AMINO ACIDS AND CEREBRAL EXCITABILITY1

Abstract— —Seizure threshold, measured with hexafluorodiethyl ether, was shown to be reduced in immature albino rats fed a diet containing excess tryptophan. Similar diets containing excess glycine, serine, leucine, valine or lysine had no effect on seizure threshold. Previous studies had shown phenylalanine, tyrosine and methionine to enhance cerebral excitability with the same technique. The increased cerebral excitability was shown to occur within 24 hr following diet supplementation with phenylalanine. Brain concentration of hexafluorodiethyl ether was identical in control and experimental animals, although the experimental animals had lowered seizure thresholds; this established an alteration in cerebral excitability rather than variation in tissue penetration by the convulsant. Alterations were found in blood and brain ammonia in serine, lysine and methionine supplemented animals. Brain glutamate and glutamine were lowered in methionine-supplemented animals; however, it was concluded that this effect was not causally related to the increased cerebral excitability.     

Immunosuppressive Activity of the 3a-1 Fraction of Papain

The maximal nonlethal dose of the 3a-1 fraction of papain, determined by use of 9-week-old white albino rabbits, was 10 mg per injection, administered intravenously. The immunosuppressive activity of the 3a-1 fraction of papain was studied by its inhibition of sheep red blood cell hemolysin, bacterial agglutinin, and horse serum precipitin. Immune suppression was observed when papain injections preceded antigen injections by 12 to 18 hr. The enzyme preparation was analyzed by use of cellulose acetate and starch-gel electrophoresis and by the micro-Kjeldahl method. Starch-gel electrophoresis of serum samples revealed qualitative alterations in the alpha(-1) region 7 hr after papain administration. Changes were also observed in the urinary aminopolysaccharide content.     

Correlation of isonicotinic acid hydrazide infiltration across the blood-brain barrier to postnatal development in rats.

Changes in the isonicotinic acid hydrazide (INH) concentration in rat blood and brain were studied in correlation to postnatal development in groups of animals aged 21 and 42 days and 3 months. In the first part of the experiments, INH was administered intravenously to all the age groups in a dose of 100 mg/kg. In the second part, the dose was related to extracellular fluid volume, so that the 3-week-old rats were given 154 mg/kg, the 6-week-old animals 129 mg/kg and the 3-month-old animals 100 mg/kg. After a dose of 100 mg/kg, INH levels in the blood of 21-day-old rats were significantly lower than in 42-day-old and adult animals. The brain INH levels did not differ significantly. On relating the dose to the amount of extracellular fluid, there were no significant differences in the blood INH levels, but the levels in the brain of 21- and 42-day-old rats were significantly higher than in 3-month-old animals. Blood volume related to body weight and brain weight did not differ in the various age groups. The authors conclude that the blood-brain barrier for isonicotinic acid hydrazide alters in rats during postnatal development. In young animals (21- and 42-day-old), more INH infiltrates into the CNS than in adult animals.     

The influence of tyrosine, phenylpyruvate and vitamin B 6 upon seizure thresholds

Abstract— Thresholds to the first appearance of a myoclonic jerk and to the appearance of tonic-clonic seizures induced by the convulsant, hexafiuorodiethyl ether, were examined in immature rats at sequential time intervals following the administration of L-tyrosine, L-phenylalanine, Na-phenylpyruvate and several forms of vitamin B₆. l -Tyrosine failed to lower either seizure threshold even though plasma and brain levels of tyrosine exceeded those obtained with a dose of phenylalanine that was effective in lowering threshold. Na-phenylpyruvate lowered both seizure thresholds at a time that correlated with elevation of brain phenylalanine. Pyridoxine hydrochloride, pyridoxal-5′-phosphate and pyridoxamine phosphate all lowered both seizure thresholds. The time course and dose dependency of the effects of B₆ vitamers were examined; the effect of phenylalanine on seizure threshold was unrelated to derived tyrosine or phenylpyruvate and vitamin B₆ was not involved. The enhanced cerebral excitability following administration of B₆ vitamers is discussed.     

