Jeno Toth

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.

Comparison of turnover rates of proteins of the brain, liver and kidney in mouse in vivo following long term labeling

Intraperitoneal injection of [14C]tyrosine suspension followed by subcutaneous implantation of a [14C]tyrosine pellet in mice produced a fairly constant specific activity of plasma free tyrosine for 5 days, and for 3-5 days in the tissue free amino acid pool. The specific activity of tyrosine in the tissue (brain, liver, and kidney) free amino acid pool was 75-90% of that in plasma. Incorporation of tyrosine into tissue proteins was followed for 5 days in brain; during this time 33% of tissue proteins were labeled. Incorporation for 68 h in liver and kidney showed labeling of over 70% of the protein of these tissues. These percentages assume a homogeneous tissue free tyrosine pool as the precursor. The rate of incorporation initially was 0.6, 2.8, and 2.0% per h in brain, liver, and kidney protein, respectively. These rates decreased in longer term experiments. The best fit to the incorporation curves was obtained by assuming the following average half-lives for tissue proteins: brain, two compartments, 5.7% with a half-life of 15 h, 94.3% with a half-life of 10 days; liver, a single compartment with a 26-h half-life; kidney, two compartments, 41% with an 18-h half-life, and 59% with a 63-h half-life.

THE BRAIN BARRIER SYSTEM—V STEREOSPECIFICITY OF AMINO ACID UPTAKE, EXCHANGE AND EFFLUX

A NUMBER of studies including some from our laboratory provide evidence that the uptake of amino acids by the brain in civo occurs mainly through mediated transport (for a recent review see LAJTHA, 1962). Recently evidence for similar transport was found also in the exit of amino acids from the brain (LAJTHA and TOTH, 1961). Since the cerebral levels of a compound will be determined by mechanisms of efflux as well as influx, it is of interest to establish the relationship of these fluxes in the two directions. It is of obvious theoretical and practical importance to establish the role that mechanisms of exit play in cerebral homeostasis. These studies were initiated as an attempt to find out whether or not the same transport mechanism participates in the uptake as that which participates in the efflux of amino acids in the brain. Because few direct approaches seem to be available for the living animal, a comparison of various properties of the two fluxes were chosen, the present paper being a comparison of the stereospecificity of the fluxes. It is hoped that comparison of the non-metabolizable with the natural analogue will elucidate the possible role of the metabolism of a compound in its uptake. The degree of stereospecificity in the transport of the various amino acids gives important information about the nature of the carrier, or carriers. If it can be shown in addition that the stereoisomers of a compound are specific for the same carrier or for the same transport mechanism, then the comparison of the stereospecificity of uptake with that of efflux may answer the question of whether uptake is mediated by the same or by a different mechanism than efflux.

THE BRAIN BARRIER SYSTEM—IV. CEREBRAL AMINO ACID UPTAKE IN DIFFERENT CLASSES

IN THE preceding papers of this series (LAJTHA and MELA 1961, LAJTHA and TOTH 1961, 1962) some of the properties of the mechanisms determining the passage of amino acids into and out of the brain in viuo have been investigated. It has been shown that this passage occurs mainly through mediated transport in both influx and efflux of the amino acids. Among the properties found were substrate and area specificity, and alterations during development. The present paper reports attempts to answer two questions; One: Is the brain barrier system for amino acids a necessary component of brain in all animals? and Two: Are the properties of the system similar in the various types of brains? The experiments were performed on only two species, representatives of two classes (mouse and carp), and with only a few amino acids. The conclusions, therefore, cannot be generalized until a number of other classes have been studied with a number of other compounds. The conclusions drawn from the present study are that the barrier system for amino acids is probably present in all brains and its properties are similar in the various brains, although some differences exist between the various classes. EXPERIMENTAL

Rates of exchange of free amino acids between plasma and brain in mice

The rate of appearance of label in the brain in mice following the intraperitoneal or intravenous injection of tracer doses of amino acids was measured in short time periods (1–8 min). Amino acid flux varied between 2 and 10 nmol/min per g brain for the amino acids used. Defining half-life as the uptake of labeled amino acid amounting to 50% of endogenous levels, a short half-life (between 3 and 30 min) was found for the essential amino acids. The half-life of the nonessential amino acids varied between 2 and 24 h, depending on their level in brain. Flux (exchange) of an amino acid was increased when the level of amino acids belonging to the same transport class was increased by intracerebral injection. Protein-free diet resulted in decrease in some amino acids, increase in others; flux was altered parallel to changes in brain levels in animals on this diet. The stercospecificity of exchange and the substrate specificity of effects of altered brain amino acids indicate that exchange occurs via mediated transport. Mediated exchange was present in immature brain. Heteroexchange (flow of one amino acid causing the counterflow of a related amino acid) may play an important part in cerebral homeostasis.

Drug-induced changes in the composition of the cerebral free amino acid pool

The effects of insulin, hydroxybutyrate, deoxypyridoxine, chlorpromazine, codeine, morphine, puromycin, and cycloheximide on the composition of the free amino acids in mouse and rat brain were tested. Significant changes occurred in a number of amino acids with most compounds tested; the largest was of alanine (a 50% increase with glucose, a 50% decrease with drugs); histidine was often increased, and the nonessential amino acids were mostly decreased. The pattern of changes was somewhat different in the mouse brain from that in the rat brain. Changes of amino acid levels may participate in the pharmacological action of a number of compounds.