amino acid metabolism and ammonia formation in brain slices
TRANSCRIPT
Journal of Neurochemistry, 1971, Vol. 18, pp. 1659 to 1672. Pergamon Press. Printed in Great Britain.
AMINO ACID METABOLISM A N D AMMONIA FORMATION IN BRAIN SLICES
H. WEIL-MALHERBE and J. GORDON Section on Neurochemistry, Laboratory of Clinical Psychopharmacology, Division of
Special Mental Health Research, IRP, MH, National Institute of Mental Health, St. Elizabeth's Hospital, Washington, D.C. 20032
(Received 30 November 1970. Accepted 5 February 1971)
Abstract-The formation of ammonia and changes in the contents of free amino acids have been investigated in slices of guinea pig cerebral cortex incubated under the following condi- tions: (1) aerobically in glucose-free saline; (2) aerobically in glucose-free saline containing 10 mM-bromofuroic acid, an inhibitor of glutamate dehydrogenase. (EC 1.4.1.2); (3) aero- bically in saline containing 11.1 mM-glucose and (4) anaerobically in glucose-free saline. Ammonia was formed at a steady rate aerobically in glucose-free medium. The formation of ammonia was largely suppressed in the absence of oxygen or in the presence of glucose whereas the inhibitor of glutamate dehydrogenase produced about 50 per cent inhibition. Other inhibitors of glutamate dehydrogenase exerted a similar effect. Ammonia formation was also inhibited by some inhibitors of aminotransferases but not by others. Inhibition was generally more pronounced during the second and third hour of incubation.
With the exception of glutamine which decreased slightly, the contents of all amino acids increased markedly during the anaerobic incubation. During aerobic incubation in a glucose- free medium, there was an almost complete disappearance of glutamic acid and GABA. Glutamine also decreased, but to a relatively smaller extent. The content of all other amino acids increased during aerobic incubation in glucose-free medium, although to a lesser extent than under anaerobic conditions. The greater increase of amino acids appearing anaerobically in comparison to the increase or decrease occurring under aerobic conditions corresponded closely to the greater amount of ammonia formed aerobically over that formed anaerobically. This finding is interpreted as indicating a similar degree of proteolysis under anaerobic and aerobic conditions; aerobically, the amino acids are partly metabolized with the concomitant liberation of ammonia.
In glucose-supplemented medium, the content of glutamine was markedly increased. The content of glutamate and aspartate remained unchanged, whereas that of some other amino acids increased but to a lesser extent than in the absence of glucose. F'roteolysis in the presence of glucose was estimated at about 65 per cent of that in its absence. In the presence of bromofuroate the rate of disappearance of glutamate was unchanged, but there was a larger increase in the content of aspartate and a smaller decrease of GABA and glutamine. Other changes did not differ significantly from those observed in the absence of bromo- furoate. We conclude that the metabolism of amino acids in general and of glutamic acid in particular differs according to whether they are already present within the brain slice or are added to the incubation medium. Only the endogenous amino acids appear to be able to serve as precursors of ammonia and as substrates for energy production.
IT IS now nearly 50 years since TASHIRO (1922) reported that nervous activity is associated with the appearance of ammonia. Yet in spite of this long history, relatively little is known about the origins of the ammonia or the mechanisms of its formation. Brain tissue forms ammonia both in vivo and in vitro but it is uncertain if the process in vitro can serve as a model of the events in vivo. Ammonia formation in brain slices is fundamentally different from that in a brain homogenate, particularly when homo- genates are prepared in a hypotonic medium (WEIL-MALHERBE and GREEN, 1955). In the latter case, the initial rate of formation is rapid, but the reaction very soon comes to a virtual standstill; moveover, the amount of ammonia formed is about the same under anaerobic as under aerobic conditions. In brain slices, on the other hand, the
1659
1660 H. WEIL-MALHERBE and J. GORDON
formation of ammonia not only continues at a fairly steady rate for several hours, but is dependent on the presence of oxygen and is inhibited by respiratory poisons and uncoupling agents. The importance of an intact cellular structure and of a functional electron transport system suggests that the process in slices is closer to the process in vivo than that in homogenates.
