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Endogenous respiration and ammonia formation in brain slices
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
66, 196-205 (1957)
Endogenous Respiration and Ammonia Formation in Brain Slices Genkichiro Takagaki, Shusuke Hirano and Yasuzo Tsukada From the Departwlent of Physiology, School of Uedicine, Keio University, Tokyo, Japan
Received July 19, 1956
INTRODUCTION Although in some cases it is possible to depress the endogenous metabolism in tissue slices by some metabolic inhibitor (1, 2), it nevertheless often persists at a fairly high level and should not be neglected. In brain cortex slices, the endogenous oxygen uptake accounts for more than half that which is observed in the presence of glucose as a substrate. Accordingly, it was considered desirable to analyze the endogenous metabolism of brain slices to determine the nature of the main substrate and to ascertain its relationship to t#he over-all metabolism in the presence of added substrate. It is well-known that ammonia is formed in large amount during the endogenous metabolism of brain slices and that it is almost completely depressed in the presence of glucose. In earlier reports (3, 4) it was pointed out that ammonia would possibly be formed from glutamic acid in brain tissue. The metabolism of glutamic acid and the formation of ammonia in living brain are physiologically significant, and suggest many interesting problems (5). This investigation was undertaken with the particular objective of analyzing the relationship between the endogenous respiration and ammonia formation in brain slices. A preliminary account of a part of this investigation was reported elsewhere (6). MATERIAW AND METHODS Experimental
Procedures in General
All animals used were guinea pigs of some 300 g. in weight. The animals were killed by decapitation, and the brain cortex slices were cut freehand with a moist 196
RESPIRATION
AND
AMMONIA
197
FORMATION
safet,y-razor blade on a refrigerated stage, and stored in ice-cold phosphate Ringer solution. Batches of SO-120 mg. in wet weight were used in 3 ml. of medium. Weighted slices with a torsion balance were introduced in the usual Warburg vessels and the vessels were attached t.o the manometers. The air in the vessels was replaced with pure oxygen by the evacuation method, and the vessels were placed in a thermostat at 37°C. It took about 30 min. from the death of the animals t.o reach this point in the procedure. Readings of manometers were taken at IO-ruin. intervals. In anaerobic experiments pure nit.rogen, freed from contaminated oxygen by exposure to yellow phosphorus, was used. Cyanide as an inhibitor was used according to the directions of Robbie (7); that is, the proper mixture of KCNKOH was placed in t.he center wells of the Warburg flasks in order to maintain the desired concentration of cyanide in the medium throughout the incubation period. The respiratory quotient was measured by the second method of Dickens and Simer (S), using the flasks possessing two side-arms, one of which was equipped with a double sack. Krebs-Ringer phosphate solution (9) at pH 7.0 was used throughout. When t.he sodium-free medium was prepared, sodium chloride was completely replaced by choline chloride, and sodium phosphate by potassium phosphate, while maintaining the correct osmotic pressure of the medium.
Chemical Estimations The specimens removed from t.he medium at the end of t,he incubation period were deproteinized with trichloroacetic acid. The clear supernatant was used for the determination of free ammonia and glutamine according to the method of Richter and Dawson (10). For the determination of glutamic acid content in brain slices, bat,ches of some 500 mg. wet weight slices were incubated in 5 ml. of medium. After 1 hr. of incubation, the slices were taken out, homogenized and extracted in 0.5 N HCI for 30 min. at O”C., and then centrifuged. The acidity of the supernatant was adjusted to pH 5.8, and then analyzed using the squash glutamic acid decarboxylase preparation (11-j. The determination of glucose was made using the “anthrone” reagent described by Morris (11) following the procedure of Koehler (12). Glucose consumption was estimated from the decrease of its cont.ent in the medium after t.he incubation period. Lactic acid formation in the medium was determined by the method of Barker and Summerson (13). For t,he determination of total carbohydrate, the slices in the medium were hydrolyzed with N HzSOI at 100°C. for 40 min., and neutralized with KOH. Carbohydrate was then determined by the “anthrone” method and calculated as glucose.
Chemicals All of the chemicals used were of “guaranteed were used without further purification.
grade”
commercial
origin
and
198
TAKAGAEI,
HIMNO
AND TSUKADA
RESULTS
Ammonia Formation in Brain Slices
Guinea-pig brain cortex slices respiring in a substrate-free medium formed ammonia at a rate of 7.60 pmoles/g./hr. In the presence of glucose or glutamic acid, the ammonia formation was depressed strongly. These data are in good agreement with the observations by Weil-Malherbe and Green (14). Although brain slices incubated in the presence of glutamic acid formed glutamine at a rate corresponding to the decreaseof ammonia formation, the inhibition of ammonia formation by the addition of glucose was not accompanied by a significant increase of amide synthesis (P > 0.1) (Table I). Moreover, on the further addition of 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation, the ammonia formation was increased and glutamine synthesis was suppressed in spite of the presence of glutamic acid. Accordingly, it is clear that the amide formation which requires adenosine triphosphate (15, 16) is responsible for the inhibition of ammonia formation due to glutamic acid. In the case of glucose the inhibition was not simply explained by the amide formation, but rather it was considered that glucose metabolism itself might be necessary for the inhibition of ammonia formation, for iodoacetic acid at a fairly low concentration increased the formation of ammonia in the presence of glucose (Table I). Endogenous Respiration
The oxygen consumption of guinea-pig brain cortex slices respiring in a substrate-free medium was measured. This endogenous oxygen uptake amounted to 41.2 pmoles/g./hr., and was about 70 % of the value for the oxygen consumption in the presence of 0.2 % glucose (Table I). With the purpose of finding out the possible main substrate for endogenous respiration, the changes of the contents of total carbohydrate and lactic acid in slices were analyzed chemically, and the respiratory quotient was determined. These results are shown in Table II. The respiratory quotient was almost unity and not different from the value for brain cortex slices’ respiration in 0.2 % glucose. The consumption of lactic acid and carbohydrate contained in brain slices could account for only some 35 % of the observed total oxygen uptake. Nevertheless, the oxygen uptake in brain slices respiring in the medium containing 0.2% glucose could be almost exactly estimated from the
(i Decrease from the init,ial glutamic shown.
