Effect of acetyl-l-carnitine on extracellular amino acid levels in vivo in rat brain regions

Acetyl-l-carnitine (ALCAR) was found to have beneficial effects in senile patients. In recent years many of its effects on the nervous system have been examined, but its mechanism(s) of action remains to be elucidated. We previously reported that it causes release of dopamine in the striatum. In the present paper we report that ALCAR, when administered at intracerebral sites via microdialysis, stimulates the release of amino acids in a concentration-dependent and regionally heterogeneous manner. The effect was strong in the striatum and cerebellum, less so in the frontal cortex, and weak in the thalamus. Seven amino acids were measured: the increase in the level of aspartate, glutamate, and taurine was substantial, and the increase in the level of glycine, serine, threonine, alanine, and glutamine in the microdialysate was minor. The stimulatory effect of ALCAR on the release of amino acids in the striatum was inhibited by the muscarinic antagonist atropine, but was not inhibited by the nicotinic antagonist mecamylamine. The effect of ALCAR on the levels of most of the amino acids tested was independent of the presence of Ca²⁺ in the perfused. These results indicate that ALCAR, when administered intracerebrally at fairly high concentrations, can affect the level and the release not only of such neurotransmitters as acetylcholine and dopamine, but also of amino acids. The mechanism of action of ALCAR on the release of cerebral amino acids may involve the participation of muscarinic receptors or may be mediated through the release of dopamine, but the lack of Ca²⁺ dependence indicates a release from the cytoplasmic amino acid pool, possibly through the effect of ALCAR on cell membrane permeability.     

Comparison of the Effects of a Convulsant Barbiturate on the Release of Endogenous and Radiolabeled Amino Acids from Slices of Mouse Hippocampus

The convulsant barbiturate 5-(2-cyclohexylidene-ethyl)-5-ethyl barbituric acid (CHEB) stimulates the spontaneous release of endogenous and radiolabeled acetylcholine (ACh) from mouse hippocampal slices in vitro. In order to determine if the ability of CHEB to release ACh was unique to this neurotransmitter, we have studied the action of this drug in vitro on the release of both radiolabeled and endogenous putative neurotransmitter and non-transmitter amino acids in the hippocampus. Although CHEB stimulated the spontaneous release of both [3H]gamma-n-aminobutyric acid (GABA) and endogenous GABA, CHEB had different effects on the spontaneous release of radiolabeled and endogenous L-glutamate and L-aspartate: L-[3H]glutamate release was inhibited by CHEB, but endogenous L-glutamate release was unaffected by CHEB, but endogenous L-aspartate release was stimulated. The spontaneous release of the amino acids L-alanine and glycine (not thought to be neurotransmitters in the hippocampus) was not affected by CHEB. The results of this study indicate that CHEB does not always stimulate the release of all putative neurotransmitters. The ability of this drug to release ACh, GABA, and L-aspartate may be the result of some specific interaction of CHEB with nerves using these neurotransmitters in the hippocampus. In addition, the results suggest some problems that may be encountered when radiolabeled substances are used to study neurotransmitter release.     

Amino acids as putative transmitters: failure to bind to synaptic vesicles of guinea pig cerebral cortex

Abstract— The levels of free amino acids in subcellular fractions of guinea pig cerebral cortex have been determined. Putative transmitter amino acids are not bound to synaptic vesicles in a manner analogous to acetylcholine, as shown by the failure of hypo-osmotic shock to cause a specific release of the excitant and depressant amino acids.     

Effect of Ouabain Applied by Intrastriatal Microdialysis on the In Vivo Release of Dopamine, Acetylcholine, and Amino Acids in the Brain of Conscious Rats

In the present study we have applied a brain microdialysis technique to investigate the effects of ouabain infusion on the release of dopamine, acetylcholine, and amino acids from striatal neurons in freely moving rats. Ouabain caused an increase in the dialysate levels of dopamine; its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC); and the amino acids glutamate, aspartate, taurine, glycine, alanine, serine, asparagine, and threonine. The ouabain-induced increase in dopamine was dose dependent and explosive (100-fold at an infusion concentration of 1 mmol/L) and contrasted strongly with the small effect of the glycoside on the output of DOPAC. We investigated the nature of ouabain-induced transmitter release by determining its sensitivity to coinfusion with tetrodotoxin or the calcium antagonist Mg2+. In the case of dopamine two mechanisms of ouabain-induced release could be established. At lower infusion concentrations ouabain induced an exocytotic type of release whereas at higher concentrations the release was probably carrier mediated. In the case of amino acids we noticed a calcium-independent release which was nerve impulse flow dependent in the case of glutamate and aspartate and impulse flow independent in the case of alanine, serine, glycine, threonine, and asparagine. Ouabain induced a decrease in the release of acetylcholine and glutamine.     

Amitriptyline and imipramine inhibit the release of acetylcholine from parasympathetic nerve terminals in the rat iris

The electrically stimulated release of [3H]acetylcholine from the parasympathetic nerve terminals of the rat iris in vitro is increased in a dose-dependent manner by scopolamine but is decreased by the tricyclic antidepressants amitriptyline and imipramine. The increased release in the presence of scopolamine seems to be due to the blockade of a presynaptic muscarinic autoreceptor that, in the drug-free state, inhibits the release of acetylcholine. However, at drug concentrations that should have comparable antimuscarinic potency, the antidepressants inhibit the release of acetylcholine. This suggests that the anticholinergic side effects of the antidepressants may be due to the reduced release of acetylcholine from parasympathetic nerve terminals as well as a possible direct postsynaptic muscarinic receptor blocking action. Whatever the mechanism of this action, the antidepressants do not have the same effect as scopolamine at the presynaptic muscarinic autoreceptor in the rat iris.     

