Georgina Rodríguez de Lores Arnaiz

Glutamic acid decarboxylase inhibition and ultrastructural changes by the convulsant drug allylglycine

Abstract The effects in vivo and vitro of the convulsant drug allylglycine on the activity of glutamic acid decarboxylase (GAD), aminobutyrate aminotransferase, glutamine synthetase and aspartate- and alanine-aminotransferases of the rat cerebral cortex were studied. The most significant result was the finding of an inhibition of GAD, during the period of convulsion, which was even greater by addition in vitro of the drug. This inhibition is not by way of the cofactor, pyridoxal phosphate. Preincubation of the homogenate with allylglycine enhanced the inhibition, while preincubation in buffer-substrate produced a protective effect. The inhibition of GAD was correlated with a decrease of 40 per cent in the concentration of γ-aminobutyric acid in the cerebral cortex. In the convulsant rat, ultra-structural alterations of some nerve endings of the cerebral cortex were observed. After cell fractionation, such altered nerve endings were preferentially found in the GAD-rich (nonaminergic) fraction of isolated nerve endings. The possible mechanism of the convulsions induced by allyglycine is discussed and a specific effect on GAD-rich inhibitory nerve endings is postulated.

Species differences in subcellular distribution of choline acetylase in the CNS. A study of choline acetylase, acetylcholinesterase, 5-hydroxytryptophan decarboxylase, and monoamine oxidase in four species.

THE isolation of ACh-rich and ACh-poor nerve endings from the CNS, and the demonstration that ACh is concentrated in a fraction rich in synaptic vesicles, made it of interest to study the subcellular localization of ChAc in those fractions. In rat brain, a direct correlation between ACh content and ChAc activity in the two nerve ending fractions was observed; this result indicates that both the transmitter and the synthesizing enzymes are topographically related within the synaptic complex. A similar close relationship was found in the fraction containing the synaptic vesicles, which were separated from osmotically disrupted nerve endings. In spite of some solubilization, ACh and ChAc were found to have 3.6and 5.6-fold concentrations, respectively, in synaptic vesicles to the total homogenate (DE ROBERTIS, PELLECRINO DE IRALDI, RODR~GUEZ DE LORES ARNAIZ and SALGANICOFF, 1962; DE ROBERTIS, RODRiCUEZ DE LORES ARNAIZ, SALGANICOFF, PELLECRINO DE IRALDI and ZIEHER, 1963). WHITTAKER, MICHAELSON and KIRKLAND (1964), using osmotic shock and a gradient technique to separate the vesicular fraction from guinea pig brain, confirmed our results for ACh but found that ChAc was not related to the synaptic vesicles. According to them, ChAc is essentially a soluble enzyme which is similar to lactate dehydrogenase and potassium in its subcellular distribution. These authors appeared to question the validity of our results without giving consideration to a possible species difference or taking into account the different technical approaches used. In view of this conflicting report, we have reinvestigated the localization of ChAc in the rat and guinea pig brain and extended this study to the rabbit and pigeon. A new and more precise technique for ChAc was used (MCCAMAN, 1963; MCCAMAN and HUNT, 1965). In this micromethod [14C]acetyl CoA and choline are used as

ALTERATION OF GABA SYSTEM AND PURKINJE CELLS IN RAT CEREBELLUM BY THE CONVULSANT 3-MERCAPTOPROPIONIC ACID

Abstract— The effect of the convulsant, 3‐mercaptopropionic acid (MP) on the content of free amino acids and on the activity of some enzymes related to their metabolism was studied in the rat cerebellum. A decrease in the activity of glutamate decarboxylase (EC 4.1.1.15) and in the level of GABA was found; at the same time, the activity of GABA‐aminotransferase (EC 2.6.1.19) was increased. These changes coincided with a profound alteration of the morphology of the Purkinje cells which was related to the dose of MP. These findings, plus some changes in the content of other free amino acids and the activities of related enzymes, suggest that 3‐mercaptopropionic acid induces in the cerebellum an imbalance among the amino acids involved in the excitation‐inhibition mechanisms.

