited at 20mg/kg (Van Gelder, 1966), was found to lower the glutamine concentration, whereas the glutamate concentration was only lowered at the higher dose (Table 1). The incorporation of acetate in the 5min period into glutamate was not decreased, whereas that into glutamine was decreased by about 50% 1h after injection of the amino-oxyacetic acid at both doses (Table 2). The 1975
556th MEETING, LONDON
529
Table 1. Concentrations of glutamate and glutamine in brain of adult mice treated with amino-oxyacetic acid, with and without NH4Cl Adult mice(24-26g) were injected intraperitoneally with amino-oxyaceticacid (in 0.1 ml of neutralized 0.9% NaCl), and N K C l (7mmol/kg) was injected intraperitoneally 1h later. The animals were killed 1h 6min after the injection of the amino-oxyacetic acid by immersion in liquid N2. Glutamate and glutamine were extracted by the method of Van den Berg et al. (1969) and the concentrations were determined by the method of Cheng & Mela (1966). Each determination was performed in duplicate. The concentrations are the means ~ s . Dand . are expressed as pmol/g of brain. Numbers in parentheses are the number of animals used. Treatment Control Amino-oxyacetic acid (20mg/kg) Amino-oxyacetic acid @Omg/kg) Control+NH&l Amino-oxyacetic acid (20mg/kg)+NH4Cl Amino-oxyacetic acid (80mg/kg)+NH4C1
Glutamate
Glutamine
9.78k0.61 (11) 9.64f 1.25 (8) 7.60 k 0.09 (3) 9.06 k 0.54 (4) 9.73 k 0.15 (3) 6.22k 0.16 (3)
4.20f0.43 (10) 3.18 f 0.61 (8) 3.44k0.33 (3) 5.80f0.83 (10) 3.10k0.33 (4) 2.43 k 0.58 (4)
Table 2. Incorporation of [3H]acetateinto glutamate and glutamine in adult mice treated with amino-oxyacetic acid and with and without NH4Cl Adult mice were injected with amino-oxyacetic acid; 1h afterwards NH4Cl (7mmol/kg) was injected where stated and after 1min IOpCi of [3H]acetatewas injected (in 0.1 ml of neutralized NaCl); the animalswere killed in liquid N2 5min later. The total radioactivity of the trichloroacetic acid-soluble fraction and the total incorporation of radioactivity into glutamate and glutamine was counted by liquid-scintillationspectroscopy (adjusted to 100% efficiency) and related to an injected dose of 2OpCi/25g body weight. Values are the means k s.D.,and the numbers in parentheses are the number of animals used. RTI is the total incorporation into glutamine relative to that into glutamate. Total radioactivity Total radioactivity of acid-soluble incorporation (d.p.m./mg) into: fraction RTIof (d.p.m./mg) Glutamate Glutamine glutamine
-
/
Treatment Control Amino-oxyacetic acid (20mg/kg) Amino-oxyaceticacid (80mg/kg) Control+NH4C1 Amino-oxyaceticacid (20mg/kg)+NH4C1 Amino-oxyaceticacid (80mg/kg)+NH4C1
176.0f 48.4 (15) 141.5f47.6 (8)
31.56k 4.60 24.61 k7.40
56.02f 6.12 27.56k9.19
1.78 f 0.31 1.09k0.10
164.8f28.4 (3)
29.70k 8.14
25.29k4.62
0.90f0.20
173.0+ 6.1 (4) 204.0k 19.6 (8)
25.23f4.62 32.85k4.71
65.90k7.56 2.61 f0.36 51.75+ 10.63 1.64f0.13
225.0f 37.7 (3)
23.17k4.60
38.32f4.26
1.68f0.26
incorporation of the labelled acetate into glutamine relative to that into glutamate was therefore greatly decreased. When NH4Cl was administered 1h after amino-oxyaceticacid, there was no increase in the glutamine concentration that occurred in the NH4Cl-treated animals (Table 1). If the ammonia acts both in the control and the amino-oxyaceticacid-treated animals by VOl. 3
530
BIOCHEMICAL SOCIETY TRANSACTIONS
inhibiting the degradation of glutamine, these data could indicate that there is a large decrease in the rate of glutamine formation in the amino-oxyacetic acid-treated animals. A possible explanation for these observations could be that the y-aminobutyric acid, which is normally converted via part of the tricarboxylic acid cycle into glutamine, no longer contributes to this pathway by the inhibition of y-aminobutyrate transferase. However, as shown in Table 2, there is no change in the labelling of glutamate from acetate. Assuming that acetate and y-aminobutyric acid are largely metabolized by the same tricarboxylic acid cycle (see Van den Berg & Garfinkel, 1971) this lack of change in the glutamate is surprising. A possible way to reconcile these differences is if y-aminobutyric acid is degraded by y-aminobutyrate transferase exclusively, there is a conversion of a-oxoglutarate into glutamate. When this reaction is inhibited by amino-oxyacetic acid, there is no further formation of glutamate and the a-oxoglutarate proceeds via succinate around the tricarboxylic acid cycle. The labelling of glutamate in this tricarboxylic acid cycle