Forty years of amino acid transmission in the brain.

This article is concerned with the discovery that amino acids, particularly L-glutamate and gamma-aminobutyrate (GABA), are central neurotransmitters. The crucial observations that lead to the conclusion that these two amino acids produce most of the synaptic excitation and inhibition in the central nervous system, were made in late 1950's. The combination of neurochemical knowledge and improved electrophysiological techniques was paramount in making these discoveries possible. In particular, the use of specific antagonists in microiontophoretic experiments provided the most decisive evidence. The relationship is also explored between these early findings and those of the present era characterised by extensive use of techniques of molecular biology and the development of drugs against targets identified 30 to 40 years ago.     

Release of homovanillic acid from the brain of children.

Cerebral arterio-venous differences of homovanillic acid (HVA) and cerebral blood flow were measured in 24 patients, 6 adults and 18 children, anaesthetized by different techniques. A statistically significant release of HVA from the brain (p < 0.001) was demonstrated in five children anaesthetized by barbiturate, nitrous oxide and droperidol/fentanyl. The release rate was 509 pmoles HVA/100 g brain tissue/minute - corresponding to approximately 1.3 mg HVA/24 hours from the whole brain.     

The effect of essential fatty acid deficiency upon fatty acid uptake by the brain.

Young adult rats, either control or essential fatty acid deficient, were administered either [3-H] oleic acid or [3-H] arachidonic acid by stomach tube. In addition, a group of control rats was given [3-H] palmitic acid. The rats were killed at various times therafter, and the radioactivity of the lipids of brain and plasma was examined. In confirmation of previous work, the blood lipid label was found to rise rapidly and then fall, wheras the activity of brain lipids increased slowly and did not show a decline through the 24-h period studied. Analysis of the brain uptake data according to first-order kinetics confirmed the impressions gained from visual inspection of the data. The initial rate of uptake of arachidonic acid was about 4.5 times that of oleic acid in control animals and in deficient animals. Essential fatty acid deficiency, however, did not induce an altered rate of uptake for either oleic acid or arachidonic acid. The rate of uptake of palmitic acid by control rats was not significantly different from that of oleic acid. Even though the initial rates of incorporation of oleic and arachidonic acids were not changed during essential fatty acid deficiency, the final levels of radioactivity obtained in brain lipids were higher in deficient rats with both fatty acids. The plateau value obtained with oleic acid was 1.5 times higher in deficient animals, while the plateau value for arachidonic acid was 1.7 times higher. An experiment in which deficient animals were allowed access to a control diet for 12 or 24 h prior to the labeling experiment suggested that the higher levels of radioactivity found in brain lipids of deficient animals was not due to an isotope dilution effect. Such animals still displayed the labeling pattern of deficient animals with arachidonic acid, while the results with oleic acid varied somewhat. Our results suggest that essential fatty acid deficiency does not alter the ability of the brain to take up the fatty acids studied. However, the fatty acids, especially arachidonic, are retained in the brain to a greater extent in the deficient animals.     

On the formation of gamma-aminobutyric acid from putrescine in brain.

Gamma-aminobutyric acid is not formed in significant amounts from putrescine by incubation with rat brain homogenates. However, it is formed if acetyl-CoA is added to the incubation medium. This is taken as further evidence for the existence of a metabolic pathway in mammalian brain which comprises acetylation of putrescine to monoacetyl putrescine and oxidative deamination of monoacetyl putrescine by MAO. Nerve cells and glia cells have comparable capacities for putrescine degradation along this pathway.     

Pipecolic acid in the dog brain

Pipecolic acid is an intermdiary metabolite of lysine and is decarboxylated to produce piperidine, an endogenous synaptotropic substance. In the present study, the existence of pipecolic acid in the dog brain was confirmed. It was present in highest concentration in the cerebellum followed by the diencephalon and caudate nucleus, and this distribution resembles that of piperidine in dog brain. It seems to be evident that pipecolic acid is a precursor of piperidine in the brain.     

Separation of acid and neutral proteinases of brain.

1. Cerebral proteinases were separated on Sephadex G-100 columns into acid and neutral fractions free from cross-contamination. Acid proteinases were more stable and were purified by additional steps with salt and pH5.0 precipitations, column chromatography on DEAE- or CM-cellulose and free-flow electrophoresis. 2. The separation made it possible to study the properties of the partially purified enzyme fractions. Some of these properties, such as K(m) with selected protein substrates, pH optima and temperature-dependence in the presence and absence of substrates, are described. 3. No requirement for metal ions or added cofactors was demonstrated. Neutral-proteinase activity was more sensitive to inhibition by heavy-metal ions; its activity could be increased by thioglycollate and glutathione, and inhibited by thiol reagents. Neutral and acid proteinases were inhibited by the chymotrypsin inhibitor chloromethyl l-2-phenyl-1-toluene-p-sulphonamidoethyl ketone. 4. In the presence of the appropriate synthetic substrates no cathepsin A activity was found, and only trace quantities of cathepsin B or C activities, which were more than 50-fold less than cathepsin D-like activity.     

