EFFLUX OF PHENYLALANINE FROM RAT CEREBRAL CORTEX SLICES AS INFLUENCED BY EXTRA- AND INTRACELLULAR AMINO ACIDS

Abstract— Effects of other amino acids on the efflux of l -[³H]phenylalanine from rat cerebral cortex slices were studied in a superfusion system. Extracellular large neutral amino acids caused a strong trans-stimulation of [³H]phenylalanine efflux. Some small neutral amino acids were less effective, whereas acidic and basic amino acids and the amino acids without an amino group in the α-position were ineffective. Any trans -inhibition was not detected. The stimulatory trans -effects of phenylalanine and tryptophan were additive, reversible and concentration-dependent. They were apparently mediated by the same mechanisms. The efflux of [³H]phenylalanine was much slower at 273 K than at 310 K, but the effects of unlabelled phenylalanine and tryptophan on it were qualitatively similar at both temperatures. Amino acids accumulated intracellularly at moderately high concentrations did not inhibit [³H]phenylalanine efflux, but phenylalanine, leucine, isoleucine and norleucine caused an enhancement. Spontaneous efflux of [³H]phenylalanine showed some similarities to physical diffusion, but its selective and specific modification by other amino acids strongly suggests the involvement of mediated processes.     

AMINO ACIDS IN SYNAPTIC VESICLES FROM MAMMALIAN CEREBRAL CORTEX: A REAPPRAISAL

Synaptic vesicles were prepared from rat cerebral cortex and separated by gel filtration from small molecular weight compounds contaminating this fraction. Electron microscopy of the vesicle suspension showed that vesicles were by far the most abundant morphological entities. The amino acid content of the purified vesicle fraction was examined and the two amino acids appearing in the most significant amounts were found to be taurine and glutamate. This amino acid pool was not osmotically sensitive as is the vesicular pool of ACh and remained attached to the vesicular protein after passage through Sephadex columns equilibrated in water. However, amino acids added to the vesicle fraction prior to passage through Sephadex did not become associated with this pool and this indicated that the vesicular pool was not likely to be an artifact due to the vesicular protein non-specifically adsorbing amino acids. The release of taurine from incubated synaptosome beds was studied and elevated medium K⁺ (56 mm ) was found to cause a small increase (36 per cent) in the amount of the taurine released to the medium. During the same experiments another physiologically active amino acid, glutamate, was released in more significant amounts, increasing in the medium by 186 per cent. The possible significance of the presence of taurine is discussed.     

BIOACTIVE PROTEINS,PEPTIDES, AND AMINO ACIDS FROM MACROALGAE1

Macroalgae are a diverse group of marine organisms that have developed complex and unique metabolic pathways to ensure survival in highly competitive marine environments. As a result, these organisms have been targeted for mining of natural biologically active components. The exploration of marine organisms has revealed numerous bioactive compounds that are proteinaceous in nature. These include proteins, linear peptides, cyclic peptides and depsipeptides, peptide derivatives, amino acids, and amino acid–like components. Furthermore, some species of macroalgae have been shown to contain significant levels of protein. While some protein‐derived bioactive peptides have been characterized from macroalgae, macroalgal proteins currently still represent good candidate raw materials for biofunctional peptide mining. This review will provide an overview of the important bioactive amino‐acid‐containing compounds that have been identified in macroalgae. Moreover, the potential of macroalgal proteins as substrates for the generation of biofunctional peptides for utilization as functional foods to provide specific health benefits will be discussed.     

EFFECTS OF GLUTAMATE AND OTHER AMINO ACIDS ON THE RETINA1

The application of unlabelled glutamate to the isolated chicken retina charged with [¹⁴C]glutamate caused an increase in the tissue transparency and a release of the label into the superfusion fluid. The processes causing the change in transparency were‘desensitized’by a prolonged application of unlabelled glutamate, whereas the release of the labelled amino acid was relatively unaffected. Mg²⁺ tended to depress the change in transparency caused by stimulation with unlabelled glutamate but had little effect on the release of labelled glutamate from the retina. The effect of a Ca²⁺-free superfusion fluid on the transparency and release of glutamate varied from retina to retina. Aspartate (in higher concentrations) elicited a change in transparency and release of the label in a manner similar to that of glutamate. Glutamine caused a change in transparency accompanied by a release of labelled glutamine and in some experiments the release of a small amount of labelled glutamate. Homocysteic acid elicited marked changes in transparency but no release of labelled glutamate. Pyroglutamate depressed both the change in transparency and the release of labelled glutamate caused by the unlabelled amino acid. Gamma-aminobutyric acid and glycine had no effect on the transparency of the tissue or on the release of amino acids. We have discussed the possibility that a release of glutamate from the intracellular compartment into the extracellular space is involved in the mechanism of spreading depression.     