The formation of ammonia by brain slices is almost completely suppressed not only in the absence of oxygen but also by the addition of glucose to the incubation medium (LOEBEL, 1925). Thus, neuronal cells, which are known to utilize glucose preferentially, apparently must resort, in its absence, to the utilization of endogenous nitrogenous substrates. Since brain contains glutamate dehydrogenase [L-glutamate : NAD oxidoreductase (deaminating); EC 1.4.1.21 and an unusually high level of free glutamic acid, glutamic acid might represent the source of cerebral ammonia forma- tion. TAKAGAKI, HIRANO and TSUKADA (1957) have indeed shown that the level of glutamic acid in brain slices decreased at a rate related to the formation of ammonia; however, the decrease of glutamic acid corresponded to only about 50 per cent of the ammonia formed.
Two objections may be raised to the hypothesis that glutamic acid is the immediate precursor of cerebral ammonia. First, the addition of glutamate to the incubation medium not only fails to promote an increased formation of ammonia by brain slices, but actually diminishes ammonia formation as a result of the simultaneous conversion to amide nitrogen (KREBS, 1935; WEIL-MALHERBE, 1936). Secondly, the reaction catalysed by glutamate dehydrogenase is reversible WON EULER, ADLER, GUNTHER and DAS, 1938) with its equilibrium strongly in favour of reductive amination (STRECKER, 1953). Hence, any accumulation of ammonia would be expected to block the forward reaction and enhance the potential for reductive amination. Several authors (BORST and SLATER, 1960; KREBS and BELLAMY, 1960; BORST, 1962; JONES and GUTFREUND, 1962; DEHAAN, TAGER and SLATER, 1967) have in fact proposed that the first step in glutamate metabolism is a transamination and thus does not involve the liberation of ammonia. In view of these uncertainties and paradoxes we decided to investigate the changes in content of the whole range of acid-soluble amino acids under those conditions which are known to affect the rate of ammonia formation in brain slices. Preliminary accounts of this work have been given (WEIL-MALHERBE, 1969a; GORDON and WEIL-MALHERBE, 1970).
M E T H O D S
Slices of guinea pig cerebral cortex were cut with a STADIE-RIGGS (1944) slicer. The slices were floated off the blade and stored at room temperature in Ringer solution (‘bicarbonate-poor’ Krebs- Ringer solution containing 4.6 m-NaHC03). Requisite quantities were blotted free of adhering moisture and weighed on a torsion balance. Batches of 100-1 50 mg per flask were used in experiments involving only the measurement of ammonia, whereas about twice this amount was used in those experiments in which amino acids were to be analysed. The slices were transferred to 25-ml conical flasks containing 3 ml of bicarbonate saline (KREBS and HENSELEIT, 1932). The flasks were closed with rubber stoppers provided with gas inlet and outlet tubes and gassed for 10 min with 0, or N, contain- ing 5 % COz, while being shaken at 37°C in a Dubnoff metabolic incubator. For anaerobic experi- ments the rubber stoppers also carried a glass cup (1.5 x 0.9 cm) sealed to the end of a glass rod (3.5 cm Long, 2 mm diameter); these cups were charged with 0.1 ml water and a suitably-sized piece of yellow phosphorus from which the surface layer of oxides had been removed mechanically.
At the end of the incubation period perchloric acid was added to a final concentration of 0.4 N when only ammonia was to be determined, whereas 25 mg of picric acid per flask were added in experiments destined for amino acid analyses. The slices and medium were then homogenized to-
Amino acids and ammonia in brain slices 1661
gether and the mixture was centrifuged, Portions (0.3-0.5 ml) for ammonia estimations were taken directly from the supernatant fluid of the perchloric acid precipitate. The picric acid precipitate was washed twice with 1 ml of saturated picric acid solution. The combined supernatant fluids were treated with about 4 g of Dowex 2, XB (C1- form), sufficient to discharge the yellow colour. The resin was removed by filtration and thoroughly washed with water. Filtrate and washings were evaporated to dryness in uacuo and the residue was dissolved in 5 ml of 0 2 M-citrate buffer (pH 2.2).
In each experiment an initial value was established by placing a batch of unincubated slices into bicarbonate saline previously mixed with deproteinizing agent. Ammonia was estimated after micro- diffusion in Seligson bottles, followed by a phenol-hypochlorite reaction (for details see WEIL- MALHERBE, 19696). Under the conditions used, less than I per cent of glutamine was hydrolysed during the estimation. Amino acid analyses were carried out in a Beckman Model 116 Amino Acid Analyser according to the manufacturer’s manual (Spinco Division, Beckman Instrument Inc., Palo Alto, Calif.).