NEUROCHEMICAL AND STRUCTURAL STUDIES ON THE MECHANISM OF ACTION OF HEMICHOLINIUM-3 IN CENTRAL CHOLINERGIC SYNAPSES

—The action of hemicholinium‐3 (HC‐3) on the cerebral cortex of the rat was studied after subarachnoidal administration. There was a marked decrease of content of ACh in nerve endings and especially in the fraction containing synaptic vesicles, despite the fact that the number of synaptic vesicles was not reduced, as judged by electron microscopy, by the rate of incorporation of ortho [32P]phosphate, and by the phosphorus content of the phospholipids of the isolated synaptic vesicles. There was a close association of [l4C]HC‐3 and of monoaminoxidase, which indicated that the drug was preferentially bound to mitochondria. Experiments indicating that HC‐3 could be acetylated suggested that this drug may compete with choline not only for entry but also for acetylation.

Characterization of synaptosomal membrane Na+, K+-ATPase inhibitors

Previous work carried out in this laboratory has led to the isolation from rat brain of an aqueous soluble fraction (peak II) inhibiting synaptosomal membrane Na+, K(+)-ATPase and possessing other ouabain-like properties. Brain peak II was subjected to several treatments or fractionation by reversed-phase or anionic exchange HPLC and the effect of resultant fractions tested on synaptosomal membrane ATPase activity. The inhibitory components proved highly hydrophilic since they were neither extracted by hexane nor retained by a C-18 HPLC column, ruling out a lipidic nature. By anionic exchange chromatography, peak II was separated into eight fractions, two of which, named II-A and II-E, presented inhibitory activity, had low molecular weight, reacted with ninhydrin and were sensitive to acid hydrolysis. Fraction II-A was further chromatographed through a C-18 column, rendering five fractions, II-A1 to II-A5, inhibitory activity being confined to the most hydrophilic one (II-A1). Fraction II-E seems non-peptidic in nature, and its inhibitory activity was completed lost by alkalinization. II-E differs from authentic ouabain in u.v. spectrum, chromatographic behaviour and alkali sensitivity. It is suggested that two small hydrophilic compounds, probably one peptidic (II-A1) and another non-peptidic (II-E) in nature are involved in the regulation of Na+, K(+)-ATPase activity at the synaptic membranes.

In search of synaptosomal Na^+, K^+ATPase regulators.

The arrival of the nerve impulse to the nerve endings leads to a series of events involving the entry of sodium and the exit of potassium. Restoration of ionic equilibria of sodium and potassium through the membrane is carried out by the sodium/potassium pump, that is the enzyme Na+,K+-ATPase. This is a particle-bound enzyme that concentrates in the nerve ending or synaptosomal membranes. The activity of Na+,K+-ATPase is essential for the maintenance of numerous reactions, as demonstrated in the isolated synaptosomes. This lends interest to the knowledge of the possible regulatory mechanisms of Na+,K+-ATPase activity in the synaptic region. The aim of this review is to summarize the results obtained in the authors laboratory, that refer to the effect of neurotransmitters and endogenous substances on Na+,K+-ATPase activity. Mention is also made of results in the field obtained in other laboratories.Evidence showing that brain Na+,K+-ATPase activity may be modified by certain neurotransmitters and insulin have been presented. The type of change produced by noradrenaline, dopamine, and serotonin on synaptosomal membrane Na+,K+-ATPase was found to depend on the presence or absence of a soluble brain fraction. The soluble brain fraction itself was able to stimulate or inhibit the enzyme, an effect that was dependent in turn on the time elapsed between preparation and use of the fraction.The filtration of soluble brain fraction through Sephadex G-50 allowed the separation of two active subfractions: peaks I and II. Peak I increased Na+,K+- and Mg2+-ATPases, and peak II inhibited Na+,K+-ATPase. Other membrane enzymes such as acetylcholinesterase and 5′-nucleotidase were unchanged by peaks I or II.In normotensive anesthetized rats, water and sodium excretion were not modified by peak I but were increased by peak II, thus resembling ouabain effects.3H-ouabain binding was unchanged by peak I but decreased by peak II in some areas of the CNS assayed by quantitative autoradiography and in synaptosomal membranes assayed by a filtration technique. The effects of peak I and II on Na+,K+-ATPase were reversed by catecholamines. The extent of Na+,K+-ATPase inhibition by peak II was dependent on K+ concentration, thus suggesting an interference with the K+ site of the enzyme. Peak II was able to induce the release of neurotransmitter stored in the synaptic vesicles in a way similar to ouabain. Taking into account that peak II inhibits only Na+,K+-ATPase, increases diuresis and natriuresis, blocks high affinity3H-ouabain binding, and induces neurotransmitter release, it is suggested that it contains an ouabain-like substance.

