- Email: [email protected]
General Discussion*
243
General Discussion*
GIARMAN : I would like to ask Dr. Aprison one question that interests me a great deal. We have been studying the schedule of appearance of amines and associated enzymes in prenatal rats following the animal into the postnatal period. We find as far as serotonin is concerned a very marked jog upward in the slope of the curve showing the appearance of serotonin 24 h before birth, at birth, and 24 h after birth and then the curve slopes again to what it was before this period. We also have an interesting suggestion that monoamine oxidase activity ceases to go upward during this period which could correlate well with this increasing appearance of serotonin during birth and I wondered if Dr. Aprison had looked at his animals during this period of time in this regard. APRISON:I do not know why your brain serotonin measurements followed the trend you report unless it reflects the differential rate of formation as well as the activity of the catabolic enzyme, MAO, in distinct brain areas. Karki et al., ( J . Neurochem., 9 (1962) 54) also find at birth a change in the slope of the curve relating serotonin in rat brain to age. It is possible that continuous ‘smooth’ curves were not obtained for serotonin because the measurements were made on total brain instead of specific parts. I have stated i n some of our publications that I do not feel that serotonin may be a neurohumor in the sense that acetylcholine is thought to be a neurohumor. At present I think that serotonin acts as a chemical modulator (Rec. Adv. Biol. Psychiat., 4 (1962) 133). Other than that, I just would not care to speculate further until we have more data. GIARMAN : I was interested in Dr. Bogdanski’s allusion to the ergotrophic-tropotrophic system of Dr. Brodie in relation to his finding differential ratios of serotonin and catecholamines in various species. Certainly the ratio would indicate, according to the theory, that amphibia are tropotrophic animals that live a life of quiet restoration. Our knowledge of 400-year-old turtles would certainly bear this out. I just wondered, however, what role is conceived of for acetylcholine in this system and whether measurements were made in amphibia and other species for acetylcholine and cholinesterase activities. Certainly, acetylcholine must figure prominently in any considerations of parasympathetic representation in the brain. BOGDANSKI : We have not made any measurements of cholinergic mechanisms in any of the species. While we do not disregard the possible importance of cholinergic
* This discussion refers to the papers of Dr. G. Guroff and Dr. S. Udenfriend, Dr. R. E. McCanian and M. H. Aprison, and Dr. B. B. Brodie and D. F. Bogdanski.
244
GENERAL DISCUSSION
systems for some aspects of brain function, we have not included these systems in our work. HIRSCH:I would like to ask Dr. Guroff the following questions: ( I ) It was remarkable to observe in your lecture that the brain tyrosine uptake decreases if the tyrosine loading is combined with other amino acids, with the exception of lysine, alanine and arginine. I wonder whether other competitive mechanisms are known to be responsible for this exceptional behavior of the three afore-mentioned amino acids? (2) Contrary to this, in your experiments, the tyrosine uptake was increased in the presence of glucose. Is there some active mechanism involved, or is it only a matter of increased energy production? (3) I did not understand the connection between the amino acid and the potassium uptake, and how the intra-cell amino acid content is connected with a decrease in cell potassium. (4) In the newborn animal, according to your experiments, the tyrosine uptake was entirely different from the conditions in the adult animal. Combination with phenylalanine even increased the tyrosine uptake, whereas combination with tryptophan reduced the tyrosine uptake to zero. This can certainly not be due to the brain barrier lacking in newborns, and I wonder whether I may have misunderstood the speaker. GUROFF:The first question was why do some amino acids inhibit and others do not? Lysine, alanine, arginine and glutamic acid are really just indicative of the classes of amino acids. That is, glutamate, aspartate, lysine and arginine do not inhibit, and histidine inhibits only slightly. Alanine and glycine and the smaller monocarboxylic monoamine acids also do not inhibit. In fact, there are a very small number of amino acids which do. The other aromatics and the three long chain aliphatics are good inhibitors. The thought is that they are competitive because they are going into the brain themselves and thus using the sites which would normally be taken by tyrosine. This is a classical competitive situation for a transport carrier, we would like to think, but we have not done the more direct experiments to measure both compounds. The second question was about glucose. This is probably due merely to an increase in the energy production of the slices. Glucose, mannose, and other hexoses which will increase the oxygen uptake of the slices were effective, but succinic, ketoglutarate, etc. which do not increase the oxygen uptake of the slice do not have any effect. Also hexose derivatives which are not metabolized do not have any effect. The third question involves the relationship between potassium and amino acids. This relationship