A 0.01 M GABA 0 0.01 M L-ALANlNE A 0.01M L-GLUTAMATE 0 GLUTAMATE + GABA V GLUTAMATE +ALANINE
I hr 3hr
FIG. 1 .-Ammonia formation by slices of guinea pig cerebral cortex in the presence of various exogenous substrates as a function of time of incubation. Means f S.E.M. of three experiments are plotted. Slices were incubated aerobically in bicarbonate saline at
37°C.
RESULTS
The effect of added L-glutamate on the ammonia formation by brain slices. The addition of 0.01 M L-glutamate to the suspension medium inhibited the formation of ammonia by slices of guinea pig brain by approximately 50 per cent (Fig. l), confirm- ing earlier results (WEIL-MALHERBE, 1936). Experiments were also carried out (Fig. 1) in which GABA or alanine was added, in the presence or in the absence of L-glutamate. These amino acids were chosen since their transamination with a-ketoglutarate might be expected to lower the steady state concentration of the latter and thereby allow the forward reaction of glutamate dehydrogenase to proceed, with a concomitant increase in ammonia formation. BUNIATIAN (1963) has indeed claimed that the addition of GABA, in the absence of glucose, increased the metabolism of glutamic acid and the formation of ammonia by slices of cat cerebral cortex, although he
1662 H. WEIL-MALHERBE and J. GORDON
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envisaged a different mechanism of action. The opposite effect, an inhibition of ammonia formation after the addition of a-ketoglutarate to brain slices, has previously been demonstrated (WEIL-MALHERBE, 1936). However, the addition of GABA or alanine had no effect on the formation of ammonia by slices of guinea pig brain or on the inhibitory effect of added glutamate (Fig. 1).
Changes in contents of amino aci&. In our experiments, brain slices were incubated for 3 h under the following conditions: (1) aerobically in a substrate-free medium, (2) anaerobically in a substrate-free medium, (3) aerobically in a substrate-free medium containing 0-01 M-bromofuroate, an inhibitor of glutamate dehydrogenase (CAUGHEY, SMILEY and HELLERMAN, 1957) and (4) aerobically in a medium containing 11.1 mM-glucose. The amounts of ammonia formed in these experiments are summarized in Fig. 2. The almost complete suppression of ammonia formation in a glucose- containing medium or under anaerobic conditions was clearly apparent. In the presence of bromofuroate, ammonia formation was inhibited by about 50 per cent. The contents of amino acids found initially in slices of guinea pig brain and the changes occurring during their incubation were examined (Table 1). The initial levels were only about 30-50 per cent of the values reported for the quick-frozen brain of the rat (KNAUFF and BOCK, 1961 ; MANDEL and MARK, 1965) or the cat (TALLAN, MOORE and STEIN, 1954; BATTISTIN, GRYNBAUM and LAJTHA, 1969). This difference may reflect partly metabolism, partly losses by diffusion during the initial storage of the slices, and partly the rapid swelling of brain slices in saline (PAPPIUS and ELLIOTT, 1956; VARON and MCILWAIN, 1961 ; BOURKE and TOWER, 1966; BOURKE, 1969). Of these three factors swelling is probably the most important quantitatively, although
-
-
02 N2 02 0.01 M 0.2OIo GLUCOSE
9 GLUCOSE BROMOFURO ABSENT ATE
T
FIG. 2.-Ammonia formation by slices of guinea pig cerebral cortex in the presence or absence of glucose under aerobic (0,) or anaerobic (N2) conditions. The bars denote means S.E.M. after 3 h incubation for the following numbers of experiments: aerobic, glucose-free (n = 54); aerobic, glucose-free, 0.01 M-bromofuroate added (n = 10); anaerobic, glucose-free (n = 8); aerobic, 11.1 mM-glUCOW present (n = 11). For details
of conditions of incubation see. Methods.
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1664 H. WEIL-MALHERBE and J. GORDON
rapid post mortem decarboxylation of glutamic acid (LOVELL, ELLIOTT and ELLIOTT, 1963) may account for the fact that the concentration of GABA was maintained at a near normal level, whereas the low levels of taurine and the virtual absence of urea may be attributed to rapid diffusion.
