Oxygen free radicals, melatonin, and aging.

The recognized peripheral and central biochemical, neuroendocrinological, behavioral, pharmacological, and clinical actions of melatonin are involved at different levels of an overall concept of the pathophysiology of aging. This conceptual approach combines the biochemistry of oxygen free radicals, the social behavior of the individual and the resulting cellular alterations. This has allowed us to propose antioxidant properties of melatonin that we have experimentally demonstrated.

Possible involvement of a gamma-hydroxybutyric acid receptor in startle disease.

Startle disease or hyperreflexia is an autosomal dominant neurological disorder, with a neonatal onset, characterized by muscular hypertonia and myoclonic jerks, exaggerated by the slightest stimulus. Low concentrations of free gamma-aminobutyric acid (GABA) have been found in the cerebrospinal fluid of two affected infants. The involvement of GABA or its receptors has been raised and the use of GABA-agonist drugs has been suggested. We report a newborn with startle disease who also had a low concentration of GABA in the cerebrospinal fluid. No clinical improvement was observed with progabide, a GABA agonist. Furthermore, a high dose (100 mg/kg) of gamma-hydroxybutyrate (GHB) did not improve muscular stiffness and failed to induce general anesthesia. GHB, currently used as an effective general anaesthetic, is a structural analogue of GABA. It is present naturally at low concentrations in the brain and is regarded as an inhibitory neurotransmitter. Two specific GHB receptors, distinct from the GABA receptors, have been identified in rat brain. Failure to induce general anesthesia with a high dose of GHB suggests that one of these receptors could be involved in startle disease.

Anxiolytic activity of melatonin in mice: involvement of benzodiazepine receptors.

The anxiolytic properties of melatonin are revealed by two behavioral studies. In a free exploratory situation, the holeboard test, melatonin decreased head-dip performance. In an unconditioned conflict test, the light/dark box choice situation, melatonin increased the time spent in the lit box as well as the number of transitions between the two compartments. Melatonin was given in a dose range from 0.5 to 5.0 mg/kg body weight i.p. 30 minutes before testing in daytime. Moreover, the anxiolytic activity of diazepam (2.5 mg/kg i.p.) was evaluated and found to be completely inhibited by the specific benzodiazepine antagonist flumazenil (10 mg/kg i.p. 30 minutes before). In the same manner flumazenil counteracted melatonin activity in the two tests. Involvement of the benzodiazepine/GABAergic system in the anxiolytic activity of melatonin is discussed.

Antioxidant activity of melatonin in mice.

Melatonin (in gum tragacanth as solvent) was administered to mice in the dose range of 100 to 450 mg/kg intraperitoneally. It prevented the increase in plasma glucose resulting from pancreatic toxicity caused by the intravenous administration of alloxan at 40 mg/kg. This action of melatonin was significant and dose-dependent. In parallel work using mouse brain homogenates, melatonin and more so its principal hepatic metabolite, 6-hydroxymelatonin, inhibited the formation of colored products reacting with thiobarbituric acid. Again, this inhibition was significant and dose-dependent. Alloxan-induced diabetes and lipoperoxidation induced by thiobarbituric acid are imputed to the production of oxygen free radicals. The consistent results obtained using these two experimental models show the antioxidant activity of melatonin, both in vivo and in vitro. This effect may be reasonably attributed to the indole structure of the molecule.

Carnitine action on neuromuscular disturbances in the fasting rat: potentiation by L-lysine.

L-Carnitine (oral route) significantly corrects the muscle hypocontractility and hypoexcitability induced in the rat after 5 consecutive days of fasting. This effect is interpreted on the basis of the dual role of L-carnitine as cofactor in the transport of long-chain fatty acids into the mitochondria and as a detoxifying agent of intracellular acyl-CoA. The activity of L-carnitine is increased with the concomitant administration (oral route) of an equimolar dose of L-lysine. In the present experimental conditions, there is a threefold potentiation by comparison with L-carnitine alone. This result is discussed on the basis of the combined effects of an exogenous supply of L-carnitine and endogenous synthesis of L-carnitine from the L-lysine administered at the same time.