IONIC BASIS FOR THE DEPOLARIZATION OF CEREBRAL TISSUES BY EXCITATORY ACIDIC AMINO ACIDS

1. Membrane potentials were measured in samples of guinea pig cerebral cortex, before and after small localized additions of neutral solutions of a number of acidic amino acids. 2. Prompt depolarization from resting membrane potentials of about ?60 mv to values of ?30 to ?45 mv occurred in response to l -aspartate, l -cysteate, l -glutamate, dl -homocysteate and l -α-aminoadipate. Slower, smaller and less frequent depolarization followed N-methyl-d -aspartate, N-methyl-dl -aspartate and N-ethyl-D-aspartate. No change was observed to follow N-n-propyl-d -aspartate, N-methyl-dl -glutamate, β-alanine or glutamine. 3. The five amino acids which promptly depolarized, were found also to lower the phosphocreatine of cerebral tissues to which they were added during in vitro metabolism. They also increased the tissues’inorganic phosphate and in some cases diminished its acid-labile nucleotide phosphate. 4. The five amino acids which promptly depolarized increased the intracellular sodium of the tissues within a minute of their addition; two of the N-alkylated acids also had this effect. None of the acids had a comparably prompt effect on the tissues’potassium content. 5. Calculation from the data presented suggests (i) that glutamate increases fivefold the tissues’permeability to sodium relative to that to potassium; and (ii) that the additional sodium entering the tissue could accelerate its Na,K-dependent adenosine triphosphatase sufficiently to account for the observed loss of energy-rich phosphate and appearance of inorganic phosphate.     

脑性瘫痪与血浆游离氨基酸

<正> 脑性瘫痪(Cerebral Hemiplegie)是一组虽发生于婴幼儿时期的先天性疾病,但也涉及后天因素。表现为出生后,出现双侧性肌张力及功能不全,导致非进行性脑性运动障碍及智力发育不全。病理特征是锥体系受损,甚至累及锥体外系、小脑、脑干以及脊髓等,按临床症状分为痉挛型、运动障碍型、共济失调型以及混合型等。     

FATTY ACIDS,AMINO ACIDS,MINERAL CONTENTS,AND PROXIMATE COMPOSITION OF SOME BROWN SEAWEEDS1

This study was conducted to create a nutritional database on brown seaweeds and to popularize their consumption and utilization in Iran. The fatty acid contents, amino acids profiles, and certain mineral elements composition of some brown seaweeds, Padina pavonica (L.) Thivy, Dictyota dichotoma (Huds.) J. V. Lamour., and Colpomenia sinuosa (Mert. ex Roth) Derbés et Solier were determined. Total lipid content ranged from 1.46 ± 0.38 to 2.94 ± 0.94 g · 100 g^?1dry weight (dwt), and the most abundant fatty acids were C16:0, C18:1, C20:4 ω6, and C20:5 ω3. The unsaturated fatty acids predominated in all species and had balanced sources of ω3 and ω6 acids. Highest total polyunsaturated fatty acid (PUFA) levels occurred in C. sinuosa. The protein content of D. dichotoma was 17.73 ± 0.29 g · 100 g^?1dwt, significantly higher than the other seaweeds examined. Among amino acids essential to human nutrition, methionine (Met; in D. dichotoma and P. pavonica) and lysine (Lys; in C. sinuosa) were present in high concentrations. The crude fiber content varied by 9.5 ± 11.6 g · 100 g^?1dwt in all species. Chemical analysis indicated that ash content was between 27.02 ± 0.6 and 39.28 ± 0.7 g · 100 g^?1dwt, and that these seaweeds contained higher amounts of both macrominerals (7,308–9,160 mg · 100 g^?1dwt; Na, K, Ca) and trace elements (263–1,594 mg · 100 g^?1dwt; Fe, Ni, Mn, Cu, Co) than have been reported for edible land plants. C. sinuosa had the highest amount of Ca, Fe, and a considerable content of Na was measured in P. pavonica.     