was first observed by Dr. Christensen some years ago in the ascites cell. When amino acids enter the cell potassium leaves and there has been a suggestion but never a confirmation that the mechanism of amino acid transport involves some gearing to a potassium flow in the opposite direction. The intimate chemistry of this gearing is not known. There is now evidence from the work of Dr. Heinz and more recently from Dr. Quastel’s laboratory and from our own that ouabain will inhibit amino acid transport. It is also clear that there is a potassium and sodium involvement in glucose transport and I think the thought is beginning to grow that all these transport mechanisms will be i n some way chemically linked (see Tables VI and VII). What we wanted to show in Tables VI and VII and what you should remember
GENERAL DISCUSSION
245
about these studies is that when we measured the brain-to-plasma ratio, the control tyrosine uptake in that particular case was, I think, 32 ,ug/g. When we had a simultaneous concentration of tryptophan there was no increase i n the tyrosine content of the brain over the endogenous and when we had p-fluorophenylalanine there was a slight increase over the endogenous, but, if you remember the blood concentration was almost three-fold higher, so that the brain-to-plasma ratio in that case was much lower which we consider to be an indication of a competitive action. GIARMAN: I would like to ask Dr. Guroff a couple of questions, too. We have been doing some studies with 5hydroxytryptophan uptake by brain slices in the whole animal, and we have very comparable data from the standpoint of the inhibitors that are involved, the kinetics, the equilibrium-concentration ratio, etc. However, we have found that with phenylalanine the effects of administered 5-hydroxytryptophan upon brain serotonin are reduced, that is, the brain serotonin does not go up so that there seems to be a block in the transport of 5-HTP. Also as Dr. Guroff showed in his slide, with phenylalanine, the endogenous levels of serotonin are somewhat reduced, and we also found that with phenylalanine, the endogenous levels of catecholamines are not changed. I was interested in whether Dr. Guroff and Dr. Udenfriend had done any determinations of the catecholamines in relation to this blockade of tyrosine uptake by phenylalanine? I have one other question. In our studies we found in some in vivo studies that glucose administration in the diet would relieve the phenylalanine block of 5-HTP uptake by the brain as well as, of course, the effect of glucose in virro on the brain slices, and I was wondering whether Dr. Guroff has done any studies with glucose in this respect? GUROFF:I might say that we are. of course. aware of your work and gratified to find that it is such a nice confirmation of ours or ours is a confirmation of yours. Thc first question is, have we done anything similar to the serotonin work in terms of catecholamines? This is almost finished. That is we have all the background work and are now involved in the key experiments. We are not going to use tyrosine, we were going to use leucine as a competitor to see if a labeled dose of tyrosine gave rise to more or less norepinephrine. We have no data about glucose in vivo which I think is a very interesting finding, and I would not even try to speculate as to the mechanism, perhaps you would. Its very unusual. GIARMAN: We have no idea about this mechanism. One thing that has come to our minds that we would like to investigate is simply that possibly with the high levels of phenylalanine in the blood of these animals and also in the brain, the metabolism of the phenylalanine may be required to go by way of transamination. The only substances available in the brain that I know of for such transamination would be a-ketoglutarate and pyruvate. These substances might be reduced under these conditions and, therefore, the brain deprived of these essential metabolites. One could perhaps replenish a-ketoglutarate and pyruvate with the glucose. FOLCH-PI:I would like to ask Dr. Withrow a question. 111 the lowering of C02 with maturation have you thought of the role played by specific lipids especially the sulphatides which appear in large amounts at the time of myelination and which
246
G E N E R A L DISCUSSION
would provide a number of acidic charges which were not present before and thus might displace a certain amount of COs? WITHROW : We have not considered this possibility. We have been concentrating more on the idea that the carbon dioxide which is released from these tissues by acid is in some form other than bicarbonate or carbonic acid, for example, carboamino compounds. We are currently interested in some of the old studies of Conway and Fearon on the barium soluble fraction. These studies were done on muscle and have been confirmed only once in the last 18 years. We are repeating this work in muscle and we will apply it to brain. Thus, we have not considered the appearance of other acid groups, but have concentrated more on changes in the nature of the carbon dioxide containing compounds. 