Abel Lajtha

AMINO ACID AND PROTEIN METABOLISM-VI CEREBRAL COMPARTMENTS OF GLUTAMIC ACID METABOLISM

IN RECENT studies of glutamic acid metabolism in uivo, the fate of tracer amounts of rqlabelled glutamic acid administered intravenously to rats and mice was investigated in experiments of short duration (LAJTHA, BERL and WAELSCH, 1959). The major portion of the amino acid apparently entered brain, liver, kidney and muscle as the acid and conversion to the amide was not an essential prerequisite for exchange across the blood-brain barrier. The metabolic changes which then ensued were quite rapid, for within 2 min after injection of [14C]glutamic acid, the glutamic acid, glutamine, and glutathione* fractions of brain, liver and kidney and the y-aminobutyric acid fraction of brain were significantly labelled; the specific activity of the organ glutamine was closest to that of the dicarboxylic acid. In many experiments with [14c]glutamic acid the specific activity of plasma glutamine was higher than that of glutamine and glutamic acid in the brain and liver. Such a result would be obtained if the plasma glutamine were being formed in organs other than those analysed, or if the glutamine were synthesized from (14C]glutamic acid of higher specific activity than that resulting from the mixing of the administered labelled amino acid with the organ glutamic acid. In addition, the newly formed amide may have been released to the blood before being equilibrated with the organ glutamine. The latter explanation suggests cellular compartmentalization of glutamine synthesis. In this report data are presented on the metabolism of [14C]glutamic acid after intracisternal administration of p4C]glutamic and aspartic acids and p4C]glutamine to rats and monkeys. As in the study of protein turnover and the previous investigations, the animals were exposed for only short periods of time to the isotopic amino acids in order to avoid equilibration of the label and to study the initial fate of the administered glutamic acid. EXPERIMENTAL

Substrate specificity of steady-state amino acid transport in mouse brain slices

Abstract The substrate specificity of transport was studied in mouse brain slices by measuring the inhibition of uptake of individual amino acids by related compounds, and was found to be similar in many ways to that of other systems. More than one amino acid transport site was found to exist, but not a specific one for each amino acid. At least six transport sites could be differentiated, each one utilized by a group of amino acids of similar charge and structure as their primary site of entry. These groups, or classes, are: (1) small neutral, (2) large neutral, (3) small basic, (4) large basic, (5) acidic, and (6) GABA. A particular amino acid seems to have an affinity to more than one transport site, a high affinity to its primary site of transport, and a lower affinity to a secondary site of entry.

AMINO ACID AND PROTEIN METABOLISM OF THE BRAIN—I

SINCE the classical study of SCHOENHEIMER and associates (1939) on the metabolism . of doubly-labelled L-leucine, a study which provided the first estimate of the halflife time of liver protein, the turnover rates of the proteins of many organs have been determined with the aid of labelled amino acids, in particular glycine and methionine (BORSOOK, 1950). Most of the estimates of protein turnover were based on the results of experiments of relatively long duration in which the appearance of the labelled amino acid in or the disappearance from the proteins was followed. However, in such experiments the average half-lifetime of mainly the relatively slowly metabolized protein will be measured; the turnover rates of the rapidly m e t a b o m organ proteins would have to be calculated from the ascending portion of activitytime curves. During long-term experiments, proteins with a short half-lifetime lose part of their label, which is diluted out by the free amino acids of the body. The earlier determinations of turnover values of proteins suffered also from the handicap of the lack of methods for the determination of the concentration and isotope content of the free amino acids from which the organ proteins are derived. Owing to this situation, the estimate of turnover rates had to be based on assumptions as to the nature and dynamics of the isotope pool from which the labelled protein constituents were drawn. Despite these limitations, the investigations demonstrated clearly the relatively high turnover rates of the proteins of organs or tissues such as liver, kidney, or intestinal mucosa; on the other hand, the turnover rates of the proteins of organs such as brain or muscle were in doubt or considered very low. The half-lifetimes of the proteins of the internal organs (spleen, heart, kidney, intestine, and testes) were estimated as close to that of liver proteins (7 days); those of skin, muscle, bone, lung, and brain were assumed to equal 158 days ( S m N and RIITENBERG, 1944; SPRINSON and RITTENBERG, 1949). In such an estimate any small organ with rapidly metabolized protein would, of course, not decisively change the average values derived for the half-lifetime of the slowly metabolized proteins.

PROTEIN METABOLISM OF THE NERVOUS SYSTEM.

Publisher Summary The chapter discusses cerebral protein metabolism, general problems of protein metabolism in other organs and organisms, and some of the recent reviews discussing the areas of importance in cerebral protein metabolism. In the brain, more than in any other organ, the functional metabolism seems to involve changes in the proteins of the organ. The controlling factors of cerebral protein metabolism are of interest, particularly because there is very little regeneration in the central nervous system. Some diseases of the nervous system are apparently caused by the lack of some enzyme proteins normally present. In other diseased states, the presence of abnormal proteins, and structural changes in normal proteins, are found. Also of interest are the reported effects of a number of drugs on cerebral protein metabolism. The control of protein metabolism in all its aspects is the most important problem. It is related to processes of growth, differentiation, degeneration, as well as maintenance of the cells and their protein complement. The growth factor discussed affects one or more of these processes. This factor is specific for a part of the nervous system, but controlling factors of decisive influence may be operative that are specific for other parts of the nervous system, or specific for a cell type in other organs. The knowledge of the mechanism of action of such factors sheds light on the control mechanisms of cellular metabolism.