takes place via glutamate dehydrogenase and/or glutamate-a-oxoglutarate transaminase. The only effect of the inhibition of y-aminobutyrate transferase is to decrease the formation of glutamate used for glutamine synthesis. The rate of incorporation of acetate into glutamine is decreased by all treatments; part of this decrease is almost certainly due to the decreased rate of formation of glutamine in the presence of amino-oxyacetic acid. As, however, there is not only formation of glutamine, but also an exchange of the label between glutamate and glutamine (Van den Berg & Garfinkel, 1971), the decrease in the amount of incorporation of the label into glutamine need not be directly related quantitatively to the decreased rate of formation. The lack of decrease of labelling of glutamate from acetate cannot be used to assign the more important role in glutamate labelling to glutamate dehydrogenase. In these and other experiments with labelled 8-hydroxybutyrate, we never foundstrikingdecreases in the labelling of glutamate and aspartate in uiuo (results not shown). We can now explain why the labelling of glutamate is not very much affected by assuming that there is still rapid labelling of glutamate, either by glutamate dehydrogenase or by glutamate-a-oxoglutarate transamination. That all transaminase enzymes are not inhibited to such an extent that they become limiting is also borne out by the observation that the labelling of aspartate is not affected (results not shown). The only pathway known by which asparate can receive the label is by a transaminase reaction. That there is some increase in the amount of the label in glutamine in animals receiving NH,CI after amino-oxyacetic acid can very well be the result of a faster exchange between glutamate and glutamine; this exchange was postulated to exist in the computer simulation study (Van den Berg and Garfinkel, 1971). One of the essential conclusions from this study is that the degradation of y-aminobutyric acid is intimately linked to the net synthesis of glutamate, which in turn is converted into glutamine. The results of these experiments are consistent with previously made postulates, but much more elaborate kinetic analysis is needed to fill in all the details. Berl, S., Clarke, D. D. & Nicklas, W. J. (1970) J. Neurochem. 17,999-1007 Cheng, S . C. & Mela, P. (1966)J. Neurochem. 13,281-287 Haber, B. (1965) Can. J. Biochem. 43,865-876 Matheson, D. F. & Van den Berg, C. J. (1975) Biochem. SOC.Trans. 3,525-528 Rognstad, R. & Katz, J. (1970) Biochem. J . 116,483491 Van den Berg, C. J. & Garfinkel, D. (1971) Biochem. J. 123,211-218 Van den Berg, C. J., Krzalic, Lj, Mela, P. & Waelsch, H. (1969) Biochem. J. 113, 281-290 Van Gelder, N. (1966) Biochem. Pharmacol. 15,533-539
1975
BIOCHEMICAL SOCIETY TRANSACTIONS
of the label in glutamine in the NIUI-treated animals (Fig. 2). From these two experiments we conclude that the primary action of ammonia on the metabolism of glutamate and glutamine in brain is an inhibition of the rate of degradation of glutamine and not an enhancement of the rate of synthesis. From the data obtained after acute injectionof NH4CI into rats (Hawkins et al., 1973), it has been concluded that there is a rapid increase in the rate of formation of glutamine from glucose; in unpublished experiments we found no change in the rate of incorporation of 8-hydroxybutyrate into brain amino acids after NH4Cl loading over the same time-period. Differences between the results of Hawkins et al. (1973) and those of ourselves could be due to species variation, but the mechanism shown to exist by us could be present in rats under different experimental conditions. It is to be noted that from 11 to 16min after injection of NH4Cl there is a definite increase in the total amino nitrogen, which is indicative of the occurrence of the mechanism postulated by Hawkins et al. (1973). The dose chosen by us did not produce spontaneous convulsions, but after 5min there were very clear signs of excitability and other neurological symptoms, such as an extreme susceptibility to sound. Cheng, S-C. & Mela, P. (1966)J. Neurochem. 13,281-287 Cremer, J. E. & Lucas, H. M. (1971) Brain Res. 35,619-621 Hawkins, R. A., Miller, A. L., Nielson, R. C. & Veech, R. (1973) Biochem.J. 134,1001-1008 Krebs, H. (1935) Biochem.J. 29,1951-1969 O”ea1, R. M. & Koeppe, R. E. (1966)J. Neurochem. 13,835-847 Richter, D. & Dawson, R. M. C. (1948)J. Biol. Chem. 176,1189-1198 Van den Berg, C. J. & Garfinkel, D. (1971) Biochem.J. 123,211-218 Van den Berg, C. J., Krzalic, Lj., Mela, P. & Waelsch, H. (1969) Biochem. J. 113, 281-290 Weil-Malherbe, H. (1950)Physiol. Rev. 30,549-568 Weil-Malherbe, H. (1974) Mol. Cell. Biochem. 4, 31-44