1s that clear? FOLCH-PI:That is clear. Now Dr. Withrow, am 1 not right in assuming that COZ which is being produced in large amounts must necessarily be removed by the blood stream at a comparatively fast rate? Thus, in brain we are dealing with very dynamic conditions, while in muscle we are dealing with much inorc static conditions. I mean by that that the amount of COZ i n muscle might depend upon local variations in inetabolism at that time. The half life of COz in the brain is very short because immediately after COZ is produced it must be removed. WITHROW: This would be consistent with what we know about brain and muscle metabolism. But this does not help us account for the tremendous amount of carbon dioxide in immature muscle and brain in an 8-day-old rat in so far as their carbon dioxide content is concerned. This puzzles us. FOLCH-PI:In considering the amount of tryptophan in the brain, I would like to call attentioil to the fact that proteolipids are especially rich in tryptophan, they contain about 2-3 % tryptophan, which is to my knowledge a much higher content than that found in other mammalian proteins. Now that would mean that at the period of myelination, which is the period Dr. Tyce is discussing, you would have the incorporation of tryptophan into those compounds and I wonder if this should not have to be taken into account in considering the variation of the concentration of tryptophan with age. TYCE:Many factors may contribute to changes in tryptophan concentration in the infant rat, including increasing activity of enzymes involved in tryptophan inetabolism and the incorporation of tryptophan into proteins and proteolipids. However, it is difficult to correlate the sharp decrease in the concentration of tryptophan in the brain in the first 3 days with any one factor: this decrease occurs earlier than the period of myelination at the 5th and 7th day. PLETSCHER : Dr. Bogdanski mentioned that the enzymes responsible for synthesis and breakdown of brain monoamines (e. g . DOPA/5HTP decarboxylase and MAO) are basically similar in all vertebrates. According to recent findings in our laboratories this seems, however, not to be the case for diamine oxidase (DAO). With a very sensitive method (according to Kobayashi and Okuyama, using 1,5-[14C]-cadaverineor 1 ,4-[14C]-putrescineassubstrates) no significant activity of the enzyme could be detected in brain of mammals, amphibia, and reptiles; in birds only very small amounts of DAO were, however, found in the brain of fishes, especially in the diencephalon,
G E N E R A L DISCUSSION
247
prosencephalon, and medulla oblongata. In most brain regions the activity of DAO was of the same order as that of 5-HTP decarboxylase, but about 100 times lower than that of MAO. The results of Dr. Bogdanski together with our findings of a unique occurrence of DAO in brains of fishes and birds possibly demonstrate that in the phylogenetic development the cerebral metabolism of diamines is more variable thin that of monoamines.
General Discussion*
GIARMAN : I would like to ask Dr. Aprison one question that interests me a great deal. We have been studying the schedule of appearance of amines and associated enzymes in prenatal rats following the animal into the postnatal period. We find as far as serotonin is concerned a very marked jog upward in the slope of the curve showing the appearance of serotonin 24 h before birth, at birth, and 24 h after birth and then the curve slopes again to what it was before this period. We also have an interesting suggestion that monoamine oxidase activity ceases to go upward during this period which could correlate well with this increasing appearance of serotonin during birth and I wondered if Dr. Aprison had looked at his animals during this period of time in this regard. APRISON:I do not know why your brain serotonin measurements followed the trend you report unless it reflects the differential rate of formation as well as the activity of the catabolic enzyme, MAO, in distinct brain areas. Karki et al., ( J . Neurochem., 9 (1962) 54) also find at birth a change in the slope of the curve relating serotonin in rat brain to age. It is possible that continuous ‘smooth’ curves were not obtained for serotonin because the measurements were made on total brain instead of specific parts. I have stated i n some of our publications that I do not feel that serotonin may be a neurohumor in the sense that acetylcholine is thought to be a neurohumor. At present I think that serotonin acts as a chemical modulator (Rec. Adv. Biol. Psychiat., 4 (1962) 133). Other than that, I just would not care to speculate further until we have more data. GIARMAN : I was interested in Dr. Bogdanski’s allusion to the ergotrophic-tropotrophic system of Dr. Brodie in relation to his finding differential ratios of serotonin and catecholamines in various species. Certainly the ratio would indicate, according to the theory, that amphibia are tropotrophic animals that live a life of quiet restoration. Our knowledge of 400-year-old turtles would certainly bear this out. I just wondered, however, what role is conceived of for acetylcholine in this system and whether measurements were made in amphibia and other species for acetylcholine and cholinesterase activities. Certainly, acetylcholine must figure prominently in any considerations of parasympathetic representation in the brain. BOGDANSKI : We have not made any measurements of cholinergic mechanisms in any of the species. While we do not disregard the possible importance of cholinergic
* This discussion refers to the papers of Dr. G. Guroff and Dr. S. Udenfriend, Dr. R. E. McCanian and M. H. Aprison, and Dr. B. B. Brodie and D. F. Bogdanski.