Variations of lipid profile in animals caused by adenosine analogs: N6 (amido-3-propyl) adenosine hydrochloride and (carboxamido-3-propylamino)-6-(triproprionyl) 2',3',5'beta (D-ribosyl)-9-purine.

N6-substituted adenosine analogues are powerful inhibitors of lipolysis in the adipose tissues of animals and humans, because of their agonist effect on A1 purine receptors. Using a model of hypertriglyceridemia provoked by intravenous injection of Triton WR 1339, we observed that Agr 529 [N6(amido-3-propyl)adenosine hydrochloride] at 2 mg.kg-1 intravenous in rabbits, and intraperitoneally and orally in rats led to a return of the levels of circulating triglycerides to normal values. In addition, Agr 529 and its prodrug, Agr 540 [(carboxamido-3-propylamino)-6-(triproprionyl)2', 3',5'beta(D-ribosyl)-9-purine] administered to rats at 3 and 30 mg.kg-1, respectively, returned plasma triglyceride concentrations to normal levels. Intravenous administration of Agr 529 to normal rats led to decreased concentrations of plasma fatty acids, phospholipids, triglycerides and total cholesterol as a function of dose. The decrease began at 0.1 mg.kg-1 and was highly significant at 3 mg.kg-1. In the same conditions, the intraperitoneal administration of Agr 529 caused a dose-dependent hypolipemia. There was no apparent effect on cholesterol and on the triglycerides of high density lipoproteins. A kinetic study showed that the antilipemic effect of Agr 529 intravenously injected at 3 mg.kg-1 began 30 minutes after the injection with a maximum effect at 2 hours. The effect persisted up to 8 hours after injection. The present results show that the administration of Agr 529 and Agr 540 to normal animals causes hypolipemia (decrease in fatty acids, phospholipids, triglycerides and cholesterol) and restores induced hypertriglyceridemia. These effects may be attributed to an interaction of the molecules with A1 purinergic receptors of adipose tissue.

Protective effects of gamma-hydroxybutyrate on alloxan induced diabetes in mice.

Gamma-hydroxybutyrate (GHB) administered to mice prior to alloxan antagonizes its diabetogenic action in a dose dependent fashion and up to 96 hours after injection. Results are discussed on the basis of free radical formation by alloxan and of the metabolic property of GHB which is known to increase the rate of operation of the pentose phosphate pathway.

Chronic stress and memory: implication of the central cholinergic system.

Restraint stress for ten days (two times two hours daily) induces a hypersensitivity of the central cholinergic system, reflected by antagonism to amnesia induced by scopolamine at 0.1 mg/kg in a passive avoidance test and by hypersensitivity to the hypothermic effect of oxotremorine at 1 mg/kg. A restraint stress for 30 days, on the other hand, diminishes animal retention in the passive avoidance test and causes a hyposensitivity to oxotremorine-induced hypothermia, reflecting a hypoactivity of the central cholinergic system. An acute 24-hour stress causes no change. The relationship between chronic stress and associated memory deficits is discussed.

Action of N6(amido-3-propyl) adenosine hydrochloride (Agr 529) on plasma corticosterone levels in rats.

N6(amido-3-propyl) adenosine (Agr 529) has central properties analogous to those of adenosine. It increases plasma corticosterone levels in rats as a function of dose and of time. A plateau is reached at the dose of 10 mg.kg-1 one hour after intraperitoneal injection. Treatment with a blocker of adenosine receptors, 8-(p-sulfophenyl)-theophylline, which does not cross the blood-brain barrier, inhibits the increase of plasma corticosterone induced by Agr 529. This increase is also absent in hypophysectomized rats and in rats receiving Agr 529 intracerebroventricularly. Thus it is consistent to believe that Agr 529 increases plasma corticosterone in normal rats by acting on adenosine A2 receptors in the pituitary.

Changes in plasma catecholamine levels after the intraperitoneal and intracerebroventricular administration of adenosine analogues and of clonidine in conscious rats.