4.69 f
Succinic acid, lo-* M
1.79 f
1.41 f
7.74 f 2.25 f
0.55 f 2.03 f 2.80 f
1.59 (3)
1.13 (3)
1.12 (6)
1.18 (6)
1.82 (5)
1.69 (5)
0.76 (4)
pmolcs/g.
Glutamine
acid content in brain slices (6.69 f 0.15 (3) pmoles/g.)
1.46 (3)
0.47 (3)
1.70 f
0.36 (5) 0.75 (6) 0.53 (6)
3.9 (5) 5.9 (4)
3.20 f
0.92 (10) 1.51 f 0.74 (5)
7.60 f
pmoles/g./hr.
Ammonia formation
1.22 f 7.70 f
67.1 f 62.5 f
41.2 z!z 4.6 (15) 58.2 f 5.4 (23) 50.9 f 2.9 (4)
imoles/g./hr.
Oxygen uptake
Glutamic acid, 1OV M Glutamic acid, 1OP M + 2,4-dinitrophenol, 5 X 10-b M Lactic acid, 10-Z M
5 X IO-6 M
None Glucose, 0.2yo Glucose, 0.2% + iodoacetic acid,
Addition
5.47 f 1.04 (3) 2.89 f 0.47 (3)
0.53 0.37
-
(4)I (4)I
3.80
1.22
1.43
4.59
is
P.mnolcs/g.
pmoles/g. 2.10 f 5.26 f
Decreasea
-8 Content
-
Glutamic acid
during 1 hr. incubation
= I
TABLE I Ammonia Formation and Glutantic Acid Content in Guinea-Pig Brain Cortex Slices From 80 to 120 mg. (wet weight) of guinea-pig brain cortex slices was incubated in 3 ml. Krebs-Ringer phosphate sohtion at 37°C. for 1 hr. under pure oxygen. For the glutamic acid determinat,ion, about 500 mg. of slices was incubated in 5 ml. Ringer solution. After 1 hr. incubation, the slices were taken out and the content of glutamic acid was determined using the squash decarboxylase preparation. All values given are means and st,andard deviations. Figures in parentheses - _ indicate numbers of experiments.
h E
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TAKAGAKI,
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TABLE
AND
TSUKADA
II
Brain Cortex Slices Guinea-pig brain cortex slices were incubated for 1 hr. without substrates under the same conditions as in Table I. Respiratory quotient was measured by the second method of Dickens and Simer. Means f standard deviations are shown. Figures in parentheses indicate numbers of experiments. Analysis
of Endogenozts
Metabolism
in Guinea-Pig
Respiratory quotient
Oxygen uptake
Lactic acid
pmoles/g./hr.
After incubation 41.2 f
p?noles/g.
pmoles/~.
Before incubation
Difference
Total carbohydrate
4.6 (15)
0.977 f 0.016 (5)
4.25 8.11 f 0.23 (5) f 0.52 (4) 2.16 6.88 z!z 0.46 (4) f 0.27 (4) 1.23 2.09
TABLE III and Lactic Acid Formation i,n Guinea-Pig Brain Cortex Slices About 150 mg. (wet weight) of guinea-pig brain cortex slices were incubated in 3.0 ml. Krebs-Ringer phosphate solution in the presence of glucose (3.0 miW) for 90 min. under pure oxygen at 37°C. All values given are means and standard deviations. Numbers of experiments are shown in parentheses. Glucose Utilization
Oxygen uptake, ,moles/g./hr.
56.3 f
2.9 (11)
Respiratory
0.981 f
quotient
0.026 (4)
Glucose utilization, pmoIes/g./hr.
19.0 f
1.8 (11)
Lactic acid formation, /.mdes/g./hr.