Distribution and persistence of kainic acid in brain.

The distribution of ³H-kainic acid in rat brain was studied as a function of time after injections of 5 nmoles into the neostriatum, substantia nigra or cerebellum. More than half of the injected material had disappeared from the injection site and the brain by 1/2 hour post injection. Under the conditions used very small amounts of radioactivity (corresponding to less than 7 pmol/ mg of tissue) were found in areas other than the injection site, suggesting that the histological damage reported in the hippocampus and pyriform cortex after striatal injections may be due to a secondary process not dependent on the presence of toxic concentrations of kainic acid in those areas. No radioactivity was found in the TCA-insoluble material nor did it appear that there was rapid metabolism of the bulk of the kainic acid.     

Accumulation of labeled gamma-aminobutyric acid into rat brain and brain synaptosomes after i.p. injection

The accumulation of labeled GABA into brain and brain nerve endings was studied in the adult rat after i.p. injection of large doses of neurotransmitter (740 mg/Kg). In the first 5–30 minutes after the injection the exogenous neurotransmitter reaches a stable plasma level of around 5 mM. The accumulation of radioactive GABA into the brain presents a latency of a few minutes from the time of the injection. Thereafter, the accumulation of the neurotransmitter is almost linear with time. Once in the brain tissue labeled GABA is in part broken down. The exogenous neurotransmitter is taken up in GABA-ergic nerve endings with a steep increase between 20 and 30 minutes after the injection. From a quantitative point of view, the data show that the brain accumulation of labeled GABA at 30 minutes post injection is minimal in the respect of the steady state average concentration of the endogenous neurotransmitter (0.014%). However, the amount of radioactive GABA which accumulates in the nerve endings, at the same post injection time, is around 7% of the endogenous neurotransmitter in that comparment. The data thus show a selective enrichment of exogenous systemic GABA in a physiologically important compartment of the brain.     

The postnatal methylation of transfer ribonucleic acid in brain. Evidence for the methylation of precursor transfer ribonucleic acid.

Incubation of 3-day-old rat brain with L-[methyl-3H]methionine resulted in the rapid labeling of low-molecular-weight cytoplasmic RNA. Electrophoresis in 15% polyacrylamide gels provided evidence for the methylation of precursor tRNA molecules, and high-performance liquid chromatography demonstrated N2-methylguanine to be the predominant methylated base formed during the first 2 min of labelling.     

Linoleic acid metabolism in brain cortex of aged rats.

The linoleic acid metabolism was examined in the brain cortex of 4 month-old and 24 month-old rats. After the injection of [1-14C]-linoleate into the lateral ventricle of the brain the animals were sacrificed at 1,3 and 6 hours from the injection. The linoleate (18:2) incorporation into lipids, the presence of fatty acid peroxidation products, as well as the 18:2 transformation into elongated and desaturated derivatives were determined. Both an age-related reduction in linoleate incorporation rate into glycerophospholipids and a decrease in fatty acid turnover were found. Furthermore, in glycerophospholipids from 24 month-old rat brain cortex a higher level of hydroperoxide derivative of linoleate was found as compared to 4 month-old animals, and this damaged fatty acid is eliminated more slowly in aged rats than in adults. Finally, unlike 4 month-old animals, a stimulation of the transformation rate of linoleate into desaturation (6,9,12-C18:3) and elongation (8,11,14,C20:3) products was found in 24 month-old rat brain cortex. On the contrary, as far as arachidonic acid (one of the most important end products of the mechanism of linoleate modification) is concerned, the differences between aged and control animals were small, making it quite difficult to attribute a physiological meaning to this phenomenon.     

Activation of acid ribonuclease from bovine brain by aluminum.

An acid ribonuclease (optimum pH 6.0) has been purified from bovine brain in a five-step procedure. The preparation appeared homogeneous on SDS-polyacrylamide gel electrophoresis. The molecular size of the acid ribonuclease is 70 kDa and it is a dimeric protein with a subunit molecular size of 35 kDa. The acid RNase was activated by aluminum at low concentration. Preincubation of the acid RNase with 10 microM increased the specific activity of the enzyme 2.3-fold at acid pH, while the effect of aluminum was much weaker at alkaline pH under otherwise the same conditions. A stoichiometry of 1: 1 for the binding aluminum to brain acid RNase was estimated. None of the enzyme-bound aluminum was dissociated by dialysis against 50 mM HEPES, pH 7.0 at 4 degrees C for 24 h. Citrate, EDTA, NaF, and apotransferrin abolished the effects of aluminum on the enzyme. Ribonucleic acid also protected the enzyme against the activation caused by aluminum. These results suggest that accumulation of aluminum in brain may change the regulation of ribonucleic acid metabolism.