When an adenosine analogue, N6-(amido-3-propyl) adenosine hydrochloride (Agr 529) is administered systemically, it causes a substantial release of epinephrine (E) by the adrenal medulla with no change in plasma norepinephrine (NE) levels. Low doses of N6-R-phenylisopropyl adenosine (L-PIA), an agonist of adenosine A1 receptors, has no significant effect on plasma epinephrine levels, which are increased by high doses of the analogue. Low doses of 5'-N-ethylcarboxamido-adenosine (NECA), an agonist of adenosine A2 receptors, however, lead to increases. The intracerebroventricular (icv) administration of Agr 529 leads to a significant decrease in plasma NE without affecting E levels. This action is synergistic with that of clonidine icv. The mechanism of central action of this effect is discussed. It can be admitted that the inhibitory effect of adenosine analogues icv occurs via an inhibition of the posterolateral hypothalamus and the locus coeruleus, although we cannot rule out an action on more rostral-situated brain regions. The authors suggest that the central effect of Agr 529 results primarily from the inhibition of central acetylcholine release caused by adenosine analogues and review experimental arguments enabling this hypothesis to be supported. On the other hand, the considerable release of plasma epinephrine seen when adenosine analogues are administered systemically can be attributed to their stimulation of ACTH secretion by the pituitary and the increase in glucocorticoids in the adrenal medulla, as a result of the existence of the cortico-medullary portal system. These glucocorticoids stimulate phenylethanolamine-N-methyltransferase, which transforms norepinephrine to epinephrine.

[New route of 2-N-(carboxypropylamino)-2-deoxy-D-glucopyranose synthesis]. / Nouvelle voie de synthèse de 2-N-(carboxypropylamino)-2-désoxy-D-glucopyranose.

A new route to 2-N-(carboxypropylamino)-2-deoxy-D-glucopyranose synthesis is reported, involving the Schiff-base derivative of beta-D-glucosamine with benzyl 4-oxobutyrate. Improvement in the synthesis of benzyl-4-oxobutyrate is also described. Biological compounds action was investigated by evaluating sedative and dopaminergic activities on male mice.

Brain tyrosine increases after treating with prodrugs: comparison with tyrosine.

After mice had been treated with L-tyrosine, O-phospho-L-tyrosine, L-tyrosine methyl ester or N-acetyl-L-tyrosine, tyrosine was assayed by HPLC coupled with fluorometric detection. O-Phospho-L-tyrosine behaved as a tyrosine prodrug after its hydrolysis by acid and alkaline phosphatases. After the intraperitoneal administration of O-phospho-L-tyrosine or the methyl ester, there was a substantial increase in bioavailability in terms of the effect of tyrosine. The two prodrugs were as powerful as tyrosine following oral administration. N-Acetyl-L-tyrosine was the least effective prodrug tested. The stability, solubility and bioavailability of O-phospho-L-tyrosine are consistent with proposing it for use as a tyrosine prodrug. In addition, it can be used parenterally. The use of a tyrosine aminotransferase inhibitor is necessary for limiting the hepatic breakdown of tyrosine and for increasing its bioavailability.

[Action of PMA (phorbol myristate acetate), scopolamine, propranolol, and oxotremorine on memorization of an active or passive avoidance test]. / Action du PMA (phorbol-myristate-acétate), de la scopolamine, du propranolol, et de l'oxotrémorine sur la mémorisation d'un test d'évitement passif ou actif.

The role of various second messengers in the learning and retention of a passive or active avoidance has been investigated in mice. Scopolamine at 3 mg/kg i.p. inhibits muscarinic M1 and M2 receptors and thus acetylcholine activation of the phosphoinositide cycle. This results in amnesia of passive avoidance but has no effect on active avoidance learning. Oxotremorine at 0.05 mg/kg i.p., whose preferential M2 muscarinic action limits acetylcholine release and also inhibits adenylate cyclase activity, causes amnesia of the retention of a passive avoidance and antagonizes the learning of an active avoidance. DL-propranolol at 40 mg/kg i.p., which inhibits cAMP formation, does not affect retention of a passive avoidance but antagonizes that of an active avoidance. Similarly, phorbol myristate acetate a 0.1 mg/kg i.p., which activates protein kinase C, has no effect on the retention of a passive avoidance but antagonizes that of an active avoidance. The results tend to show a distinct role for cAMP-dependent protein kinase, which would participate in memorization processes of an active avoidance, and for protein kinase C, which would participate in that of a passive avoidance. The authors discuss the involvement of different neurophysiological mechanisms as a function of the type of behavior, depending on whether or not it is related to the control of environmental situations.