16.6 f
3.0 (11)
utilization of added glucose and the formation of lactic acid in the medium, as shown in Table III. These facts showed that the oxygen consumption observed in the presence of glucose did not include the respiration due to endogenous metabolism. Therefore, it should be considered that the endogenous metabolism would be almost completely suppressed by the addition of glucose. But the clarification of the mechanism of endogenous metabolism itself could not be expected from the respiratory quotient measured, and it was suggested that it would be of a composite nature. Ammonia Formation and Glutamic Acid Content Based on the previous results (4), the analyses of the changes of content of glutamic acid in brain slices were t.hought to be of interest. Bs shown in Table I, in a substrate-free medium, the glutamic acid in brain slices was decreased significantly during 1 hr. incubation. But in the
RESPIRATION
AND
AMMONIA
201
FORMATION
presence of glucose or lactic acid, under which conditions the ammonia formation was suppressed strongly, the glutamic acid content in brain slices was maintained at a fairly high level. Succinic acid, which did not inhibit the ammonia formation very much, suppressed the decrease of glutamic acid to a moderate extent. That is, there was found to be a good parallel relationship between the decrease of glutamic acid content and the formation of ammonia in brain cortex slices. From all these data, it was assumed that the ammonia formation and the endogenous oxygen uptake, to at least 50%, could be explained as due to the oxidation of glutamic acid in brain slices. Eflect of Some Metabolic Inhibitors on E.ndogen0u.s Oxygen Uptake and Ammonia Format&m To confirm further the above considerations, the effect of various metabolic inhibitors on the endogenous oxygen uptake and, at the same time, on the ammonia formation was studied (Table IV). Because t,he endogenous oxygen uptake may have a multiple origin, and also because the concentration of metabolic inhibitors used was rather high in comparison with the endogenous substrate concentration, an easy interpreTABLE
IV
Effect of Some Metabolic Inhibitors on. the Ammonia of Gu,inea-Pig Brain Slices
Conditions
of incubation
Formation
were same as in Table I.
Ammonia formation Inhibitor
Lxxentration
-
I
-
Oxygen uptake
-
C haye
:hyge
--
M
Anaerobic Cyanide Arsenite 2,4-DNP Sodium-free Iodoacetate Sodium fluoride Malonate Sodium azide
pmoles/g./kr.
x 10-b 10-z 10-z 10-S
7.75 f 8.24 f
5 x lo-’ IO-3 5 x 10-h 5
f zk f f f f f
3.90 4.22 4.87 5.36 5.72 6.60 7.68
-
0.52 0.50 0.86 0.60 0.47 1.56 0.45
%
(5j (4) (4) (3) (3) (6) (4)
-48 -44 -35 -29 -24 -13 0
0.72 (4) 1.38 (4)
+2 $8
-
4,o
ptrroles/g./kr.
11.8 7.7 22.9 20.2 37.8 22.7
f f f f f f
40.8 f 34.9 f
0.73 1.25 3.67 0.87 7.83 4.45
- 71
(4) (3) (4) (3) (4) (3)
-81 -44 -51 -8 -45
4.04 (4j 4.28 (4)
-15
-1
0 Increase or decrease of ammonia formation and oxygen uptake from the control value (Table I) is shown as plus or minus change in per cent.
202
TAKAGAKI,
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AND
TSUKADA
tation of data may not be readily expected. At any rate, significant inhibition of oxygen uptake was produced by those agents that inhibited the ammonia formation strongly, that is, by arsenite, cyanide, and 2,4dinitrophenol. On the other hand, those agents that did not significantly inhibit the ammonia formation, i.e., malonate, sodium azide, and iodoacetate, did not inhibit the respiration either. The ammonia formation was depressed strongly under anaerobic conditions. When sodium ions in the usual Krebs-Ringer phosphate solution were omitted, the oxidation of lactic acid in brain cortex slices was almost completely depressed.’ The effect on the ammonia formation and the endogenous oxygen uptake was consequently tested. Both were inhibited significantly to the same degree (Table IV). All of these results indicated a parallel relationship between ammonia formation and endogenous oxygen uptake under the influence of various metabolic inhibitors. It might therefore be considered that ammonia formation and endogenous oxygen consumption have the same origin. However, sodium fluoride, an exception among t,he inhibitors studied, inhibited the oxygen uptake but did not inhibit ammonia formation. DISCUSSION
The above results suggest that the origin of endogenous oxygen uptake is not single, but composite. The decrease of carbohydrate and lactic acid in brain slices following the incubation without added substrates could only be accounted for in terms of less than half of the oxygen consumption, while the remaining part could be explained by the decrease of glutamic acid. The oxygen uptake and the respiratory quotient in brain slices respiring in a glucose-containing medium could be exactly calculated from the amounts of glucose utilization and lactic acid formation, and, therefore, it should be considered that the added glucose inhibits the endogenous metabolism almost completely. On the basis of this consideration, the fact that added glucose inhibits the ammonia formation strongly and, at the same time, inhibits the consumption of endogenous glutamic acid, is quite naturally understandable. Under anaerobic conditions the formation of ammonia decreased to about a half of the amount formed aerobically. Presumably this anaerobic part could be formed autolytically. In the presence of cyanide which inhibits the oxygen uptake almost completely, the surviving ammonia formation probably arises similarly to that under anaerobic formation. 1 G. Takagaki,
to be published.