TRANSAMINATIONS BETWEEN AMINO ACIDS AND KETO ACIDS ELEVATED IN PHENYLKETONURIA AND MAPLE SYRUP URINE DISEASE

Abstract— The transamination between amino acids and aliphatic and aromatic keto acids has been investigated in homogenates of human and rat brain. Tryptophan, phenylalanine and 3,4-dihydroxyphenylalanine (DOPA) at concentrations of 3.6 min and below trans-aminated aromatic keto acids more rapidly than α-ketoglutarate; lower K_m values were found for tryptophan and phenylalanine in the presence of the aromatic keto acid. Rat brain and liver arninotransferases exhibited similar affinities for tryptophan in the presence of different keto acids. Branched chain keto acids were also acceptors of the amino groups of tryptophan and DOPA. In brain homogenates α-ketoglutarate and p -hydroxyphenyl-pyruvate were transaminated by tyrosine and 5-hydroxytryptophan at about equal rates, whereas a-ketoglutarate was transaminated more rapidly with aliphatic amino acids. At concentrations of 1.6 m DOPA and 0.8 mM p -hydroxyphenylpyruvate, transamination was 6-fold greater than the rate of formation of dopamine. The dihydroxyphenylpyruvate formed during arninotransfer from DOPA by brain tissue was not readily decarboxylated, whereas 65–70 per cent of the indolepyruvate formed from tryptophan was decarboxylated. We suggest that an increased rate or degree of transamination between tryptophan and aromatic and branched chain keto acids may explain the increased excretion of non-hydroxylated indolic acids in phenylketonuria and'maple syrup urine'disease, respectively. Increased aminotransfers from tryptophan and DOPA may reduce the amounts of precursors available for the synthesis of serotonin and catecholamines, both of which are at low levels in the sera of untreated phenylketonurics.     

EFFECTS OF TETRODOTOXIN, CALCIUM AND MAGNESIUM ON THE RELEASE OF AMINO ACIDS FROM SLICES OF GUINEA-PIG CEREBRAL CORTEX

Abstract— Tetrodotoxin, Ca²⁺-deprivation and high-Mg²⁺ were used in an effort to identify the portion of the evoked release of endogenous amino acids, labelled via metabolism of [¹⁴C]-glucose, and several exogenous labelled amino acids, that came from nerve terminals when slices of guinea pig cerebral cortex were superfused with glucose-free solutions and stimulated electrically. With some exceptions, spontaneous release of labelled amino acids was decreased by 2 μm -tetrodotoxin but increased in Ca²⁺-free medium and in solutions containing an extra 24 mm -MgCl₂. Tetrodotoxin suppressed 85–90% of the stimulated release of almost all labelled amino acids, but had a smaller effect on the release of endogenous ¹⁴C-labelled threonine-serine-glutamine (unseparated). In Ca²⁺-free solution, the stimulated release of endogenous ¹⁴C-labelled glutamate, aspartate and GABA was suppressed by 80–90%, but that of endogenous ¹⁴C-labelled threonine-serine-glutamine was unaffected as was most of the release of the other labelled amino acids. In medium containing an extra 24mM-MgCl₂, the stimulated release of endogenous ¹⁴C-labelled glutamate, aspartate and GABA was suppressed by 75-85%, that of exogenous labelled aspartate and GABA by 50–65%, but the release of the other labelled amino acids was unaffected. The control stimulated releases of endogenous ¹⁴C-labelled glutamate, aspartate and GABA were much larger than those of other labelled amino acids but were reduced by tetrodotoxin, Ca²⁺-deprivation and high-Mg²⁺ to a level similar to that of the control stimulated releases of the other labelled amino acids. These results suggest that almost all of the stimulated release of endogenous ¹⁴C-labelled glutamate, aspartate and GABA came from nerve terminals while those of the other labelled amino acids came from other tissue elements. In addition, they are in accord with a transmitter role for glutamate, aspartate and GABA in cerebral cortex.     

HETEROTROPHY AND PHOTOHETEROTROPHY BY ANTARCTIC MICROALGAE: LIGHT-DEPENDENT INCORPORATION OF AMINO ACIDS AND GLUCOSE 1

Diatoms isolated from the benthic, planktonic and sea ice microbial communities in Mc Murdo Sound, Antarctica assimilated ambient concentrations of dissolved amino acids and glucose in both the light and dark. Uptake of amino acids but not glucose was influenced by the iucubation irradiance and amino acid uptake rates were up to 250 times greater than those of glucose. Amino acids were incorporated into proteins and other complex polymers and the rates of assimilation and patterns of polymer synthesis were similar to those of the light-saturated photosynthetic incorporation of inorganic carbon. This suggests that these diatoms can use exogenous amino acids to synthesize the essential macromolecules for heterotrophic growth. The assimilation of dissolved organic substrates could supplement light-limited growth during the austral spring and summer as well as potentially support the heterotrophic growth of these diatoms throughout the aphotic polar winter.     