The changes in levels of amino acids during a 3-h incubation of brain slices under different conditions are summarized graphically in Fig. 3, for those changes which
1.4r N2 4 Br-F GLUCOSE I TYR
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FIG. 3 . 4 a n g e s in levels of free amino acids during 3-h incubation of slices of guinea pig cerebral cortex. The columns represent (from left to right) results after 3 h of incu- bation: anaerobically (Nz) in glucose-free medium; aerobically (0,) in glucose-free medium; aerobically in the presence of 0.01 M-bromofuroate (Br-F, no glucose); and aerobically in the presence of 11.1 mM-glucose. The height of the blocks above 0 denotes increases in content and below 0 decreases in content. The blocks are arranged in the order of their height. Consult Table 1 for numbers of experiments and standard errors.
were statistically significant ( P S 0.05). For other changes, S.E.M. and numbers of observations, reference should be made to Table 1. During anaerobic incubation the only decrease observed was a slight decrease of glutamine content. The contents of all other amino acids increased to varying extents. During aerobic incubation in a glucose-free medium, there was a large and almost complete disappearance of glutamic acid and GABA. The level of glutamine also decreased although to a relatively lesser extent. The contents of the other amino acids increased, but these increases were
Amino acids and ammonia in brain slices 1665
smaller than those observed in the anaerobic experiments. The addition of 0.01 M-bromofuroate did not alter the disappearance of glutamic acid. However, there was a significantly larger rise in the level of aspartic acid and a significantly smaller disappearance of glutamine and GABA than in the absence of the inhibitor. The inhibitor did not significantly affect the changes in content of other amino acids. When brain slices were incubated in the presence of glucose, glutamic acid was maintained at its initial level and the disappearance of GABA was greatly reduced. The most striking change was the marked increase in the level of glutamine. Other amino acids also increased but generally to a lesser extent than in the absence of glucose. Aspartic acid and proline, like glutamic acid, did not exhibit significant changes of content.
The effect of inhibitors of glutamate dehydrogenase (EC 1.4.1.2) and of amino- transferases (EC 2.6.1) on the formation of ammonia by brain slices. For reasons fully discussed below, our results have led us to conclude that endogenous glutamic acid was indeed a major source of ammonia formed during the incubation of brain slices and that other endogenous amino acids also contributed, after transamination with
z N
h
FIG. 4.-Effect of inhibitors of glutamate dehydrogenase on the formation of ammonia by slices of guinea pig cerebral cortex. The slices were incubated aerobically in glucose- free medium. Open bars: first hour; shaded bars: second plus third hours. The results (means f s.E.M.) are expressed as per cent of controls for the numbers of experiments
given in brackets. See text for details.
1666 H. WEIL-MALHERBE and J. GORDON
a-ketoglutarate. The terminal enzyme responsible for the evolution of ammonia in both cases was, therefore, glutamate dehydrogenase. This conclusion was supported by the effects of inhibitors of glutamate dehydrogenase and aminotransferases on the formation of ammonia by brain slices. Of the inhibitors of glutamate dehydrogenase (Fig. 4), D-glutamic acid, glutaric acid, a-bromofuroic acid and tri-iodothyronine inhibit the activity of the enzyme in beef liver homogenates (CAUGHEY et al., 1957); the inhibitory effect of D-glutamic acid on the formation of ammonia by brain slices has been demonstrated by TAKAGAKI, HIRANO and NAGATA (1959). In view of the
I r n
I P
5) (3)
't; d cu z
!J
FIG. 5.-Effect of inhibitors of aminotransferases on the formation of ammonia by slices of guinea pig cerebral cortex. The conditions and method of depicting data are the
same as described in the legend to Fig. 4.