The Formation of Glutamine in Mouse Brain: Effect of Amino-oxyacetic Acid and Ammonia C. J. VAN D E N BERG and D. F. MATHESON Study Group: Inborn Errors and Brain, Department of Biological Psychiatry, Psychiatric University Clinic, Oostersingel59, Groningen, The Netherlands
In the foregoing paper (Matheson &Van den Berg, 1975) we have provided evidence for the hypothesis that the effect of acute ammonia intoxication is initially to inhibit the degradation of the glutamine labelled by acetate. It has been previously postulated from an analysis of data on the incorporation of labelled glucose and acetate into brain amino acids that the degradation of y-aminobutyric acid was coupled with the formation of glutamine(Vanden Berg & Garfinkel, 1971).As amino-oxyacetic acid has been shown to inhibit transaminases, particularly y-aminobutyrate transaminase (EC 2.6.1.19) (Van Gelder, 1966; Rognstad & Katz, 1970), we decided to investigate the effect of amino-oxyacetic acid, withand without ammonia, on theconcentrations and thelabelling by radioactive acetate of glutamate and glutamine in adult mouse brain. In addition we wanted to see if the relative contributions of glutamatea-oxoglutarate transaminase and glutamate dehydrogenase activities to the labelling could be discerned. It has been shown already that amino-oxyacetic acid in oitro decreased the labelling of glutamate and glutamine by various labelled precursors (Haber, 1965; Berl et al., 1970). Amino-oxyacetic acid, administered at a dose of both 20 and 80mgIkg because y-aminobutyrate transaminase is fully inhibited at 20mg/kg (Van Gelder, 1966), was found to lower the glutamine concentration, whereas the glutamate concentration was only lowered at the higher dose (Table 1). The incorporation of acetate in the 5min period into glutamate was not decreased, whereas that into glutamine was decreased by about 50% 1h after injection of the amino-oxyacetic acid at both doses (Table 2). The 1975
556th MEETING, LONDON
529
Table 1. Concentrations of glutamate and glutamine in brain of adult mice treated with amino-oxyacetic acid, with and without NH4Cl Adult mice(24-26g) were injected intraperitoneally with amino-oxyaceticacid (in 0.1 ml of neutralized 0.9% NaCl), and N K C l (7mmol/kg) was injected intraperitoneally 1h later. The animals were killed 1h 6min after the injection of the amino-oxyacetic acid by immersion in liquid N2. Glutamate and glutamine were extracted by the method of Van den Berg et al. (1969) and the concentrations were determined by the method of Cheng & Mela (1966). Each determination was performed in duplicate. The concentrations are the means ~ s . Dand . are expressed as pmol/g of brain. Numbers in parentheses are the number of animals used. Treatment Control Amino-oxyacetic acid (20mg/kg) Amino-oxyacetic acid @Omg/kg) Control+NH&l Amino-oxyacetic acid (20mg/kg)+NH4Cl Amino-oxyacetic acid (80mg/kg)+NH4C1
Glutamate
Glutamine
9.78k0.61 (11) 9.64f 1.25 (8) 7.60 k 0.09 (3) 9.06 k 0.54 (4) 9.73 k 0.15 (3) 6.22k 0.16 (3)
4.20f0.43 (10) 3.18 f 0.61 (8) 3.44k0.33 (3) 5.80f0.83 (10) 3.10k0.33 (4) 2.43 k 0.58 (4)
Table 2. Incorporation of [3H]acetateinto glutamate and glutamine in adult mice treated with amino-oxyacetic acid and with and without NH4Cl Adult mice were injected with amino-oxyacetic acid; 1h afterwards NH4Cl (7mmol/kg) was injected where stated and after 1min IOpCi of [3H]acetatewas injected (in 0.1 ml of neutralized NaCl); the animalswere killed in liquid N2 5min later. The total radioactivity of the trichloroacetic acid-soluble fraction and the total incorporation of radioactivity into glutamate and glutamine was counted by liquid-scintillationspectroscopy (adjusted to 100% efficiency) and related to an injected dose of 2OpCi/25g body weight. Values are the means k s.D.,and the numbers in parentheses are the number of animals used. RTI is the total incorporation into glutamine relative to that into glutamate. Total radioactivity Total radioactivity of acid-soluble incorporation (d.p.m./mg) into: fraction RTIof (d.p.m./mg) Glutamate Glutamine glutamine
-
/
Treatment Control Amino-oxyacetic acid (20mg/kg) Amino-oxyaceticacid (80mg/kg) Control+NH4C1 Amino-oxyaceticacid (20mg/kg)+NH4C1 Amino-oxyaceticacid (80mg/kg)+NH4C1
176.0f 48.4 (15) 141.5f47.6 (8)
31.56k 4.60 24.61 k7.40
56.02f 6.12 27.56k9.19
1.78 f 0.31 1.09k0.10