RESPIRATION AND AMMONIA FORMATION
203
The fact that the amount of ammonia formed in a glucose-containing medium aerobically is lower than that formed in a glucose-free medium anaerobically shows clearly that added glucose suppresses the autolysis as well. It might be thought that the ammonia formation in brain slices would consist of at least two components, one of them being autolytic, the other aerobic component arising from the oxidative deamination of glutamic acid. Weil-Malherbe and Green have extensively studied the ammonia formation in brain slices and suggested that the ammonia arises in a reaction intimately linked with proteolysis (14). They assume some single origin of ammonia formation in brain slices. Furthermore, in studying the effect of various inhibitors on the ammonia formation, they incubated the brain slices for 4 hr. This would probably be the main reason for some differences between their results and the present ones (Table IV). Because it was considered that the ammonia formation after incubation for as long as 4 hr. might be the result of a highly complicated series of reactions, the greater part due to proteolysis, the present authors invariably limit,ed t,he incubation time to 1 hr. During the early period, the oxidative part may be observed predominantly. Certainly the equilibrium of the glutamic dehydrogenase reaction in brain slices is rather in favor of the reductive amination of a-ketoglutaric acid (l4), but in the absence of some energy source the condit,ions may be quite different. Of course, t,he present considerations are not conclusive, but only suggestive, and t,he large ammonia formation of brain slices, which reaches as much as 30 pmoles/g. in 5 hr. (14), is quit,e difficult to interpret. There is further the problem as to whether the ammonia formation by brain slices in vitro is related to the ammonia formation induced by the increased activity of nervous tissue. Though it cannot be decided as yet, there are some experimental data in favor of a close connection between the two processes. These data suggest the physiological significance of glutamic acid metabolism in brain tissue (17). The energy requirement of brain is usually obtained by the oxidation of glucose, while brain slices in vitro can also oxidize glucose and support the formation of the energyrich phosphates only in the presence of glucose. But in emergencies such as excit,ation, in which the glucose supply is insufficient, glutamic acid may probably be oxidized and donate cr-ketoglutaric acid to the tricarboxylic acid cycle with the consequent production of ammonia. These conditions in tivo may correspond to the brain slices incubated without
204
TAKAGAKI,
HIRANO
AND
TSUKADA
substrate. Therefore, the ammonia formation reflect the state of affairs in the living brain.
in brain slices may well
SUMMARY
1. Ammonia formation in brain cortex slices of guinea pig respiring in a glucose-free medium was analyzed. 2. The formation of ammonia was accompanied with a decrease of glutamic acid content, and an appreciable oxygen consumption. In the presence of oxidizable substrates, ammonia formation and t,he decrease of glutamic acid content in brain slices mere suppressed. Therefore, it was thought that glucose or ot,her oxidizable substrates depressed ammonia formation through the inhibition of an appreciable part of the endogenous metabolism in brain slices. 3. More than half of the endogenous oxygen consumption could be related to a decrease of glutamic acid content, and the remaining part to the carbohydrate and lactic acid consumption in brain slices. 4. It was thought that ammonia formation in brain slices had two components, one of them being presumably autolytic, the other depending upon the oxidation of glutamic acid. 5. The effect, of certain metabolic inhibitors on ammonia formation and endogenous oxygen uptake was studied. Generally, inhibitors which inhibited the oxygen uptake also inhibited the ammonia formation, and those which did not inhibit the former did not affect the latter. 6. The relations between the ammonia formation in brain slices in vitro and the increase of ammonia in living brain in excited conditions were discussed. REFERENCES 1. TERNER, C., Riochem. J. 60, 145 (1951). 2. MELROSE, D. R., AND TERNER, C., Biochem. J. 63, 296 (1953). 3. TSUKADA, Y., AND TAKAQAKI, G., Nature 173, 1138 (1954). 4. TAKAGAKI, G., J. Biochem. (Japan) 42, 131 (1955).
5. WAELSCH, H., in “Neurochemistry” (Elliott, Ii. A. C., Page, I. H., and Quastel, J. H.), p. 173. C. C Thomas, Springfield, Ill., 1955. 6. TAKAGAKI, G., HIRANO, S., AND TSUKADA, Y., Symposia on Enzyme Chemistry, 12,283 (1957). 7. ROBBIE, W. A., in “Methods in Medical Research” (Potter, V. R.), Vol. I, p. 307. The Year Book Publishers, Inc., Chicago, Ill., 1948. 8. DICKENS, F., AND SIMER, F., Biochem. J. 26, 973 (1931). 9. COHEN, P. P., in “Manometric Techniques and Tissue Metabolism” (Umbreit, W. W., Burris, R. H., and Stauffer, J. F.), p. 119. Burgess Publishing Co., Minneapolis, Minn., 1949.
RESPIRATION
10. 11. 12. 13. 14. 15. 16.