effect of D-glutamic acid we examined a series of other D-amino acids. The following, at 0.01 M, had no effect on the formation of ammonia by brain slices: D-alanine- D-leucine, D-valine, D-norvaline, D-serine, D-threonine, D-methionine, D-lysine, D- arginine, D-histidine, D-phenylalanine, D-tyrosine (sat. sol.), D-3 : 4-dihydroxyphenyl, alanine (sat. sol.), D-tryptophan and D-asparagine. The only D-amino acids with an inhibitory effect on ammonia formation were D-aspartic acid, D-glutamine and D- cysteine. In the case of D-cysteine the inhibitory effect was not stereospecific since L- cysteine was equally inhibitory (Fig. 5), but the effect was probably attributable to the capacity of cysteine and similar SH-compounds to form stable cyclic condensation products (thiazolidines) with pyridoxal phosphate and thereby to act as inhibitors of
Amino acids and ammonia in brain slices 1667
aminotransferases (BRAUNSTEIN, 1960). On the other hand, the inhibitory effect of D-aspartic acid and D-glutamine was strictly stereospecific. The inhibitory effect of D-aspartic acid and D-glutamic acid could be reversed by the addition of L-aspartic acid (Table 2), and we assumed therefore that D-aspartic acid like D-glutamic acid acted by inhibiting glutamate dehydrogenase. Like most other L-amino acids examined, L-aspartic acid alone did not significantly alter the formation of ammonia by brain slices. L-Aspartic acid, however, did not seem to affect the inhibition produced by D-glutamine (Table 2).
TABLE 2.-EFFECTS OF SOME D-AMINO AClDS ON THE FORMATION OF AMMONIA BY SLICES OF GUINEA PIG CEREBRAL CORTEX : REVERSAL OF INHIBITION BY L-ASPARTATE
Additions Number of experiments
Ammonia formation (% of control) First hour Second & third hours
0.01 M-D-ASpartate
5 mM-D-ASpartate + 5 mM-L-aspartate 0.01 M-D-Aspartate + 0.01 M-L-aspartate 0.01 M-D-Aspartate + 0.02 M-L-aspartate 0.01 M-D-Glutamate 5 mM-D-Glutamate 5 mM-D-Glutamate + 0.01 M-L-aspartate 5 mM-D-Glutamate + 0.01 M-L-alanine 0.01 M-D-Ghtamine 0.01 M-D-Glutamine + 0.01 M-L-aspartate
3 mM-D-Aspartate 4 2 2 2 2
1 3 2 2 2 6 2
94 f 11.2 loo, 95 113, 101 111,99 105, 91 68 & 4.7 70, 52 114, 96 74, 62 88 i: 8.8 115, 81
48 & 7.6 44, 24 98, 86 103,95 98, 88 37 f 6.7 32,22 112, 86 45, 35 22 i 8.0 64, 10
Slices were incubated aerobically in glucose-free medium for the times specified. Values are individual values for two experiments or mean k S.E.M. for the numbers of experiments tabulated. See footnotes to Table 1 and text for further details.
The effects of a series of inhibitors of aminotransferases (Fig. 5) were more variable than the effects of glutamate dehydrogenase inhibitors and were insignificant in some cases. With both types of inhibitors the effects were usually more marked during the second and third hours of incubation. In view of the possibility that proteo- lysis is linked energetically to protein synthesis in tissue slices (WALTER, 1960), we studied the effect on the formation of ammonia by brain slices of a series of anti- biotics known to be inhibitors of protein synthesis. The following (at concentrations of 1 mM or 0.1 mM) were without effect: puromycin, cycloheximide, chloramphenicol, tetracycline, antimycin A and actinomycin D. Puromycin (40 mg/kg) was also ineffective when injected into rats at 2-4 h before death. Those antibiotics known to be inhibitors of electron transport, i.e. oligomycin and rotenone, exerted some inhi- bitory effect, in agreement with the inhibitory effect of other respiratory poisons (WEIL-MALHERFIE and Green, 1955).
DISCUSSION
What are the implications of these observations insofar as the origin of the ammonia formed by brain slices is concerned? The increases in the levels of free amino acids during the incubation of brain slices presumably indicate proteolysis, and this interpretation is confirmed by the fact that ninhydrin-positive substances which
1668 H. WEIL-MALHEREIE and J. GORDON
are not constituents of protein, such as taurine and phosphoethanolamine, did not exhibit significant changes of content in any of our experiments. For purposes of discussion we have assumed that, in the absence of glucose, proteolysis in brain slices is the same aerobically and anaerobically. Proteolysis is, however, somewhat reduced in the presence of glucose. It has previously been demonstrated that brain slices form less acid-soluble amino nitrogen in the presence than in the absence of glucose, a finding also pointing to a reduction of proteolysis in the presence of glucose (WEIL-MALHERBE and GREEN, 1955). Such an effect of glucose may reflect, wholly or partly, a resynthesis of protein which can occur in brain slices incubated in a glucose- supplemented medium (ORREGO and LIPMANN, 1967). In the absence of glucose, the synthesis of protein, like other synthetic activities, is presumably greatly reduced or completely abolished.