Acute and Long-Term Effects of Chlorpromazine on Glutamine Synthetase and Glutaminase in Rat Brain

The effect of administration of chlorpromazine on the activity of glutamine synthetase and glutaminase and the content of glutamate and gamma-aminobutyric acid (GABA) in different regions of rat brain was studied in an investigation of the possible role of these amino acids in the lowering of the seizure threshold following prolonged administration of chlorpromazine. Chlorpromazine was administered at a dose of 20 mg/kg of body weight s.c. For the acute study, the animals were killed 20 min after a single injection. For the long-term study, the animals were treated every day with the same dose for 21 days and were killed 20 min after the last injection. The results showed an increase in glutamate level in each brain region investigated following long-term administration, but only in the cerebral cortex after a single dose. GABA levels showed an increase in the brainstem only in acute experiments. Glutamine synthetase activity was increased in all three regions after a single dose and only in cerebral cortex after long-term administration. Glutaminase activity showed a decrease in cerebral cortex only after long-term administration of the drug. These results suggest the possible occurrence of a state of increased excitability in the brain as a result of long-term administration of chlorpromazine, thus contributing to the known complication of seizures.     

Acute effects of aspartame on large neutral amino acids and monoamines in rat brain

The dipeptide aspartame (APM; aspartylphenylalanine methylester), an artificial sweetener, was studied $\text{in vivo}$ for its ability to influence brain levels of the large neutral amino acids and the rates of hydroxylation of the aromatic amino acids. The administration by gavage of APM (200 mg/kg) caused large increments in blood and brain levels of phenylalanine and tyrosine by 60 minutes. Brain tryptophan level was occasionally reduced significantly, but the brain levels of the branched-chain amino acids were always unaffected. Smaller doses (50, 100 mg/kg) also raised blood and brain tyrosine and phenylalanine, but did not reduce brain tryptophan levels. At the highest dose (200 mg/kg), APM gavage caused an insignificant increase in dopa accumulation (after NSD-1015), and a modest reduction in 5-hydroxytryptophan accumulation. No changes in the brain levels of serotonin, 5-hydroxyindoleacetic acid, dopamine, dihydroxyphenylacetic acid, homovanillic acid, or norepinephrine were produced by APM administration (200 mg/kg). These results thus indicate that APM, even when administered in amounts that cause large increments in brain tyrosine and phenylalanine, produce minimal effects on the rates of formation of monoamine transmitters.     

Duration of induced seizures during selective pharyngeal brain cooling]

Whole body hypothermia can be used to treat the injured brain (e.g. after hypoxic events). Side effects include hemodynamic instability, coagulopathy and infection. Because of these side effects it appears reasonable to cool the brain selectively (selective brain cooling, SBC) without changing the core temperature. A new animal model was used to demonstrate SBC from the pharynx and to examine effects of SBC on the duration of pharmacologically induced seizure activity. Sprague-Dawley rats (n=18, 12 successful experiments) were sedated and mechanically ventilated. Invasive blood pressure monitoring was instituted and blood gases were drawn to evaluate the arterial blood gas status. Electrical brain activity was recorded using a microneedle in the extracellular compartment of the rat brain cortex. Cooled water was circulated through a small tubing into and out of the pharynx of the animals. The cortical as well as the rectal temperature were recorded. After the injection of a single dose of bicuculline (1 mg/kg i.v.) per animal the duration of the induced seizure activity was measured and compared with the temperature prior to the induction of seizure activity. The cortical blood flow (CBF) was detected using intra tissue Doppler signals in the rat cortex in the same location as the EP-study. The influence of a brain temperature reduction between 36.5 degrees to 31.5 degrees C on the seizure duration was examined. There was a positive correlation between the seizure duration and the cortical temperature (r=0.64). Also the CBF was increased during seizure activity (p=0.02) and the increase correlated weakly with cortical temperature (r=0.18). The core temperature remained in the normothermic range (36.9+/-0.7 degrees C) Conclusion: The duration of induced seizures correlates with local brain temperature. In the future further studies should examine the efficiency of induced (selective) brain cooling to treat prolonged seizure activity.     

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.     

BRAIN SEROTONIN CHANGES IN PHENYLALANINE-FED RATS: SYNTHESIS STORAGE AND DEGRADATION1

The brain serotonin levels of rats maintained on a 5 % phenylalanine diet rose more slowly (0.18 μ g/g brain/hr) after administration of a monoamine oxidase inhibitor than did serotonin levels of controls (0.41 μ g/g brain/hr). The rate of brain serotonin decline following reserpine or dimethylaminobenzoyl methyl reserpate was the same for both groups as was basal monoamine oxidase activity. Brain uptake of monoamine oxidase inhibitor was also the same for both groups. It was concluded that the decrease in brain serotonin levels in phenylalanine-fed animals was due to decreased serotonin formation rather than enhanced degradation. On the basis of available data it was concluded that both hydroxylase inhibition and inhibited precursor transport were involved.     

