Sidney Roberts

REGULATION OF CEREBRAL METABOLISM OF AMINO ACIDS—IV. INFLUENCE OF AMINO ACID LEVELS ON LEUCINE UPTAKE, UTILIZATION AND INCORPORATION INTO PROTEIN IN VZVO*

THE relationship between intracerebral levels of free amino acids and the synthesis of brain proteins has not been elucidated. Evidence has been provided from experiments in ciuo that protein synthesis in the central nervous system may be limited by restricted passage of amino acids through the blood-brain barrier (GAITONDE and RICHTER, 1956; WAELSCH and LAJTHA, 1961 ; RICHTER, 1962). In support of this view is the recent observation that the protein synthetic activities of microsomal and ribosomal preparations from rat cerebral cortex were equivalent under optimal conditions to those of similar preparations from rat liver (ZOMZELY, ROBERTS and RAPAPORT, 1964), whereas hepatic proteins were renewed much more rapidly in viva than brain proteins (LAJTHA, FURST, GERSTEIN and WAELSCH, 1957; LAJTHA, 1959). Studies with isolated cerebral microsomes and ribosomes (ZOMZELY et al., 1964) further indicated that protein synthesis in the brain may be unusually sensitive to alterations in levels of amino acids, ions, and other regulatory substances. Similar conclusions may be derived from studies of amino acid incorporation into proteins of brain cortex slices (FOLBERGROVA, 1961 ; MASE, TAKAHASHI and OGATA, 1962). Alterations in the free amino acid composition of cerebral cortex were induced in young adult rats fed diets differing in amino acid content for 4 days (ROBERTS, 1963). In some instances, the cerebral variations reflected differences in plasma levels of amino acids. Concomitant changes were not observed in the total protein content of the cerebral cortex or in the amino acidcomposition of totalcerebral protein. Although these results suggested that protein synthesis in the brain might be relatively independent of alterations in the intracellular pool of free amino acids, differences may have been produced in the rates of synthesis of individual proteins which were not detectable by the methods employed, The present investigations reveal that differences in the distribution of free amino acids in plasma and cerebral cortex, produced by dietary variations or amino acid injection, were accompanied by variations in cerebral protein turnover. METHODS Dietary dejciency. Male rats of an inbred Sprague-Dawley strain were maintained on Purina laboratory chow until they were about 6 weeks old and weighed 160-17Og. They were then fed

Serotonin-sensitive adenylate cyclase activity in immature rat brain

Cell-free preparations from superior and inferior colliculi of very young rats (1-3 days old) contained adenylate cyclase systems which were highly responsive to serotonin. The response to serotonin declined markedly during early development and was very low at maturity. Adenylate cyclase activity in the 10,000 times g particulate fraction from colliculi of newborn rats was significantly stimulated by 0.05 muM serotonin. Half-maximal activation was produced with less than 1 muM serotonin. Maximal stimulation of collicular adenylate cyclase was about 80% above basal enzyme activity and occurred with approximately 50 muM serotonin. Tryptamine and several derivatives of serotonin produced responses which were comparable to that obtained with serotonin; 5-methoxytryptamine was uniformly the most active compound tested. Norepinephrine or dopamine produced much smaller increases in adenylate cyclase activity. Stimulation of collicular adenylate cyclase by serotonin was significantly but incompletely blocked by serotonin antagonists, including d-lysergic acid diethylamide (d-LSD), 2-bromo-d-lysergic acid diethylamide, methysergide, 1-methyl-8 beta-carbobenzyloxy-aminomethyl-10 alpha-ergoline and cyproheptadine. Chlorpromazine also produced partial blockade. In contrast, l-lysergic acid diethylamide, haloperidol, propranolol, phenoxybenzamine and morphine were ineffective as serotonin blocking agents. Of the compounds which produced a partial blockage of serotonin action, d-LSD, cyproheptadine and chlorpromazine were themselves capable of stimulating adenylate cyclase activity. These results are consisent with the existence of multiple receptors in rat brain which are capable of interacting with indoleamines.