AND
AMMONIA
FORMATION
205
RICHTER, D., AND DAWSON, R. M. C., J. Biol. Chem. 176, 1199 (1948). MORRIS, D. L., Science 107, 254 (1948). KOEHLER, L. H., Anal. Chew 24, 1576 (1952). BARKER, S. B., AND SUMMERSON,W. H., J. Biol. Chem. 136, 535 (1941). WELL-MALHERBE, H., AND GREEN, R. H., Biochem. J. 61,210 (1955). ELLIOTT, W. H., Nature 161, 128 (1948). BRAGANCA, B.M., FATJLKNER,~., AND QUASTEL, J.H., Biochim! et Biophys. Acta 10, 83 (1953). 17. WEIL-MALEIERBE, H., Physiol. Revs. 30, 549 (1950).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
66, 196-205 (1957)
Endogenous Respiration and Ammonia Formation in Brain Slices Genkichiro Takagaki, Shusuke Hirano and Yasuzo Tsukada From the Departwlent of Physiology, School of Uedicine, Keio University, Tokyo, Japan
Received July 19, 1956
INTRODUCTION Although in some cases it is possible to depress the endogenous metabolism in tissue slices by some metabolic inhibitor (1, 2), it nevertheless often persists at a fairly high level and should not be neglected. In brain cortex slices, the endogenous oxygen uptake accounts for more than half that which is observed in the presence of glucose as a substrate. Accordingly, it was considered desirable to analyze the endogenous metabolism of brain slices to determine the nature of the main substrate and to ascertain its relationship to t#he over-all metabolism in the presence of added substrate. It is well-known that ammonia is formed in large amount during the endogenous metabolism of brain slices and that it is almost completely depressed in the presence of glucose. In earlier reports (3, 4) it was pointed out that ammonia would possibly be formed from glutamic acid in brain tissue. The metabolism of glutamic acid and the formation of ammonia in living brain are physiologically significant, and suggest many interesting problems (5). This investigation was undertaken with the particular objective of analyzing the relationship between the endogenous respiration and ammonia formation in brain slices. A preliminary account of a part of this investigation was reported elsewhere (6). MATERIAW AND METHODS Experimental
Procedures in General
All animals used were guinea pigs of some 300 g. in weight. The animals were killed by decapitation, and the brain cortex slices were cut freehand with a moist 196
RESPIRATION
AND
AMMONIA
197
FORMATION
safet,y-razor blade on a refrigerated stage, and stored in ice-cold phosphate Ringer solution. Batches of SO-120 mg. in wet weight were used in 3 ml. of medium. Weighted slices with a torsion balance were introduced in the usual Warburg vessels and the vessels were attached t.o the manometers. The air in the vessels was replaced with pure oxygen by the evacuation method, and the vessels were placed in a thermostat at 37°C. It took about 30 min. from the death of the animals t.o reach this point in the procedure. Readings of manometers were taken at IO-ruin. intervals. In anaerobic experiments pure nit.rogen, freed from contaminated oxygen by exposure to yellow phosphorus, was used. Cyanide as an inhibitor was used according to the directions of Robbie (7); that is, the proper mixture of KCNKOH was placed in t.he center wells of the Warburg flasks in order to maintain the desired concentration of cyanide in the medium throughout the incubation period. The respiratory quotient was measured by the second method of Dickens and Simer (S), using the flasks possessing two side-arms, one of which was equipped with a double sack. Krebs-Ringer phosphate solution (9) at pH 7.0 was used throughout. When t.he sodium-free medium was prepared, sodium chloride was completely replaced by choline chloride, and sodium phosphate by potassium phosphate, while maintaining the correct osmotic pressure of the medium.
Chemical Estimations The specimens removed from t.he medium at the end of t,he incubation period were deproteinized with trichloroacetic acid. The clear supernatant was used for the determination of free ammonia and glutamine according to the method of Richter and Dawson (10). For the determination of glutamic acid content in brain slices, bat,ches of some 500 mg. wet weight slices were incubated in 5 ml. of medium. After 1 hr. of incubation, the slices were taken out, homogenized and extracted in 0.5 N HCI for 30 min. at O”C., and then centrifuged. The acidity of the supernatant was adjusted to pH 5.8, and then analyzed using the squash glutamic acid decarboxylase preparation (11-j. The determination of glucose was made using the “anthrone” reagent described by Morris (11) following the procedure of Koehler (12). Glucose consumption was estimated from the decrease of its cont.ent in the medium after t.he incubation period. Lactic acid formation in the medium was determined by the method of Barker and Summerson (13). For t,he determination of total carbohydrate, the slices in the medium were hydrolyzed with N HzSOI at 100°C. for 40 min., and neutralized with KOH. Carbohydrate was then determined by the “anthrone” method and calculated as glucose.
Chemicals All of the chemicals used were of “guaranteed were used without further purification.
grade”
commercial
origin
and
198
TAKAGAEI,
HIMNO
AND TSUKADA
RESULTS
Ammonia Formation in Brain Slices
Guinea-pig brain cortex slices respiring in a substrate-free medium formed ammonia at a rate of 7.60 pmoles/g./hr. In the presence of glucose or glutamic acid, the ammonia formation was depressed strongly. These data are in good agreement with the observations by Weil-Malherbe and Green (14). Although brain slices incubated in the presence of glutamic acid formed glutamine at a rate corresponding to the decreaseof ammonia formation, the inhibition of ammonia formation by the addition of glucose was not accompanied by a significant increase of amide synthesis (P > 0.1) (Table I). Moreover, on the further addition of 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation, the ammonia formation was increased and glutamine synthesis was suppressed in spite of the presence of glutamic acid. Accordingly, it is clear that the amide formation which requires adenosine triphosphate (15, 16) is responsible for the inhibition of ammonia formation due to glutamic acid. In the case of glucose the inhibition was not simply explained by the amide formation, but rather it was considered that glucose metabolism itself might be necessary for the inhibition of ammonia formation, for iodoacetic acid at a fairly low concentration increased the formation of ammonia in the presence of glucose (Table I). Endogenous Respiration
The oxygen consumption of guinea-pig brain cortex slices respiring in a substrate-free medium was measured. This endogenous oxygen uptake amounted to 41.2 pmoles/g./hr., and was about 70 % of the value for the oxygen consumption in the presence of 0.2 % glucose (Table I). With the purpose of finding out the possible main substrate for endogenous respiration, the changes of the contents of total carbohydrate and lactic acid in slices were analyzed chemically, and the respiratory quotient was determined. These results are shown in Table II. The respiratory quotient was almost unity and not different from the value for brain cortex slices’ respiration in 0.2 % glucose. The consumption of lactic acid and carbohydrate contained in brain slices could account for only some 35 % of the observed total oxygen uptake. Nevertheless, the oxygen uptake in brain slices respiring in the medium containing 0.2% glucose could be almost exactly estimated from the
(i Decrease from the init,ial glutamic shown.