Catechol-o-methyltransferase in nerve endings of rat brain

Abstract The distribution of COMT was studied in subcellular fractions of rat brain. About 50 percent of the enzyme is particulate and concentrated in the nerve ending fractions. The solubility of the enzyme after the osmotic rupture of the nerve ending makes impossible a finer localization of the enzyme within the synaptic complex. The difference in localization with MAO and the probable physiological action of the two enzymes on brain catecholamines are discussed.

Regulation of (Na+, K+) adenosinetriphosphatase of nerve ending membranes: action of norepinephrine and a soluble factor.

Norepinephrine added in vitro to nerve ending membranes from rat cerebral cortex stimulates the activity of (Na+, K+) adenosinetriphosphatase (ATPase) only in the presence of the soluble brain fraction. In its absence norepinephrine inhibits the enzyme. (Mg2+)ATPase also showed stimulation by norepinephrine in the presence of the soluble fraction, but of lesser magnitude. The activation of (Na+, K+)ATPase by norepinephrine is not reproduced by cyclic AMP and is not antagonized by either α- or β-adrenergic blocking agents. These results suggest that the stimulation caused by norepinephrine is a direct effect on the enzyme and is not mediated by cyclic AMP or adrenergic receptors.Norepinephrine added in vitro to nerve ending membranes from rat cerebral cortex stimulates the activity of (Na+, K+) adenosinetriphosphatase (ATPase) only in the presence of the soluble brain fraction. In its absence norepinephrine inhibits the enzyme. (Mg2+)ATPase also showed stimulation by norepinephrine in the presence of the soluble fraction, but of lesser magnitude. The activation of (Na+, K+)ATPase by norepinephrine is not reproduced by cyclic AMP and is not antagonized by either α- or β-adrenergic blocking agents. These results suggest that the stimulation caused by norepinephrine is a direct effect on the enzyme and is not mediated by cyclic AMP or adrenergic receptors.

STRUCTURAL COMPONENTS OF THE SYNAPTIC REGION

The synaptic region may be defined as the site of contact between two excitable cells having a specific structural, biochemical, and functional differentiation for the transmission of nerve impulses. As suggested by Du Bois Raymond in 1877, synaptic transmission may be either chemical or electrical, and both mechanisms have been found to occur in the peripheral and central nervous systems. However, chemical synapses are by far the more common and are the only ones that will be considered here from the point of view of their structural and biochemical organization.

Effect of angiotensin-(1–7) on ATPase activities in several tissues

The present investigation was undertaken to determine whether Ang-(1-7) is able to modify ATPase activities in membrane fractions prepared from several tissues. In the presence of 10(-6) M Ang-(1-7), total (Na , K+, Mg2+)-ATPase activity decreased 31% in rat atrium and 13% in sheep atrium but was unmodified in sheep liver, rat ventricle or crude brain membranes. In rat brain synaptosomal membranes, Ang-(1-7) at 10(-8) and 10(-7) M concentrations activated Na+, K+-ATPase 20 and 24%, respectively. Rat kidney Na+, K+-ATPase activity decreased roughly 40-70% with 10(-10)-10(-6) M Ang-(1-7)), but increased 22% with 10(-12) M peptide concentration, thus indicating a biphasic effect. Our findings showing that ATPase from several tissues responds differently to Ang-(1-7) are attributable to enzyme tissue specificity.