THE DEVELOPMENT OF THE BLOOD-BRAIN BARRIER*

SOME years ago, it was found in this laboratory that the intravenous injection of glutamic acid into rats and mice did not lead to an increase of the concentration of the dicarboxylic acid in brain, while the administration of glutamine resulted in a significant rise of the amide concentration in this organ (SCHWERIN er al., 1950; WAELSCH, 1951). This observation impressed upon us the necessity for a more extensive investigation of the properties of the blood-brain barrier to supplement our in oioo study of amino acid and protein metabolism in the central nervous system (L,NTHA and FURSF, 1955). Some information on the properties of the blood-brain barrier may be obtained by studyiag the intluence of metabolic inhibitors on the rate of uptake of substances by the brain in the hope that such experiments would lead to an insight into those enzymatic proces9cs, if any, which are responsible for the dynamics of the blood-brain barrier. One may also search for a physiological situation in which the action of the blood-brain barrier is decreased or absent and compare the enzymatic status in such a brain with that in an organ in which the blood-brain barrier is fully operative. Both approaches pressupose that the mechanism responsible for the blood-brain harrier is enzymatic in nature. The immature brain appears to offer a physiological situation in which it may be expected to find the blood-brain barrier absent or not fully effective. Evidence which may be interpreted as indicating a decreased function of the bloodbrain barrier in the immature brain has been summarized and discussed elsewhere ( W ~ C H , 1955) and, therefore, only observations of direct bearing on our findings, presented below, will be considered here. It was found (BEHNSEN, 1927) that larger areas of the brain were stained with trypan blue in younger mice than in adult animals, a finding, however, that has been challenged recently (BROMAN, 1949; GR~NTOFT, 1954). Furthermore, it was found that ferrocyanide was taken up by the brain of the newborn rabbits but not by the brain of newborn guinea-pigs (STWN and PEYROT, 1927). This was interpreted as an indication of the state of maturation of the central nervous system of the respective animal at birth. Of more recent evidence, there may be cited the observation that the rate of entrance into the brain of ammonium hippuratc decreasar by a factor of 4 in rabbits from the sixth to the twenty-first week after birth (DAVSON, 1955). It has also been shown (FRIES and CHAIKOFF, 1941; B-Y, 1953) that the rate of entrance of phosphate (P) decreases during

Control of cerebral metabolite levels: II. Amino acid uptake and levels in various areas of the rat brain

Abstract The distribution of the free amino acid pool was determined in several areas of the rat brain. The regional heterogeneity of the level of amino acids was compared with the regional heterogeneity of amino acid uptake by the same brain areas. The distribution pattern of the free pool varied with the amino acid and with the area analyzed. The regional variation of amino acid uptake in living animals was fairly similar to the variations in the physiological pool. Uptake of amino acids by brain slices also showed regional heterogeneity, with similar behavior within a transport class. It was concluded that regional differences in uptake by slices were due to differences in transport mechanisms rather than differences in cell distribution or available energy. Transport mechanisms seem to have an important, but not exclusive role in determining the regional distribution of the amino acids in the living brain.

Control of cerebral metabolite levels: I. Amino acid uptake and levels in various species

Abstract This study confirmed previous work which indicated that steady-state uptake levels of amino acids in brain slices are primarily regulated by influx and efflux. To determine whether such fluxes also have a controlling role in living brain, levels of brain amino acids from various animals were compared with the uptake of the same amino acids by brain slices. For these studies, brains of newborn and adult mouse and adult rat, guinea pig, hen, and frog were used. In the composition of the cerebral amino acid pool, differences were found between newborn and adult mice, with adult brain containing lower levels of most amino acids except glutamic acid and its related compounds. There was great similarity in the composition of the free amino acid pool of the brain in all species, glutamic acid being the highest in all brains, followed by taurine; compounds at high level were high in all species, and those at low level were low in all the brains examined. There was also similarity between the various species in the uptake of amino acid by brain slices. In each species the order of uptake was phenylalanine Brain slice uptake and physiological brain levels showed parallelism in that amino acids at high levels in the living brain were usually taken up by slices to a higher degree than amino acids at low levels in the brain. In a comparison of newborn and adult mice, and in a comparison of adults of the various species, physiological brain concentrations and uptake by slices were not parallel in all cases, and it was concluded that although factors that can be measured in slices play an important role, the level of amino acids in the living brain is determined by other mechanisms as well as by those that determined the steady-state uptake levels of amino acids in brain slices.

Amino acid and protein metabolism of the brain. IV. The metabolism of glutamic acid.