4.69 f
Succinic acid, lo-* M
1.79 f
1.41 f
7.74 f 2.25 f
0.55 f 2.03 f 2.80 f
1.59 (3)
1.13 (3)
1.12 (6)
1.18 (6)
1.82 (5)
1.69 (5)
0.76 (4)
pmolcs/g.
Glutamine
acid content in brain slices (6.69 f 0.15 (3) pmoles/g.)
1.46 (3)
0.47 (3)
1.70 f
0.36 (5) 0.75 (6) 0.53 (6)
3.9 (5) 5.9 (4)
3.20 f
0.92 (10) 1.51 f 0.74 (5)
7.60 f
pmoles/g./hr.
Ammonia formation
1.22 f 7.70 f
67.1 f 62.5 f
41.2 z!z 4.6 (15) 58.2 f 5.4 (23) 50.9 f 2.9 (4)
imoles/g./hr.
Oxygen uptake
Glutamic acid, 1OV M Glutamic acid, 1OP M + 2,4-dinitrophenol, 5 X 10-b M Lactic acid, 10-Z M
5 X IO-6 M
None Glucose, 0.2yo Glucose, 0.2% + iodoacetic acid,
Addition
5.47 f 1.04 (3) 2.89 f 0.47 (3)
0.53 0.37
-
(4)I (4)I
3.80
1.22
1.43
4.59
is
P.mnolcs/g.
pmoles/g. 2.10 f 5.26 f
Decreasea
-8 Content
-
Glutamic acid
during 1 hr. incubation
= I
TABLE I Ammonia Formation and Glutantic Acid Content in Guinea-Pig Brain Cortex Slices From 80 to 120 mg. (wet weight) of guinea-pig brain cortex slices was incubated in 3 ml. Krebs-Ringer phosphate sohtion at 37°C. for 1 hr. under pure oxygen. For the glutamic acid determinat,ion, about 500 mg. of slices was incubated in 5 ml. Ringer solution. After 1 hr. incubation, the slices were taken out and the content of glutamic acid was determined using the squash decarboxylase preparation. All values given are means and st,andard deviations. Figures in parentheses - _ indicate numbers of experiments.
h E
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TAKAGAKI,
HIRANO
TABLE
AND
TSUKADA
II
Brain Cortex Slices Guinea-pig brain cortex slices were incubated for 1 hr. without substrates under the same conditions as in Table I. Respiratory quotient was measured by the second method of Dickens and Simer. Means f standard deviations are shown. Figures in parentheses indicate numbers of experiments. Analysis
of Endogenozts
Metabolism
in Guinea-Pig
Respiratory quotient
Oxygen uptake
Lactic acid
pmoles/g./hr.
After incubation 41.2 f
p?noles/g.
pmoles/~.
Before incubation
Difference
Total carbohydrate
4.6 (15)
0.977 f 0.016 (5)
4.25 8.11 f 0.23 (5) f 0.52 (4) 2.16 6.88 z!z 0.46 (4) f 0.27 (4) 1.23 2.09
TABLE III and Lactic Acid Formation i,n Guinea-Pig Brain Cortex Slices About 150 mg. (wet weight) of guinea-pig brain cortex slices were incubated in 3.0 ml. Krebs-Ringer phosphate solution in the presence of glucose (3.0 miW) for 90 min. under pure oxygen at 37°C. All values given are means and standard deviations. Numbers of experiments are shown in parentheses. Glucose Utilization
Oxygen uptake, ,moles/g./hr.
56.3 f
2.9 (11)
Respiratory
0.981 f
quotient
0.026 (4)
Glucose utilization, pmoIes/g./hr.
19.0 f
1.8 (11)
Lactic acid formation, /.mdes/g./hr.