Correlation between alterations in brain GABA metabolism and seizure excitability following administration of GABA aminotransferase inhibitors and valproic acid—a re-evaluation

The time course of the effects of aminooxyacetic acid, γ-vinyl GABA, γ-acetylenic GABA, gabaculine, ethanolamine-O-sulphate (EOS) and valproic acid (VPA) on brain GABA content and the activities of glutamic acid decarboxylase (GAD) and GABA aminotransferase (GABA-T), the enzymes involved in biosynthesis and degradation of GABA, was re-determined and compared with the action on the electroconvulsive threshold in mice. All drugs caused significant increases in the seizure threshold, and the temporal pattern of this effect correlated rather well with the induced elevation of brain GABA. However, no clear relationship was found between the extent of GABA increase and the relative increase of seizure threshold. Except for VPA, the time course of the increment in brain GABA followed closely the inhibition of GABA-T. The activity of GAD was gradually decreased by γ-acetylenic GABA and a slow decline of GAD activity was also observed after γ-vinyl GABA. EOS and gabaculine suggesting a feedback repression of GAD synthesis by highly elevated GABA concentrations. Concomitant with significant reduction of GAD activity, a decrease in seizure threshold occurred though brain GABA levels remained markedly elevated. On the other hand, following administration of VPA the effect of GABA levels was paralleled by an increase in GAD activity indicating that the GABA-elevating action of this drug can be attributed at least in part to an activation of GABA synthesis. The data suggest that reduction of GAD activity may be an inevitable consequence of increasing brain GABA concentrations over a certain extent and this effect seems to limit the anticonvulsant efficacy of GABA-T inhibitors.     

Brain 2-phenylethylamine as a major mediator for the central actions of amphetamine and methylphenidate.

2-Phenylethylamine (PEA) was measured in rabbit brain by gas-liquid chromatography. D-Amphetamine sulfate (0.65 mg/Kg) initially reduced brain PEA levels to one-third of its usual content (30 min) and subsequently doubled brain PEA (4 hr). Brain PEA levels were reduced (30 min) and subsequently increased (ten-fold at 4 hr) by D-amphetamine sulfate (13 mg/Kg); tolerance to these two effects was observed in rabbits treated for three days with D-amphetamine. Methylphenidate HCl (30 mg/Kg) but not L-amphetamine sulfate (0.65 mg/Kg and 13 mg/Kg) induced a small, non-significant lowering of brain PEA (30 min) followed by a marked augmentation (4 hr) of brain PEA content. D-Amphetamine (30 min or 4 hr prior) increased the recovery of labeled PEA from the brain of rabbits injected intraventricularly with labeled phenylalanine, and reduced the recovery of labeled PEA after its intraventricular injection, suggesting that D-amphetamine accelerates both the synthesis and the disposition of brain PEA. Pretreatment with α-methyldopa (which depletes PEA and other brain amines) or with α-methyldopa hydrazine (which selectively reduces brain PEA content by inhibiting decarboxylase in peripheral tissues only) markedly reduced the CNS effects of D-amphetamine (behavioral stimulation in mice and rabbits, anti-convulsant effect in mice); these decarboxylase inhibitors enhanced the amphetamine-like effects induced by PEA in mice pretreated with a monoamine oxidase inhibitor. The ability of PEA depleters to selectively block the stimulant effects of D-amphetamine, together with the close structural and pharmacological similarities between amphetamine and PEA, and marked influence of amphetamine administration upon PEA brain levels, synthesis and metabolism, suggest to us that many of the central actions of amphetamine may be mediated by endogenous PEA.     

Acceleration of metabolism of stress-related substances by L-carnosine]

Liver dysfunction was produced in the rat by injecting CCl4 subcutaneously in the back twice a week, and the effects of L-carnosine (CAR) on the resulting liver injury were examined. When CCl4 was administered to 6-week-old rats for 9 weeks, GOT and GPT values increased, but these changes were suppressed in the group concomitantly treated with CAR, indicating a protective effect of the agent on liver function. No such preventive effects of CAR was observed in 40-week-old rats, but when the CCl4 administration was discontinued after 4 weeks, GOT and GPT decreased to normal levels within 1 week of discontinuation, indicating a therapeutic effect of CAR on hepatopathy. Based on these findings, we determined the cortisone beta-reductase activity in the rat liver. The increase in this enzyme activity in the group treated with CAR indicated acceleration of cortisone metabolism. Changes of blood cortisol level and cerebral and blood noradrenaline (NA) levels were studied by exposing 6-week-old rats to electric shocks at 30 V. Cortisol released into the circulation after the stress was quickly metabolized in the CAR group and the blood level normalized after 3 hours. Following the release of NA from the brain into the circulation, the NA concentration rapidly returned to the normal level both in the brain and the blood. CAR enhanced the liver function and accelerated the metabolism of stress-related substances also in aged animals. CAR, moreover, restored the RNA contents of the mouse spleen and the immunological abilities represented by PFC reaction, which are reduced by stresses such as forced immersion, fasting, and administration of MMC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypoglycemic activity of bio-tea in mice