[The inhibition of action. Interdisciplinary approach of its mechanisms and physiopathology]. / L'inhibition de l'action. Approche interdisciplinaire de ses mécanismes et physiopathologie.

The author recalls his group's work during the three past decades and how, starting from the study of traumatic states, he established the distinction between shock and stress, physiologically and biologically speaking. According to him, stress supposes a memory process, i.e. the learning of the inefficiency of action in controlling the environmental characteristics. This learning involves cerebral areas and nerve tracks which, as behavioural consequences produce action inhibition. In the neurophysiological field, he underlines the importance, for these mechanisms, of the cortex, dorsal Ammon's horn, lateral tonsil and subthalamic nucleus. He shows why it seems obvious that the mediators of the action inhibiting system are acetylcholine and serotonin. He also recalls the many experimental facts which allow to say that the action inhibiting system is responsible for the release of the hypothalamic-hypophyseal-adrenocortical reaction and of the peripheric sympathetic adrenergic reaction to aggression. He finally shows the function of these two systems where the main pathological accidents originate. He also stresses the discovery and the introduction in the therapeutical paraphernalia of a new molecule minaprine, which he considers as inhibiting action inhibition, and as such, as an indirect anti-depressive drug. The pathology, which is said psychosomatic, thus becomes a pathology of action inhibition, where unconscious memory of past interdictions and failures, relates the subject and his present reaction to environment, to his whole psycho-social past.

Hepatic tyrosine aminotransferase inhibitor affects the bioavailability of exogenous tyrosine.

This study demonstrates the importance of controlling the activity of hepatic tyrosine aminotransferase when tyrosine is administered orally. The specific inhibition of this enzyme is proposed in the clinical use of tyrosine in syndromes related to deficient catecholaminergic transmission.

Action of adenosine on energy metabolism and on glucose-6-phosphate dehydrogenase in rat brains.

The Warburg method was used to study the action of adenosine on several phases of rat cerebral cortex metabolism, using cortex slices or homogenates. In the presence of exogenous glucose in vitro, oxygen consumption and lactate production are not affected by adenosine in sections. In vivo, there is an increase of oxygen consumption and of lactate production but which are not significant. Adenosine may activate metabolic pathways, since the observed metabolic changes remain constant during the period of activity of adenosine (30 to 60 min) and disappear concomitantly with adenosine. The action of adenosine is much more evident in sections from the brains of injected animals, where the increase of lactate production becomes significant. This suggests that in this case adenosine favors a better utilization of glycogen via an activation of adenylate cyclase. The increased activity of G-6-PDH was observed in vitro but was not significant in vivo. These observations were confirmed with homogenates from the in vivo series by the significant decrease of inorganic phosphate levels, consistent with an increased formation of nucleotide phosphates. The increased cerebral glucose concentration is perhaps a result of increased blood glucose levels, in turn resulting from the known depression of insulin release by adenosine, or from a preferential utilization of glycogen, resulting from the activation of adenylate cyclase.

Effect of 3-(2-morpholinoethylamino)-4-methyl-6-phenyl pyridazine dihydrochloride (minaprine) on plasma cortisone levels of physostigmine treated rats.

3-(2- morpholinoethylamino )-4-methyl-6-phenyl pyridazine dihydrochloride (minaprine), has recognized clinical activity against inhibition of action. In rats, this substance antagonizes the increase of plasma corticosterone levels caused by physostigmine injection. The mechanism of this action is discussed.