16.6 f
3.0 (11)
utilization of added glucose and the formation of lactic acid in the medium, as shown in Table III. These facts showed that the oxygen consumption observed in the presence of glucose did not include the respiration due to endogenous metabolism. Therefore, it should be considered that the endogenous metabolism would be almost completely suppressed by the addition of glucose. But the clarification of the mechanism of endogenous metabolism itself could not be expected from the respiratory quotient measured, and it was suggested that it would be of a composite nature. Ammonia Formation and Glutamic Acid Content Based on the previous results (4), the analyses of the changes of content of glutamic acid in brain slices were t.hought to be of interest. Bs shown in Table I, in a substrate-free medium, the glutamic acid in brain slices was decreased significantly during 1 hr. incubation. But in the
RESPIRATION
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AMMONIA
201
FORMATION
presence of glucose or lactic acid, under which conditions the ammonia formation was suppressed strongly, the glutamic acid content in brain slices was maintained at a fairly high level. Succinic acid, which did not inhibit the ammonia formation very much, suppressed the decrease of glutamic acid to a moderate extent. That is, there was found to be a good parallel relationship between the decrease of glutamic acid content and the formation of ammonia in brain cortex slices. From all these data, it was assumed that the ammonia formation and the endogenous oxygen uptake, to at least 50%, could be explained as due to the oxidation of glutamic acid in brain slices. Eflect of Some Metabolic Inhibitors on E.ndogen0u.s Oxygen Uptake and Ammonia Format&m To confirm further the above considerations, the effect of various metabolic inhibitors on the endogenous oxygen uptake and, at the same time, on the ammonia formation was studied (Table IV). Because t,he endogenous oxygen uptake may have a multiple origin, and also because the concentration of metabolic inhibitors used was rather high in comparison with the endogenous substrate concentration, an easy interpreTABLE
IV
Effect of Some Metabolic Inhibitors on. the Ammonia of Gu,inea-Pig Brain Slices
Conditions
of incubation
Formation
were same as in Table I.
Ammonia formation Inhibitor
Lxxentration
-
I
-
Oxygen uptake
-
C haye
:hyge
--
M
Anaerobic Cyanide Arsenite 2,4-DNP Sodium-free Iodoacetate Sodium fluoride Malonate Sodium azide
pmoles/g./kr.
x 10-b 10-z 10-z 10-S
7.75 f 8.24 f
5 x lo-’ IO-3 5 x 10-h 5
f zk f f f f f
3.90 4.22 4.87 5.36 5.72 6.60 7.68
-
0.52 0.50 0.86 0.60 0.47 1.56 0.45
%
(5j (4) (4) (3) (3) (6) (4)
-48 -44 -35 -29 -24 -13 0
0.72 (4) 1.38 (4)
+2 $8
-
4,o
ptrroles/g./kr.
11.8 7.7 22.9 20.2 37.8 22.7
f f f f f f
40.8 f 34.9 f
0.73 1.25 3.67 0.87 7.83 4.45
- 71
(4) (3) (4) (3) (4) (3)
-81 -44 -51 -8 -45
4.04 (4j 4.28 (4)
-15
-1
0 Increase or decrease of ammonia formation and oxygen uptake from the control value (Table I) is shown as plus or minus change in per cent.
202
TAKAGAKI,
HIRAN
AND
TSUKADA
tation of data may not be readily expected. At any rate, significant inhibition of oxygen uptake was produced by those agents that inhibited the ammonia formation strongly, that is, by arsenite, cyanide, and 2,4dinitrophenol. On the other hand, those agents that did not significantly inhibit the ammonia formation, i.e., malonate, sodium azide, and iodoacetate, did not inhibit the respiration either. The ammonia formation was depressed strongly under anaerobic conditions. When sodium ions in the usual Krebs-Ringer phosphate solution were omitted, the oxidation of lactic acid in brain cortex slices was almost completely depressed.’ The effect on the ammonia formation and the endogenous oxygen uptake was consequently tested. Both were inhibited significantly to the same degree (Table IV). All of these results indicated a parallel relationship between ammonia formation and endogenous oxygen uptake under the influence of various metabolic inhibitors. It might therefore be considered that ammonia formation and endogenous oxygen consumption have the same origin. However, sodium fluoride, an exception among t,he inhibitors studied, inhibited the oxygen uptake but did not inhibit ammonia formation. DISCUSSION
The above results suggest that the origin of endogenous oxygen uptake is not single, but composite. The decrease of carbohydrate and lactic acid in brain slices following the incubation without added substrates could only be accounted for in terms of less than half of the oxygen consumption, while the remaining part could be explained by the decrease of glutamic acid. The oxygen uptake and the respiratory quotient in brain slices respiring in a glucose-containing medium could be exactly calculated from the amounts of glucose utilization and lactic acid formation, and, therefore, it should be considered that the added glucose inhibits the endogenous metabolism almost completely. On the basis of this consideration, the fact that added glucose inhibits the ammonia formation strongly and, at the same time, inhibits the consumption of endogenous glutamic acid, is quite naturally understandable. Under anaerobic conditions the formation of ammonia decreased to about a half of the amount formed aerobically. Presumably this anaerobic part could be formed autolytically. In the presence of cyanide which inhibits the oxygen uptake almost completely, the surviving ammonia formation probably arises similarly to that under anaerobic formation. 1 G. Takagaki,
to be published.