244
GENERAL DISCUSSION
systems for some aspects of brain function, we have not included these systems in our work. HIRSCH:I would like to ask Dr. Guroff the following questions: ( I ) It was remarkable to observe in your lecture that the brain tyrosine uptake decreases if the tyrosine loading is combined with other amino acids, with the exception of lysine, alanine and arginine. I wonder whether other competitive mechanisms are known to be responsible for this exceptional behavior of the three afore-mentioned amino acids? (2) Contrary to this, in your experiments, the tyrosine uptake was increased in the presence of glucose. Is there some active mechanism involved, or is it only a matter of increased energy production? (3) I did not understand the connection between the amino acid and the potassium uptake, and how the intra-cell amino acid content is connected with a decrease in cell potassium. (4) In the newborn animal, according to your experiments, the tyrosine uptake was entirely different from the conditions in the adult animal. Combination with phenylalanine even increased the tyrosine uptake, whereas combination with tryptophan reduced the tyrosine uptake to zero. This can certainly not be due to the brain barrier lacking in newborns, and I wonder whether I may have misunderstood the speaker. GUROFF:The first question was why do some amino acids inhibit and others do not? Lysine, alanine, arginine and glutamic acid are really just indicative of the classes of amino acids. That is, glutamate, aspartate, lysine and arginine do not inhibit, and histidine inhibits only slightly. Alanine and glycine and the smaller monocarboxylic monoamine acids also do not inhibit. In fact, there are a very small number of amino acids which do. The other aromatics and the three long chain aliphatics are good inhibitors. The thought is that they are competitive because they are going into the brain themselves and thus using the sites which would normally be taken by tyrosine. This is a classical competitive situation for a transport carrier, we would like to think, but we have not done the more direct experiments to measure both compounds. The second question was about glucose. This is probably due merely to an increase in the energy production of the slices. Glucose, mannose, and other hexoses which will increase the oxygen uptake of the slices were effective, but succinic, ketoglutarate, etc. which do not increase the oxygen uptake of the slice do not have any effect. Also hexose derivatives which are not metabolized do not have any effect. The third question involves the relationship between potassium and amino acids. This relationship was first observed by Dr. Christensen some years ago in the ascites cell. When amino acids enter the cell potassium leaves and there has been a suggestion but never a confirmation that the mechanism of amino acid transport involves some gearing to a potassium flow in the opposite direction. The intimate chemistry of this gearing is not known. There is now evidence from the work of Dr. Heinz and more recently from Dr. Quastel’s laboratory and from our own that ouabain will inhibit amino acid transport. It is also clear that there is a potassium and sodium involvement in glucose transport and I think the thought is beginning to grow that all these transport mechanisms will be i n some way chemically linked (see Tables VI and VII). What we wanted to show in Tables VI and VII and what you should remember
GENERAL DISCUSSION
245
about these studies is that when we measured the brain-to-plasma ratio, the control tyrosine uptake in that particular case was, I think, 32 ,ug/g. When we had a simultaneous concentration of tryptophan there was no increase i n the tyrosine content of the brain over the endogenous and when we had p-fluorophenylalanine there was a slight increase over the endogenous, but, if you remember the blood concentration was almost three-fold higher, so that the brain-to-plasma ratio in that case was much lower which we consider to be an indication of a competitive action. GIARMAN: I would like to ask Dr. Guroff a couple of questions, too. We have been doing some studies with 5hydroxytryptophan uptake by brain slices in the whole animal, and we have very comparable data from the standpoint of the inhibitors that are involved, the kinetics, the equilibrium-concentration ratio, etc. However, we have found that with phenylalanine the effects of administered 5-hydroxytryptophan upon brain serotonin are reduced, that is, the brain serotonin does not go up so that there seems to be a block in the transport of 5-HTP. Also as Dr. Guroff showed in his slide, with phenylalanine, the endogenous levels of serotonin are somewhat reduced, and we also found that with phenylalanine, the endogenous levels of catecholamines are not changed. I was interested in whether Dr. Guroff and Dr. Udenfriend had done any determinations of the catecholamines in relation to this blockade of tyrosine uptake by phenylalanine? I have one other question. In our studies we found in some in vivo studies that glucose administration in the diet would relieve the phenylalanine block of 5-HTP uptake by the brain as well as, of course, the effect of glucose in virro on the brain slices, and I was wondering whether Dr. Guroff has done any studies with glucose in this respect? GUROFF:I might say that we are. of course. aware