164.8f28.4 (3)
29.70k 8.14
25.29k4.62
0.90f0.20
173.0+ 6.1 (4) 204.0k 19.6 (8)
25.23f4.62 32.85k4.71
65.90k7.56 2.61 f0.36 51.75+ 10.63 1.64f0.13
225.0f 37.7 (3)
23.17k4.60
38.32f4.26
1.68f0.26
incorporation of the labelled acetate into glutamine relative to that into glutamate was therefore greatly decreased. When NH4Cl was administered 1h after amino-oxyaceticacid, there was no increase in the glutamine concentration that occurred in the NH4Cl-treated animals (Table 1). If the ammonia acts both in the control and the amino-oxyaceticacid-treated animals by VOl. 3
530
BIOCHEMICAL SOCIETY TRANSACTIONS
inhibiting the degradation of glutamine, these data could indicate that there is a large decrease in the rate of glutamine formation in the amino-oxyacetic acid-treated animals. A possible explanation for these observations could be that the y-aminobutyric acid, which is normally converted via part of the tricarboxylic acid cycle into glutamine, no longer contributes to this pathway by the inhibition of y-aminobutyrate transferase. However, as shown in Table 2, there is no change in the labelling of glutamate from acetate. Assuming that acetate and y-aminobutyric acid are largely metabolized by the same tricarboxylic acid cycle (see Van den Berg & Garfinkel, 1971) this lack of change in the glutamate is surprising. A possible way to reconcile these differences is if y-aminobutyric acid is degraded by y-aminobutyrate transferase exclusively, there is a conversion of a-oxoglutarate into glutamate. When this reaction is inhibited by amino-oxyacetic acid, there is no further formation of glutamate and the a-oxoglutarate proceeds via succinate around the tricarboxylic acid cycle. The labelling of glutamate in this tricarboxylic acid cycle takes place via glutamate dehydrogenase and/or glutamate-a-oxoglutarate transaminase. The only effect of the inhibition of y-aminobutyrate transferase is to decrease the formation of glutamate used for glutamine synthesis. The rate of incorporation of acetate into glutamine is decreased by all treatments; part of this decrease is almost certainly due to the decreased rate of formation of glutamine in the presence of amino-oxyacetic acid. As, however, there is not only formation of glutamine, but also an exchange of the label between glutamate and glutamine (Van den Berg & Garfinkel, 1971), the decrease in the amount of incorporation of the label into glutamine need not be directly related quantitatively to the decreased rate of formation. The lack of decrease of labelling of glutamate from acetate cannot be used to assign the more important role in glutamate labelling to glutamate dehydrogenase. In these and other experiments with labelled 8-hydroxybutyrate, we never foundstrikingdecreases in the labelling of glutamate and aspartate in uiuo (results not shown). We can now explain why the labelling of glutamate is not very much affected by assuming that there is still rapid labelling of glutamate, either by glutamate dehydrogenase or by glutamate-a-oxoglutarate transamination. That all transaminase enzymes are not inhibited to such an extent that they become limiting is also borne out by the observation that the labelling of aspartate is not affected (results not shown). The only pathway known by which asparate can receive the label is by a transaminase reaction. That there is some increase in the amount of the label in glutamine in animals receiving NH,CI after amino-oxyacetic acid can very well be the result of a faster exchange between glutamate and glutamine; this exchange was postulated to exist in the computer simulation study (Van den Berg and Garfinkel, 1971). One of the essential conclusions from this study is that the degradation of y-aminobutyric acid is intimately linked to the net synthesis of glutamate, which in turn is converted into glutamine. The results of these experiments are consistent with previously made postulates, but much more elaborate kinetic analysis is needed to fill in all the details. Berl, S., Clarke, D. D. & Nicklas, W. J. (1970) J. Neurochem. 17,999-1007 Cheng, S . C. & Mela, P. (1966)J. Neurochem. 13,281-287 Haber, B. (1965) Can. J. Biochem. 43,865-876 Matheson, D. F. & Van den Berg, C. J. (1975) Biochem. SOC.Trans. 3,525-528 Rognstad, R. & Katz, J. (1970) Biochem. J . 116,483491 Van den Berg, C. J. & Garfinkel, D. (1971) Biochem. J. 123,211-218 Van den Berg, C. J., Krzalic, Lj, Mela, P. & Waelsch, H. (1969) Biochem. J. 113, 281-290 Van Gelder, N. (1966) Biochem. Pharmacol. 15,533-539
1975