REGULATION OF CEREBRAL METABOLISM OF AMINO ACIDS—III

RECENT studies have provided evidence which suggests that the brain is a more active site of protein metabolism than was previously supposed. Thus, the turnover rates in vivo of the more actively metabolizable cerebral proteins may approach those of other extrahepatic tissue proteins (GAITONDE and RICHTER, 1956 ; LAJTHA, FURST, GERSTEIN and WAELSCH, 1957). Nevertheless, amino acid flux into proteins of the adult brain appears to occur at an appreciably slower rate than into liver proteins, both in vivo (WAELSCH and LAJTHA, 1961) and in surviving tissue slices (QUASTEL and BICKIS, 1959 ; ROBERTS, unpublished observations) or cell-free homogenates (GREENBERG, FRIEDBERG, SCHULMAN and WINNICK, 1948 ; LEEPER, TULANE and FRIEDBERG, 1953). Differential rates of protein turnover in different organs may be due primarily to variations in (1) protein metabolism (e.g., associated with tissue renewal, enzyme synthesis, extracellular secretion, etc.), (2) substrate and cofactor availability, or (3) inherent capacity of the protein-synthesizing system. Specifically, the brain would seem to differ significantly from other tissues in the first two characteristics. Thus, protein metabolism in the adult central nervous system is obviously limited by the absence of neuronal replacement (LEBLOND and WALKER, 1956); however, this effect may be partially counterbalanced by continual synthesis of proteins and peptides of an enzymic (HYDBN and PIGON, 1960) and neurohumoral (SACHS, 1963) nature. Additionally, unique permeability barriers appear to restrict cerebral uptake of amino acids and other metabolites required for optimal protein synthesis (WAELSCH and LAJTHA, 1961). Adequate data have not previously been available to permit firm conclusions to be drawn regarding the relative activities and requirements of the protein-synthesizing systems of different tissues. The present investigations reveal that the capacities of microsomal and ribosomal preparations from brain to incorporate amino acid into protein are comparable to those of the corresponding preparations from liver (see also Acs, NEIDLE and WAELSCH, 1961 ; BONDY and PERRY, 1963), but that the requirements of the two systems for optimal activity, and their sensitivities to alterations in the environment, differ.

Interactions between lysergic acid diethylamide and dopamine-sensitive adenylate cyclase systems in rat brain

Investigations were carried out on the interactions of the hallucinogenic drug, D-lysergic acid diethylamide (D-LSD), and other serotonin antagonists with catecholamine-sensitive adenylate cyclase systems in cell-free preparations from different regions of rat brain. In equimolar concentration, D-LSD, 2-brono-D-lysergic acid diethylamide (BOL), or methysergide (UML) strongly blocked maximal stimulation of adenylate cyclase activity by either norepinephrine or dopamine in particulate preparations from cerebral cortices of young adult rats. D-LSD also eliminated the stimulation of adenylate cyclase activity of equimolar concentrations of norepinephrine or dopamine in particulate preparations from rat hippocampus. The effects of this hallucinogenic agent on adenylate cyclase activity were most striking in particulate preparations from corpus striatum. Thus, in 10 muM concentration, D-LSD not only completely eradicated the response to 10 muM dopamine in these preparations but also consistently stimulated adenylate cyclase activity. L-LSD (80 muM) was without effect. Significant activation of striatal adenylate cyclase was produced by 0.1 muM D-LSD. Activation of striatal adenylate cyclase of either D-LSD or dopamine was strongly blocked by the dopamine-blocking agents trifluoperazine, thioridazine, chlorpromazine, and haloperidol. The stimulatory effects of D-LSD and dopamine were also inhibited by the serotonin-blocking agents, BOL, 1-methyl-D-lysergic acid diethylamide (MLD), and cyproheptadine, but not by the beta-adrenergic-blocking agent, propranolol. However, these serotonin antagonists by themselves were incapable of stimulating adenylate cyclase activity in the striatal preparations. Several other hallucinogens, which were structurally related to serotonin, were also inactive in this regard, e.g., mescaline, N,N-dimethyltryptamine, psilocin and bufotenine. Serotonin itself produced a small stimulation of adenylate cyclase activity in striatal preparations and, in relatively high concentration (100 muM), partially blocked the activation by 10 muM dopamine, but was without effect on the stimulation by 10 muM D-LSD. The present results indicate that serotonin antagonists, in general, are potent inhibitors of catecholamine-induced stimulation of adenylate cyclase systems in brain cell-free preparations. In addition, these results, coupled with earlier findings on the capacity of D-LSD to interact with serotonin-sensitive adenylate cyclase systems from rat brain23,24 and other neural systems16, strongly suggest that this hallucinogenic agent is capable of acting as an agonist at central dopamine and serotonin receptors, as well as functioning as an antagonist at dopamine, norepinephrine, and serotonin receptors in the brain.