If protein synthesis is excluded, then the fact that, in the absence of glucose, the increase of amino acids is smaller under aerobic than under anaerobic conditions must reflect increased metabolism of amino acids in the presence of oxygen. It is probable that this metabolism proceeds by transamination with a-ketoglutarate and subsequent oxidation by glutamate dehydrogenase of the glutamate formed. In Fig. 6 is represented a balance sheet in which the excess of ammonia formed aerobically over that formed anaerobically has been compared with the excess of amino acids formed anaerobically over those formed aerobically. The correspondence is remark- ably close and provides strong support for the validity of our assumption. About one- half of the ammonia formed is apparently derived from glutamic acid and GABA. Not all the amino acids in brain slices contributed to ammonia formation: histidine, valine, tyrosine and methionine increased as much in the presence of oxygen as in nitrogen and thus were presumably not metabolized. The box on top of the right-hand column (dashed lines, Fig. 6) takes account of the fact that one molecule of glutamine can release two molecules of ammonia. On the other hand, it is unlikely that more than one molecule of ammonia would be formed from the lysine or arginine in brain slices.
When brain slices are respiring in glucose-supplemented medium there is a marked increase in the level of glutamine, as previously noted (WEIL-MALHERBE, 1936; WEIL-MALHE~RBE and GREEN, 1955). The synthesis of glutamine represents a mechan- ism for the binding of free ammonium ions and is largely responsible for the absence of a significant formation of ammonia in the presence of glucose. However, the increase in the level of glutamine indicates that considerable amounts of endogenous amino acids are metabolized by brain slices even when glucose is available. Since two molecules of ammonia are required for the synthesis of one molecule of glutamine, the apparent proteolysis in brain slices maintained in a glucose medium can be estimated at about 65 per cent of the proteolysis occurring in the absence of glucose. Whether the remainder is accounted for partly or entirely by the simultaneous syn- thesis of protein cannot be decided on present evidence. If glutamate dehydrogenase were the terminal enzyme for the formation of the major part of ammonia in brain slices, one would expect the addition of an inhibitor of glutamate dehydrogenase to produce changes intermediate between those observed aerobically and those observed anaerobically when the oxidative activity of the enzyme is fully inhibited. In our experiments with bromofuroic acid, this expectation has been realized insofar as the formation of ammonia is concerned; ammonia formation was inhibited by about
Amino acids and ammonia in brain slices 1669
1.2-
1.1
ID
0.9
Q8
0.7 0 0 > 0.6- a E 3. Q5-
a4
03
02-
ai
0-
50 per cent over a 3-h period. However, contrary to expectation, the inhibitor had little effect on the aerobic metabolism of most endogenous amino acids. In particular, it did not affect the rate of disappearance of glutamic acid. On the other hand, the significant changes produced by the inhibitor on the levels of aspartate, GABA and
- - - -
-
-
-
-
0" z z g m I z
PHENYULANINE ARGlNlNE
. . .. . . . . - , GWTAMINE
GLYCINE I AWRTC ACID I SERINE I LEUCINE I ALANINE I I
PROLINE
I1 GUITAMIC ACI A
FIG. 6.-Comparison of the formation of ammonia with the utilization of amino acids during the incubation of slices of guinea pig cerebral cortex. The f o l d height of the bar at the left represents the mean of formation of ammonia aerobically in glucose-free medium; the top section of the column denotes the ammonia formed under anaerobic, glucose-free conditions and the bottom section of the column represents the difference between the two. The bar at the right is composed of blocks each of which represents the difference between the increase in level of the respective amino acids under anaero- bic, glucose-free conditions and the increase or decrease aerobically. The top block enclosed by the dashed line represents the second amino group of glutamine. See text
for discussion.
glutamine suggest that glutamate was partly converted into these substances and was therefore partly metabolized through channels other than glutamate dehydrogenase. No satisfactory explanation can be offered at present why we did not find the expected increase in the levels of other amino acids. However, the interpretation of the effect of inhibitors is not always straightforward, especially in a heterogeneous system, since they rarely have a single point of action.