RESPIRATION AND AMMONIA FORMATION
203
The fact that the amount of ammonia formed in a glucose-containing medium aerobically is lower than that formed in a glucose-free medium anaerobically shows clearly that added glucose suppresses the autolysis as well. It might be thought that the ammonia formation in brain slices would consist of at least two components, one of them being autolytic, the other aerobic component arising from the oxidative deamination of glutamic acid. Weil-Malherbe and Green have extensively studied the ammonia formation in brain slices and suggested that the ammonia arises in a reaction intimately linked with proteolysis (14). They assume some single origin of ammonia formation in brain slices. Furthermore, in studying the effect of various inhibitors on the ammonia formation, they incubated the brain slices for 4 hr. This would probably be the main reason for some differences between their results and the present ones (Table IV). Because it was considered that the ammonia formation after incubation for as long as 4 hr. might be the result of a highly complicated series of reactions, the greater part due to proteolysis, the present authors invariably limit,ed t,he incubation time to 1 hr. During the early period, the oxidative part may be observed predominantly. Certainly the equilibrium of the glutamic dehydrogenase reaction in brain slices is rather in favor of the reductive amination of a-ketoglutaric acid (l4), but in the absence of some energy source the condit,ions may be quite different. Of course, t,he present considerations are not conclusive, but only suggestive, and t,he large ammonia formation of brain slices, which reaches as much as 30 pmoles/g. in 5 hr. (14), is quit,e difficult to interpret. There is further the problem as to whether the ammonia formation by brain slices in vitro is related to the ammonia formation induced by the increased activity of nervous tissue. Though it cannot be decided as yet, there are some experimental data in favor of a close connection between the two processes. These data suggest the physiological significance of glutamic acid metabolism in brain tissue (17). The energy requirement of brain is usually obtained by the oxidation of glucose, while brain slices in vitro can also oxidize glucose and support the formation of the energyrich phosphates only in the presence of glucose. But in emergencies such as excit,ation, in which the glucose supply is insufficient, glutamic acid may probably be oxidized and donate cr-ketoglutaric acid to the tricarboxylic acid cycle with the consequent production of ammonia. These conditions in tivo may correspond to the brain slices incubated without
204
TAKAGAKI,
HIRANO
AND
TSUKADA
substrate. Therefore, the ammonia formation reflect the state of affairs in the living brain.
in brain slices may well
SUMMARY
1. Ammonia formation in brain cortex slices of guinea pig respiring in a glucose-free medium was analyzed. 2. The formation of ammonia was accompanied with a decrease of glutamic acid content, and an appreciable oxygen consumption. In the presence of oxidizable substrates, ammonia formation and t,he decrease of glutamic acid content in brain slices mere suppressed. Therefore, it was thought that glucose or ot,her oxidizable substrates depressed ammonia formation through the inhibition of an appreciable part of the endogenous metabolism in brain slices. 3. More than half of the endogenous oxygen consumption could be related to a decrease of glutamic acid content, and the remaining part to the carbohydrate and lactic acid consumption in brain slices. 4. It was thought that ammonia formation in brain slices had two components, one of them being presumably autolytic, the other depending upon the oxidation of glutamic acid. 5. The effect, of certain metabolic inhibitors on ammonia formation and endogenous oxygen uptake was studied. Generally, inhibitors which inhibited the oxygen uptake also inhibited the ammonia formation, and those which did not inhibit the former did not affect the latter. 6. The relations between the ammonia formation in brain slices in vitro and the increase of ammonia in living brain in excited conditions were discussed. REFERENCES 1. TERNER, C., Riochem. J. 60, 145 (1951). 2. MELROSE, D. R., AND TERNER, C., Biochem. J. 63, 296 (1953). 3. TSUKADA, Y., AND TAKAQAKI, G., Nature 173, 1138 (1954). 4. TAKAGAKI, G., J. Biochem. (Japan) 42, 131 (1955).
5. WAELSCH, H., in “Neurochemistry” (Elliott, Ii. A. C., Page, I. H., and Quastel, J. H.), p. 173. C. C Thomas, Springfield, Ill., 1955. 6. TAKAGAKI, G., HIRANO, S., AND TSUKADA, Y., Symposia on Enzyme Chemistry, 12,283 (1957). 7. ROBBIE, W. A., in “Methods in Medical Research” (Potter, V. R.), Vol. I, p. 307. The Year Book Publishers, Inc., Chicago, Ill., 1948. 8. DICKENS, F., AND SIMER, F., Biochem. J. 26, 973 (1931). 9. COHEN, P. P., in “Manometric Techniques and Tissue Metabolism” (Umbreit, W. W., Burris, R. H., and Stauffer, J. F.), p. 119. Burgess Publishing Co., Minneapolis, Minn., 1949.
RESPIRATION
10. 11. 12. 13. 14. 15. 16.
AND
AMMONIA
FORMATION
205
RICHTER, D., AND DAWSON, R. M. C., J. Biol. Chem. 176, 1199 (1948). MORRIS, D. L., Science 107, 254 (1948). KOEHLER, L. H., Anal. Chew 24, 1576 (1952). BARKER, S. B., AND SUMMERSON,W. H., J. Biol. Chem. 136, 535 (1941). WELL-MALHERBE, H., AND GREEN, R. H., Biochem. J. 61,210 (1955). ELLIOTT, W. H., Nature 161, 128 (1948). BRAGANCA, B.M., FATJLKNER,~., AND QUASTEL, J.H., Biochim! et Biophys. Acta 10, 83 (1953). 17. WEIL-MALEIERBE, H., Physiol. Revs. 30, 549 (1950).