Administration of bio-tea (1.71 ml/kg) to normal albino mice caused hypoglycemia after 30 min which reached to maximum after 2 hr with a significant decrease in blood sugar level (BSL) and became normal beyond 8 hr. In alloxan-induced diabetic albino mice, repeated treatments of bio-tea for 3 days (five doses) brought about a significant fall in mean BSL. Continuous decrease in BSL was observed after 4 hr of administration of last dose of bio-tea. Hypoglycemic effect was persistent in alloxan-induced diabetic mice. Effect on glucose tolerance test showed a significant fall in BSL of bio-tea treated animals after 1 hr of glucose treatment indicating hypoglycemic effect of bio-tea.     

Formation of taurine and amino acid pools in rat tissues upon stimulation of NAD synthesis

A single intraperitoneal injection of nicotinamide (500 mg/kg) to mongrel albino rats causes a 6-hour increase in the 2-oxoglutarate level and the free NAD+/NADH ratio in liver mitochondria. The levels of taurine and taurocholates as well the activity of cysteine oxidase in liver tissues remains thereby unchanged, whereas the cysteine transaminase activity diminishes. In the heart and brain of experimental animals the activity of both enzymes is decreased. In the liver, blood plasma and heart of experimental animals, the Ala and Ser levels are low, whereas the taurine content is elevated both in blood plasma and brain. Nicotinamide administration eliminates positive correlations between the levels of taurine, its precursors and metabolically bound amino acids. In the liver the negative correlations between the activities of cysteine oxidase and cysteine transaminase observed in the control group disappear in the experimental group. Apparently, one of regulatory mechanisms of the taurine pool formation in the liver is the ratio of activities of the both enzymes as well as their competition at the substrate level. This emphasizes the importance of the transamination reactions in the metabolism of sulphur-containing amino acids.     

Uptake and Storage of Choline by Rat Brain: Influence of Dietary Choline Supplementation

In order to elucidate the regulation of the levels of free choline in the brain, we investigated the influence of chronic and acute choline administration on choline levels in blood, CSF, and brain of the rat and on net movements of choline into and out of the brain as calculated from the arteriovenous differences of choline across the brain. Dietary choline supplementation led to an increase in plasma choline levels of 50% and to an increase in the net release of choline from the brain as compared to a matched group of animals which were kept on a standard diet and exhibited identical arterial plasma levels. Moreover, the choline concentration in the CSF and brain tissue was doubled. In the same rats, the injection of 60 mg/kg choline chloride did not lead to an additional increase of the brain choline levels, whereas in control animals choline injection caused a significant increase; however, this increase in no case surpassed the levels caused by chronic choline supplementation. The net uptake of choline after acute choline administration was strongly reduced in the high-choline group (from 418 to 158 nmol/g). Both diet groups metabolized the bulk (greater than 96%) of newly taken up choline rapidly. The results indicate that choline supplementation markedly attenuates the rise of free choline in the brain that is observed after acute choline administration. The rapid metabolic choline clearance was not reduced by dietary choline load. We conclude that the brain is protected from excess choline by rapid metabolism, as well as by adaptive, diet-induced changes of the net uptake and release of choline.     

Pharmacological effects of phenylalanine on seizure susceptibility: An overview

The effects of excessive doses of phenylalanine on seizure susceptibility were examined in animal models in the past, primarily because of their relevance to phenylketonuria. It was thought that such effects might involve brain monoaminergic mechanisms. Recently, this issue has been pursued with a renewed interest but for a different reason. The dipeptide sweetener, aspartame, contains a phenylalanine residue. In the last three years, a number of studies involving as many as nine animal models of seizures have reexamined the effects of phenylalanine (and aspartame) on seizure thresholds. Data from these studies are in general aggreement that aspartame at dosage levels below 1,000 mg/kg, or phenylalanine at equimolar doses, is without an effect on seizure susceptibility in animals. When the dosage level of aspartame reaches 1,000 mg/kg, the findings between various laboratories and from different animal models of seizures are inconsistent, showing either no effect or a proconvulsant effect. The Acceptable Daily Intake of aspartame in humans set by the Food and Drug Administration is 50 mg/kg/day. Thus, the data from the excessive bolus doses in rodents do not appear to be relevant to human use. This article provides a detailed review of the data from both early and recent studies and points out the methodological problems apparent at such high doses.     