DEVELOPMENTAL AND REGIONAL VARIATIONS IN NEUROTRANSMITTER-SENSITIVE ADENYLATE CYCLASE SYSTEMS IN CELL-FREE PREPARATIONS FROM RAT BRAIN

Investigations have been carried out on regional and developmental variations in the properties of adenylate cyclase systems in participate preparations from rat brain. EGTA was routinely included in the assay medium to minimize differences in the state of activation of these systems resulting from variations in their exposure to endogenous Ca2+. At birth, adenylate cyclase activity was much higher in the hindbrain‐medullary preparations than in comparable fractions from cerebellum, cerebral cortex or subcortex (including midbrain, corpus striatum, hypothalamus and hippocampus). Adenylate cyclase activity increased during early development in preparations from all areas of the brain. Maximal levels were reached at 14 days of age or later. These levels were not greatly altered in the young adult animal, except in the hindbrain‐medullary area, where a decrease in activity was observed. Adenylate cyclase systems in cerebral cortical and subcortical preparations were activated by norepinephrine and dopamine throughout development. Serotonin also stimulated adenylate cyclase activity in these preparations from young animals but was much less effective in comparable fractions from adult rats. The response to dopamine was diminished with age in cerebral cortical preparations, but not in subcortical fractions. The responses to norepinephrine increased in both brain regions during early development. Adenylate cyclase systems in particulate preparations from the cerebellum and hindbrain‐medullary areas exhibited relatively poor responses to the biogenic amines. Detailed studies of the properties of the cerebral cortical adenylate cyclase systems revealed enhancement of activity by Ca2+ and F− at all stages of development with the maximal activation at 2–3 weeks of age. The results suggest that developmental differences in hormonal sensitivity of adenylate cyclase systems from diverse areas of the brain are related to changes in the proportions of the receptor‐enzyme complexes responsive to the different biogenic amines.

Cerebral protein synthesis. I. Physical properties of cerebral ribosomes and polyribosomes.

Ribonucleoprotein preparations from rat cerebral cortex, isolated in 0·25 M -sucrose, 4 m M -MgCl2, 25 m M -KCl and 50 m M -tris-HCl buffer (pH 7·4), exhibited 3 major sedimenting species in the analytical ultracentrifuge. The S 20,w values and percentage distribution of these cerebral mixed ribosomes averaged 56% of 78 s (monomer), 33% of 114 s (dimer), and 11% of 147 s (trimer). Occasionally, small amounts of tetrameric ribosomes appeared. Little dependence of S 20,w values on ribosomal concentration was noted. Ultraviolet absorption analyses and RNA : protein ratios revealed that this preparation was relatively free of contamination with non-ribosomal protein. When cerebral mixed ribosomes were suspended in media low in Mg 2+ (1 m M or less) and high in K + (100 m M ), a 57 s RNP† component appeared. An additional, smaller RNP particle was noted after dialysis against buffer of low ionic strength (35 s) and after treatment with EDTA (26 s). Inasmuch as the larger and smaller RNP components could be caused to reassociate to RNP particles with normal S 20,w values (79 sand 121 s), they probably represented normal subunits of cerebral ribosomes. When cerebral ribosomes were prepared by alternate methods which produced relatively large amounts of polyribosomes with rat liver, the proportions of trimers and larger aggregates increased. However, unlike hepatic preparations, large polyribosomes predominated in cerebral ribosomal pellets only when the isolation media contained relatively high concentrations of Mg 2+ (10 to 12 m M ) and K + (100 m M ). Resuspension of polyribosomal pellets in buffer containing 1 m M -Mg 2+ and 25 m M -K + markedly increased the proportion of mono ribosomes in cerebral preparations, but had little effect on the physical properties of hepatic polyribosomes. The maximum yield of ribosomal aggregates from cerebral tissue was very low (about 0·1 mg RNA/g tissue). This value was about one-sixth the recovery value for cerebral mixed ribosomes and only about 10% of the maximum recovery value for liver polyribosomes. The results indicate that the ribosomal population of cerebral cortical tissue is very low compared to that of liver, and suggest that cerebral polyribosomes are relatively unstable.