THE AMINO ACIDS OF SOME MISTLETOES AND THEIR HOSTS

The following mistletoes and their hosts were studied: Phoradendron serotinum (Raf.) M. C. Johnst. (P. flavescens [Pursh] Nutt. var. macrophyllum Engelm.) on Juglans hindsii Jeps. (California black walnut), Arceuthobium campylopodum Engelmn. f. abietinum (Engelm.) Gill on Abies concolor (Gord. and Glend.) Lindl. (white fir) and A. magnifica A. Murr (red fir), Arceuthobium campylopodum Engelm. f. campylopodum (Engelm.) Gill on Pinus sabiniana Dougl. (Digger pine), and Amyema pendula (Spleng) Tiegh. on Eucalyptus macrorhyncha F. Muell. ex Benth. (stringybark). With the exception of the Amyema leaves, the mistletoes are richer in free and bound amino acids than their hosts; moreover, their amino acid composition closely resembles that of their hosts. Nevertheless, there is no satisfactory evidence that mistletoe specificity depends on the amino acid composition of the hosts. Some mistletoes contain γ-aminobutyric acid and asparagine, not found in all hosts. All the above mistletoes but no hosts contain free hydroxyproline. These results are discussed in relation to (1) the region in the mistletoes where hydroxyproline is formed and (2) the possibility of using certain amino acid analogs as selective poisons for mistletoes.     

DEVELOPMENTAL TRANSITIONS IN UPTAKE OF AMINO ACIDS BY SYNAPTOSOMAL FRACTIONS ISOLATED FROM RAT CEREBRAL CORTEX

Developmental changes in mechanisms of synaptosomal amino acid transport have been studied in rat cerebral cortex. Well-defined changes over an age continuum could be observed in both the rates of amino acid accumulation and the effects of Na⁺ on the accumulation. The uptakes of five amino acids (threonine, serine and valine in Na⁺-free medium, aspartic acid and proline in Na⁺-containing medium) increased progressively with the age of the animal, whereas the uptakes of leucine and arginine (in Na⁺-free medium) decreased steadily. The uptake of serine or threonine by synaptosomal fractions prepared from newborn rats was markedly dependent on the presence of Na⁺in the incubation media. Na⁺exerted progressively less effect on the accumulation process with continuing postnatal development and to some extent inhibited uptake by fractions obtained from rats older than about 15 days. Na⁺significantly enhanced the accumulation of glycine in fractions from newborn and adult rats, but had only a slight effect in fractions prepared from 12 to 17-day old rats. A detailed study of the accumulation of glycine indicated that the synaptosomal transport of this amino acid proceeded by two independent systems, one of which was totally dependent on external Na⁺and the and adult animals than in fractions from 12 to 17-day-old rats, wheras the Na⁺-independent system was most active during this latter period of development. The decline in the Na⁺-independent accumulation of glycine from about the 15th day to adulthood was characterized by a decrease in the V_max. and an increase in the K_m.     

INCREASED CEREBRAL EXCITABILITY CAUSED BY p-CHLOROPHENYLALANINE IN YOUNG RATS

—Single intraperitoneal doses of p-chlorophenylalanine (p-CP; 100 mg/kg or more), lowered the threshold of flurothyl-induced seizures in 1- and 2-week old rats after 24 h, but not after 4 or 72 h. In older rats there was no change in cerebral excitability after a single dose, but the seizure threshold was lowered after several daily doses. Rats given p-CP in lower doses daily from birth exhibited lowered seizure thresholds only until 3-4 weeks of age. Depletion of brain serotonin and inhibition of liver phenylalanine hydroxylase by p-CP exhibited time courses and dose responses similar to those reported for older animals. Activity of phenylalanine hydroxylase returned to normal more rapidly than the content of serotonin after a dose of p-CP, and recovery of normal seizure threshold seemed to be more closely associated with recovery of the hepatic enzyme than with restoration of the brain amine. It appears, therefore, that hyperphenylalanemia or some consequence of it may be an important factor contributing to increased cerebral excitability in p-CP intoxication and possibly in clinical phenylketonuria and that depletion of brain serotonin may not be the only or even the predominant cause of the changes in cerebral excitability in these conditions.