Transport of alovudine (3'-fluorothymidine) into the brain and the cerebrospinal fluid of the rat, studied by microdialysis

Extracellular unbound concentrations of alovudine were sampled by microdialysis in order to study the transport of alovudine between the blood and the brain and the cerebrospinal fluid (CSF) in the rat. The AUC (area under the curve) ratio CSF/blood was higher than the brain/blood ratio after i.v. infusion of alovudine 25mg/kg/hr after a loading dose of 25 mg/kg in 5 minutes (n=4). Neither i.v. infusion of thymidine (25 mg/kg/hr, n=5; 100 mg/kg/hr, n=2) nor acetazolamide (50 mg/kg i.p. bolus followed by 25 mg/kg i.p. every second hour, n=3) influenced the brain/blood AUC ratio after alovudine 25 mg/kg s.c. injection compared to controls (n=5). Finally, perfusion through the microdialysis probe with thymidine (1000 microM, n=3) had also no effect on the brain/blood AUC ratio after alovudine 25 mg/kg s.c. Because neither thymidine nor acetazolamide has significant influence on the ability of alovudine to penetrate the blood-brain barrier in the rat, neither thymidine transport nor carboanhydrase dependent CSF production appear to be major determinants of the blood-brain concentration gradient. Thus, it is concluded that alovudine reaches the extracellular fluid of the brain not by cerebrospinal fluid, but via the cerebral capillaries and that the existence of a concentration gradient over both blood-brain and CSF-brain barrier can probably be explained by the presence of an active process pumping alovudine out from the brain.     

The effect of pentobarbital on the antinociceptive action of morphine in morphine-tolerant and non-tolerant rats

The interaction of sodium pentobarbital with morphine sulfate in both morphine-tolerant and non-tolerant rats was investigated using the tail-compression test for analgesia. Male Sprague-Dawley rats (300–350 g) were given pentobarbital (4, 8, or 16 mg/kg) 5 min before morphine (2, 4, 6, or 8 mg/kg). Control animals received two saline injections, or pentobarbital plus saline, or saline plus morphine. All injections were subcutaneous. Prior to the first injection, a baseline nociceptive threshold was determined for each rat by applying a modified micrometer to its tail and increasing the pressure until a squeak was elicited. Test readings were taken every half-hour for 2 hr beginning 30 min after the second injection. For the chronic studies, animals were first made tolerant to morphine by the administration of the narcotic twice a day for 3 days, increasing the dose from 10 to 50 mg/kg/injection. Identical testing procedures were then followed with these rats except that the test dose of morphine given on day 4 was in the range 8–128 mg/kg. It was found that Na pentobarbital, in the subanesthetic doses used, had neither antinociceptive nor hyperalgesic properties. Furthermore, the barbiturate had no effect on the antinociceptive action of morphine in either morphine-tolerant or non-tolerant rats.     

Study of the thrombolytic and fibrinolytic properties of thiol- dependent serine proteinase (TSP) from Thermoactinomyces vulgaris in vivo

Experiments on male albino rats showed that the thyol-dependent serine proteinase (TSP) dissolved the thrombus in the jugular vein for 2-5 hr. Intravenous injection of TSP activated the fibrinolytic system of intact animals by increasing the levels of the plasminogen activator and plasmin in the euglobulin fraction. The response of the fibrinolytic system on the intravenous injection of TSP (2mg/200 g) was different: in some rats, fibrinolysis was activated, while on others, it was inhibited. TSP in high doses caused the death of 60% of experimental animals.     

Regional accumulation of aluminium in the rat brain is affected by dietary vitamin E.

The regional accumulation of aluminium in the brain of male albino Wistar rats was investigated following 4 weeks of administration by intraperitoneal injection of aluminium lactate (10mg aluminium/kg body weight). The consequences of concomitant dietary vitamin E (5, 15, or 20 mg vitamin E/g of food) were also studied. Rat brains were dissected into functional regions, for the measurement of aluminium and markers of oxidative stress. Plasma aluminium levels were increased in all groups of animals receiving aluminium lactate (p < 0.01), and these levels were significantly reduced in rats receiving concomitant vitamin E (p < 0.05). In the group of rats receiving aluminium alone, levels of brain tissue aluminium were increased in all regions of brain examined (p< 0.01). Brain tissue aluminium levels were reduced by concomitant dietary vitamin E. Catalase and reduced glutathione levels were both reduced in several regions of brain in animals treated with aluminium (p < 0.05). Aluminium treatment was not associated with a significant increase in reactive oxygen species (ROS) generation (p > 0.05), although ROS production was attenuated by dietary vitamin E (p < 0.05) in some regions.