Influence of Elevated Circulating Levels of Amino Acids on Cerebral Concentrations and Utilization of Amino Acids

Publisher Summary The relationship between circulating and tissue levels of various amino acids does not follow any consistent pattern partly as a consequence of: competitive interactions in membrane transport, variations in intracellular metabolism, and differential effects of other regulatory factors. If the restriction is great, parenteral administration of large quantities of an amino acid results in elevated concentrations of this substance in cerebral tissues. Under these circumstances, cerebral concentrations of certain other amino acids may be acutely depressed. Data in the literature suggests that two principal mechanisms might be responsible for competition among amino acids for brain transport systems. The investigations summarized in this report provide evidence that both of these processes were responsible for the acute lowering of cerebral levels of several amino acids in the presence of elevated circulating concentrations of another amino acid. Methods for carrying out the experiments are described in this chapter. The chapter discusses about the dependence of normal brain activity and mental performance on the proper functioning of amino acid metabolism.

ROLE OF RIBONUCLEASE ACTION IN PHENYLALANINE-INDUCED DISAGGREGATION OF RAT CEREBRAL POLYRIBOSOMES

—l‐phenylalanine (1 mg/g body wt) or physiological saline (0.9% NaCl) was given intraperitoneally to infant (7‐day old), immature (14‐day old), and adult (42‐day old) rats. The state of ribosomal aggregation was determined in the cerebral postmitochondrial supernatant and purified polyribosome fractions prepared in the presence of rat liver ribonuclease inhibitor. Polyribosomes isolated from cerebral cortices of infant and immature rats 30 or 60 min after administration of phenylalanine were partially disaggregated, whereas the state of aggregation of polyribosomes from mature cerebrum was unchanged. In contrast, little or no evidence of phenylalanine‐induced polyribosome disruption was noted in the postmitochondrial supernatant fractions, from which the cerebral polyribosomes were prepared, in any of the animals. Omission of the ribonuclease inhibitor resulted in polyribosome disaggregation in the postmitochondrial supernatant fractions prepared from saline‐treated as well as phenylalanine‐treated infant rats, but the disruption was more profound in the latter group. Ribonuclease activities in cerebral postmitochondrial supernatant preparations from infant and immature rats were higher than the corresponding values in preparations from adult animals. In addition, the administration of phenylalanine resulted in increases in ribonuclease activities in cerebral postmitochondrial supernatant preparations from the younger animals, but had no effect on these activities in adult animals. These results suggest that alterations in structure and function of polyribosomes from the infant rat cerebrum following a loading dose of phenylalanine were related to exposure of the polyribosomes during isolation to elevated activities of cerebral ribonucleases resulting from this treatment. This hypothesis was supported by the finding that phenylalanine treatment had no effect on the incorporation in vivo of intracisternally‐administered radioactive lysine into total, soluble or ribosomal protein of infant cerebrum. However, when cerebral ribosomal RNA was differentially labelled in phenylalanine‐treated and saline‐treated infant rats by the intracisternal administration of [3H] or [14C]uridine, and polyribosome fractions were then prepared from the pooled cerebral cortices of both groups, radioactive ribosomes derived from saline‐treated rats were more highly aggregated than those derived from phenylalanine‐treated animals. It is concluded that gross alterations in cerebral polyribosome structure and function do not occur in vivo in young rats given a large amount of phenylalanine intraperitoneally. However, this treatment, in addition to increasing ribonuclease activity in cerebral cell‐free preparations, also sensitizes cerebral polyribosomes to subsequent breakdown upon exposure to ribonucleases during isolation.