of your work and gratified to find that it is such a nice confirmation of ours or ours is a confirmation of yours. Thc first question is, have we done anything similar to the serotonin work in terms of catecholamines? This is almost finished. That is we have all the background work and are now involved in the key experiments. We are not going to use tyrosine, we were going to use leucine as a competitor to see if a labeled dose of tyrosine gave rise to more or less norepinephrine. We have no data about glucose in vivo which I think is a very interesting finding, and I would not even try to speculate as to the mechanism, perhaps you would. Its very unusual. GIARMAN: We have no idea about this mechanism. One thing that has come to our minds that we would like to investigate is simply that possibly with the high levels of phenylalanine in the blood of these animals and also in the brain, the metabolism of the phenylalanine may be required to go by way of transamination. The only substances available in the brain that I know of for such transamination would be a-ketoglutarate and pyruvate. These substances might be reduced under these conditions and, therefore, the brain deprived of these essential metabolites. One could perhaps replenish a-ketoglutarate and pyruvate with the glucose. FOLCH-PI:I would like to ask Dr. Withrow a question. 111 the lowering of C02 with maturation have you thought of the role played by specific lipids especially the sulphatides which appear in large amounts at the time of myelination and which
246
G E N E R A L DISCUSSION
would provide a number of acidic charges which were not present before and thus might displace a certain amount of COs? WITHROW : We have not considered this possibility. We have been concentrating more on the idea that the carbon dioxide which is released from these tissues by acid is in some form other than bicarbonate or carbonic acid, for example, carboamino compounds. We are currently interested in some of the old studies of Conway and Fearon on the barium soluble fraction. These studies were done on muscle and have been confirmed only once in the last 18 years. We are repeating this work in muscle and we will apply it to brain. Thus, we have not considered the appearance of other acid groups, but have concentrated more on changes in the nature of the carbon dioxide containing compounds. 1s that clear? FOLCH-PI:That is clear. Now Dr. Withrow, am 1 not right in assuming that COZ which is being produced in large amounts must necessarily be removed by the blood stream at a comparatively fast rate? Thus, in brain we are dealing with very dynamic conditions, while in muscle we are dealing with much inorc static conditions. I mean by that that the amount of COZ i n muscle might depend upon local variations in inetabolism at that time. The half life of COz in the brain is very short because immediately after COZ is produced it must be removed. WITHROW: This would be consistent with what we know about brain and muscle metabolism. But this does not help us account for the tremendous amount of carbon dioxide in immature muscle and brain in an 8-day-old rat in so far as their carbon dioxide content is concerned. This puzzles us. FOLCH-PI:In considering the amount of tryptophan in the brain, I would like to call attentioil to the fact that proteolipids are especially rich in tryptophan, they contain about 2-3 % tryptophan, which is to my knowledge a much higher content than that found in other mammalian proteins. Now that would mean that at the period of myelination, which is the period Dr. Tyce is discussing, you would have the incorporation of tryptophan into those compounds and I wonder if this should not have to be taken into account in considering the variation of the concentration of tryptophan with age. TYCE:Many factors may contribute to changes in tryptophan concentration in the infant rat, including increasing activity of enzymes involved in tryptophan inetabolism and the incorporation of tryptophan into proteins and proteolipids. However, it is difficult to correlate the sharp decrease in the concentration of tryptophan in the brain in the first 3 days with any one factor: this decrease occurs earlier than the period of myelination at the 5th and 7th day. PLETSCHER : Dr. Bogdanski mentioned that the enzymes responsible for synthesis and breakdown of brain monoamines (e. g . DOPA/5HTP decarboxylase and MAO) are basically similar in all vertebrates. According to recent findings in our laboratories this seems, however, not to be the case for diamine oxidase (DAO). With a very sensitive method (according to Kobayashi and Okuyama, using 1,5-[14C]-cadaverineor 1 ,4-[14C]-putrescineassubstrates) no significant activity of the enzyme could be detected in brain of mammals, amphibia, and reptiles; in birds only very small amounts of DAO were, however, found in the brain of fishes, especially in the diencephalon,
G E N E R A L DISCUSSION
247
prosencephalon, and medulla oblongata. In most brain regions the activity of DAO was of the same order as that of 5-HTP decarboxylase, but about 100 times lower than that of MAO. The results of Dr. Bogdanski together with our findings of a unique occurrence of DAO in brains of fishes and birds possibly demonstrate that in the phylogenetic development the cerebral metabolism of diamines is more variable thin that of monoamines.