1670 H. WEIL-MALXERBE and J. GORDON
We must also consider how our proposed mechanism can be reconciled with the two objections raised in the introduction to this paper. There are obvious differences between the metabolism of glutamate added to the medium and the metabolism of endogenous glutamate. Whereas the oxidation of added glutamate is apparently limited by the simultaneous synthesis of glutamine (WEIL-MALHERBE, 1962) and therefore proceeds without the liberation of free ammonia, the oxidation of endo- genous glutamate does not appear to be limited in this way. In unpublished experi- ments we have found that over a 3 h incubation period the disappearance of endo- genous glutamate from brain slices follows a smooth curve which, at any point, accounts for 20-30 per cent of the ammonia formed. Thus, there was no sign of any increasing inhibition with the increasing concentrations of ammonia. We may assume that added glutamic acid does not mix readily with the endogenous pool of glutamic acid. The compartmentation of brain glutamic acid into at least two pools has been demonstrated conclusively (BERL, LAJTHA and WAELSCH, 1961 ; BERL, TAKAGAKI, CLARKE and WAELSCH, 1962, a,b; O’NEAL and KOEPPE, 1966). According to current concepts, a small pool of glutamic acid located mainly in the neuropil is more readily accessible to intermediates of the tricarboxylic acid cycle and is more active in the synthesis of glutamine than a large pool associated with neurons which is more closely associated with glucose and intermediates of glycolysis (O’NEAL and KOEPPE, 1966; GARFINKEL, 1966; VAN DEN BERG, KRZALIC, MELA and WAELSCH, 1969; NICKLAS, CLARKE and BERL, 1969; ROSE, 1970). We suggest further that added (exogenous) glutamic acid is metabolized mainly in the ‘small pool’, whereas the bulk of endogenous glutamic acid which is a precursor of free ammonia is associated with the ‘large pool’. Other amino acids must exhibit a similar compartmentation; other- wise it would be difficult to understand why the addition of L-amino acids (other than L-glutamate) to brain slices fails to augment oxygen uptake (WEIL-MALHERBE, 1936) and also fails to increase the formation of ammonia. On the other hand, in the present study we found that endogenous amino acids are metabolized in a reaction consuming oxygen and liberating ammonia.
One might also object that the accumulation of ammonia would be expected to stop the forward reaction of glutamate dehydrogenase and reverse it in favour of reductive amination. This is undoubtedly what happens in the presence of glucose. In the absence of glucose, as endogenous substrates are depleted, there will be an increase of the NAD+/NADH ratio and a decrease of the ATP/ADP ratio. Both these changes would favour the forward reaction of glutamate dehydrogenase, perhaps sufficiently to compensate for the increase of ammonia levels. Moreover, it is difficult to apply conclusions based on reactions occurring in homogeneous solutions to the much more complicated cellular systems. The actual concentrations of reactants in the microenvironment of the enzymic site within the cell may be quite different from those expected in a closed system at or near equilibrium. The ammonium ion which is liberated from the metabolism of endogenous substrates diffuses rapidly out of the brain slice and may in fact be actively removed from the reaction mixture. Doubts have previously been expressed regarding the utilization by brain of glutamic acid as a fuel (WEIL-MALHERBE, 1950). The conclusion which emerges from our present experiments is that glutamic acid, GABA, and, to a lesser extent, many other amino acids are indeed utilized as substrates in the absence of glucose. This conclusion is consistent with many previous observations demonstrating a decrease in the brain
Amino acids and ammonia in brain slices 1671
levels of glutamate in animals rendered hypoglycaemic with insulin (DAWSON, 1950; CRAVIOTO, MASSIEU and IZQUIERDO, 1951 ; JACOBSON, 1959; DE ROPP and SNEDEKER, 1961; KNAUFF and BOCK, 1961; FLOCK, TYCE and OWEN, 1969). It is possible that glutamate and other amino acids also serve as energy-yielding substrates in those states of increased nervous activity in vivo which are known to be associated with an increased production of ammonia. Under these conditions the energy demands of the nerve cell may exceed the limits which can be satisfied by the metabolism of glucose, and auxiliary sources of energy may be called upon.
Acknowledgement-We are grateful to Dr. E. COSTA for permitting us to use the Beckman Amino Acid Analyser in his laboratory.
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