THE evidence for a significant role of glutamic acid in the metabolism of nervous tissue rests almost exclusively on results obtained with isolated tissue preparations and o n the fact of the high concentration ofglutamic acid in this tissue (for reviews see WAELSCH, 1952, 1957; STRECKER, 1957). Although changes of glutamic acid concentration in insulin hypoglycemia (DAWSON, 1950; HIMWICH and SULLIVAN, 1956) and the results of perfusion experiments (BOWLANGER et al., 1950) are highly suggestive of a rapid glutamic acid metabolism, the rate of the conversion of the dicarboxylic acid has not been studied. Only very recently data on the rate of decarboxylation of glutamic acid t o y-aminobutyric acid* in rico have become available (ROBERTS et al., 1958). In order t o contribute t o the understanding of the in vico metabolism of glutamic acid in the brain, the penetration of 14C-labelled glutamic acid into this organ a s well as its conversion to glutamine, Gaba, and GSH in brain, liver, kidney and muscle was studied. Previous work in this laboratory showed no significant increase of the concentration of glutamic acid in the brains of animals in short time experiments when the concentration of the amino acid in the blood of rats or mice was raised from thirty to fifty times. The glutamic acid concentration of liver and muscle was moderately increased while a large increase was found in the kidneys. On the other hand, when glutamine was administered, the amide concentration increased in all organs, a n increase which was accompanied by an elevation of glutamic acid concentration in liver, kidney and blood. It appears, therefore, that glutamine penetrates into the organs, and especially into brain, a t a higher rate than glutamic acid, and that glutamine is deamidated rapidly to glutamic acid (SCHWERIN, BESSMAN and WAELSCH, 1950). The possibility cannot be dismissed that the failure to observe a net increase of glutamic acid in brain after the administration of the amino acid is the result of rapid metabolism and removal of any additional amounts, although it should be remembered that both in liver and in kidney increases in glutamic acid could be demonstrated after the administration of either the acid or its amide. Recently, in further studies of the amino acid and protein metabolism of the brain, it was found that after systemic administration of minute amounts of the 14C

THE BRAIN BARRIER SYSTEM‐II UPTAKE AND TRANSPORT OF AMINO ACIDS BY THE BRAIN*

THE UPTAKE and release of amino acids by the brain has only recently attracted the interest of the neurochemists. It has been shown that increase in concentration of most amino acids in the brain, after their elevation in plasma, is inhibited. The penetration into the brain of glutamic acid is strongly restricted (SCHWERIN, BESSMAN and WAELSCH, 1950), that of lysine (LAJTHA, 1958) and proline (DINGMAN and SPORN, 1959) fairly strongly restricted, that of glutamine (SCHWERIN et al., 1950) and tyrosine (CHIRIGOS, GREENGARD and UDENFRIEND, 1960) not as much. The restriction is not absolute. Cerebral methionine, histidine, lysine and arginine were found to increase during continuous infusion (KAMIN and HANDLER, 1951). The restriction to uptake, measured with glutamic acid (HIMWICH, PETERSON and ALLEN, 1957) and lysine (LAJTHA, 1958), is not as strong in newborn as in adult animals. Apart from these data, very little is known about the mechanism and the rate of uptake and release under physiological conditions. As part of a study of the mechanism of passage of metabolites into and from the brain, this paper reports changes in the concentrations, of amino acids in the brain after their administration to the intact animal in various ways. It was found that part of the administered lysine and leucine leaves the adult brain against a concentration gradient of elevated plasma levels. Phenylalanine in the adult and leucine in the newborn brain did not show such behaviour under the experimental conditions employed.

The brain barrier system. III. The efflux of intracerebrally administered amino acids from the brain.

THE more closely the brain barrier system is examined, the more its complexity is recognized. I t now appears to be an intricate homeostatic mechanism with metabolitespecific controls that can be influenced, rather than just a passive membrane (see LAJTHA, 1961). Although the uptake of acidic, neutral, and basic amino acids by the brain is impeded when plasma levels are elevated, a rapid exchange of these amino acids occurs between plasma and brain. Such results were obtained with glutamic acid (SCHWERIN, BESSMAN and WAELSCH, 1950; LAJTHA, BERL and WAELSCH, 1959), lysine (LAJTHA, FURST, GERSTEIN and WAELSCH, 1957; LAJTHA, 1958) and leucine (LAJTHA, 1959; LAJTHA and TOTH, 1961). The above findings together with the evidence of competitive inhibition of cerebral tyrosine uptake by other amino acids (CHIRIC~OS, GREENGARD and UDENFRIEND, 1960) and the finding of an increased exchange rate of an amino acid between plasma and brain when the brain level of the amino acid was elevated (LAJTHA and MELA, 1961) pointed to the participation of carrier-mediated rather than passive diffusion processes in the passage of amino acids into and from the brain. This possibility was further underlined by the finding (LAJTHA and TOTH, 1961) of a transport of amino acids from the brain against an elevated plasma level. Further information about the mechanism of passage of cerebral amino acids was hoped to be gained by a study of the egress of amino acids from the brain. In the processes of exit as well as those of uptake, carrier mediated and active transport processes may have a controlling role. This paper presents the results of measurements of the levels of amino acids in the brain at various times after their intracerebral administration.