Stimulation of protein synthesis in vitro by elevated levels of amino acids

Abstract Alterations in net uptake and exchange of free amino acids and in their subsequent incorporation into protein were produced in rat-liver slices by addition of varying amounts of one amino acid to the incubation medium. When the initial extracellular concentration of l -phenylalanine was changed from subnormal (2 μM) or normal (50 μM) levels to a supranormal level (155 μM), hepatic uptake and flux of this amino acid, as well as the flux of free valine, were increased. Concomitantly, hepatic exchange of leucine was depressed and that of lysine was unchanged. The effects of elevated intracellular levels of phenylalanine on net uptake of the other essential amino acids were masked by a general increase in their accumulation and overall utilization. Elevated levels of phenylalanine were associated with enhanced incorporation or flux of leucine, valine, and lysine into hepatic protein, even though the effect on hepatic exchange of each of these amino acids differed. Graded increases in extracellular levels of l -threonine of l -valine also enhanced incorporation of leucine into hepatic protein. The results indicate that protein synthesis and degradation were stimulated in the presence of elevated intracellular levels of one amino acid.

REGULATION OF CEREBRAL METABOLISM OF AMINO ACIDS—II INFLUENCE OF PHENYLALANINE DEFICIENCY ON FREE AND PROTEIN‐BOUND AMINO ACIDS IN RAT CEREBRAL CORTEX: RELATIONSHIP TO PLASMA LEVELS*

NUMEROUS investigations have revealed a dependence of circulating levels of amino acids upon the previous diet (FRAME, 1958; SCHREIER, 1962). The relationship between dietary and blood levels of amino acids is complicated by variations in intestinal absorption and transport into tissues due to competitive interactions (HIER, 1947; CHRISTENSEN, STREICHER and ELBINGER, 19481, differential requirements (ALMQUIST, 1954; LONCENECKER and HAUSE, 1959), and the influence of other essential food factors (CHARKEY et al., 1953). Furthermore, levels of free amino acids in plasma appear to be profoundly affected by non-dietary factors, including hepatic function (BOLLMAN, MANN and MAGATH, 1924), age (SOUPART, 1962), and hormonal state (RUSSELL, 1955). Even more complex is the interrelationship between blood and tissue levels of amino acids (LUCK, 1928; Wu, 1954). Acute increases in certain free amino acids of cerebral tissues have been produced by parenteral administration of large doses of these substances (SCHWERIN, BWMAN and WAELSCH, 1950; KAMIN and HANDLER, 1951; TIGERMAN and MACVICAR, 1951; LAJTHA, 1958; DINGMAN and SPORN, 1959; CHIRIGOS, GREENCARD and UDENFRIEND, 1960; LAJTHA and Tom, 1961 ; GUROFF and UDENFRIEND, 1962). More subtle changes which may occur as a result of relatively small, chronic, variations in blood amino acid levels have not been systematically investigated. However, cerebral levels of amino acids other than the dicarboxylic amino acids and their derivatives have generally been reported to be relatively resistant to change following deprivation of food (ROBERTS and SIMONSEN. I962), administration of psychotropic drugs (see review by TALLAN, 1962a), or removal of endocrine organs (ROBERTS and SIMONSEN, 1962). The purpose of the present investigation was to determine whether or not cerebral levels of free amino acids were altered in rats fed a phenylalanine-deficient diet which was previously found to result in diminished oxidative utilization of phenylalanine and leucine by cerebral cortical slices in vitro (ROBERTS, ETO and HANKING, 1962). In addition, a study was made of the influence of previous diet on the distribution of free amino acids between plasma and cerebral cortex and the relationship between free and protein-bound amino acids in cerebral cortex.