Beneficial effect(s) of n-3 fatty acids in cardiovascular diseases: but, why and how?

Low rates of coronary heart disease was found in Greenland Eskimos and Japanese who are exposed to a diet rich in fish oil. Suggested mechanisms for this cardio-protective effect focused on the effects of n-3 fatty acids on eicosanoid metabolism, inflammation, beta oxidation, endothelial dysfunction, cytokine growth factors, and gene expression of adhesion molecules; But, none of these mechanisms could adequately explain the beneficial actions of n-3 fatty acids. One attractive suggestion is a direct cardiac effect of n-3 fatty acids on arrhythmogenesis. N-3 fatty acids can modify Na+ channels by directly binding to the channel proteins and thus, prevent ischemia-induced ventricular fibrillation and sudden cardiac death. Though this is an attractive explanation, there could be other actions as well. N-3 fatty acids can inhibit the synthesis and release of pro-inflammatory cytokines such as tumor necrosis factoralpha (TNFalpha) and interleukin-1 (IL-1) and IL-2 that are released during the early course of ischemic heart disease. These cytokines decrease myocardial contractility and induce myocardial damage, enhance the production of free radicals, which can also suppress myocardial function. Further, n-3 fatty acids can increase parasympathetic tone leading to an increase in heart rate variability and thus, protect the myocardium against ventricular arrhythmias. Increased parasympathetic tone and acetylcholine, the principle vagal neurotransmitter, significantly attenuate the release of TNF, IL-1beta, IL-6 and IL-18. Exercise enhances parasympathetic tone, and the production of anti-inflammatory cytokine IL-10 which may explain the beneficial action of exercise in the prevention of cardiovascular diseases and diabetes mellitus. TNFalpha has neurotoxic actions, where as n-3 fatty acids are potent neuroprotectors and brain is rich in these fatty acids. Based on this, it is suggested that the principle mechanism of cardioprotective and neuroprotective action(s) of n-3 fatty acids can be due to the suppression of TNFalpha and IL synthesis and release, modulation of hypothalamic-pituitary-adrenal anti-inflammatory responses, and an increase in acetylcholine release, the vagal neurotransmitter. Thus, there appears to be a close interaction between the central nervous system, endocrine organs, cytokines, exercise, and dietary n-3 fatty acids. This may explain why these fatty acids could be of benefit in the management of conditions such as septicemia and septic shock, Alzheimer's disease, Parkinson's disease, inflammatory bowel diseases, diabetes mellitus, essential hypertension and atherosclerosis.     

Release of Neurotransmitter Amino Acids from Synaptosomes: Enhancement of Calcium-Independent Efflux by Oleic and Arachidonic Acids

Abstract: The release of preloaded [¹⁴C]neuroactive amino acids (glutamic acid, proline, γ-aminobutyric acid) from rat brain synaptosomes can occur via a time-dependent, Ca²⁺ -independent process. This Ca²⁺-independent efflux is increased by compounds that activate Na⁺ channels (veratridine, scorpion venoms), by the ionophore gramicidin D, and by low concentrations of unsaturated fatty acids (oleic acid and arachidonic acid). Saturated fatty acids have no effect on the efflux process. Neither saturated nor unsaturated fatty acids have an effect on the release of [¹⁴C]leucine, an amino acid not known to possess neurotransmitter properties. The increase in the efflux of neuroactive amino acids by oleic and arachidonic acids can also be demonstrated using synaptosomal membrane vesicles. Under conditions in which unsaturated free fatty acids enhance amino acid efflux, no effect on ²²Na⁺ permeability is observed. Since Na⁺ permeability is not altered by fatty acids, the synaptosomes are not depolarized in their presence and, thus, the Na⁺ gradient can be assumed to be undisturbed. We conclude that unsaturated fatty acids represent a potentially important class of endogenous modulators of neuroactive amino acid transport in nerve endings and further postulate that their action is the result of an uncoupling of amino acid transport from the synaptosomal Na⁺ gradient.     

Effects of amino acids on the isolated chicken retina, and on its response to glutamate stimulation

Abstract— The effect of a number of amino acids on the transparency and on the release of of [¹⁴C]glutamate from isolated chicken retinae charged with this compound was investigated. Also the effect of various amino acids on the response of the retina to stimulation with unlabelled glutamate, which causes an increase in transparency and a release of the label, was examined. In parallel experiments the effect of these same amino acids on the transparency and spreading depression (SD) was investigated in preparations consisting of the posterior part of the eye. A number of amino acids such as L-leucine, L-phenylalanine, L-tryptophan, L-lysine, L-histidine, L-arginine and others had little or no effect on these preparations. DL-valine and DL-homoserine caused an increase in transparency but no release of the label and did not affect the response to glutamate. Another group of amino acids comprising DL-a-alanine, L-serine, L-threonine, L-proline and glycine also caused an increase in the transparency of the retina without a release of labelled glutamate, but prevented the increase in transparency resulting from glutamate stimulation without affecting the release of the label. A final group of amino acids which included L-glutamic acid diethyl ester, DL-a-methyl glutamate, L-glutamine, L-asparagine, DL-homocysteate and L-cysteine caused a change in transparency of the retina accompanied by a release of the label; they prevented the change in transparency as well as the release of the label during stimulation by glutamate. Some amino acids, L-serine, L-threonine, DL-a-methyl glutamate, L-asparagine, DL-homocysteate and L-cysteine, caused wrinkling and folding of the retinae which furthermore became opaque. Of the amino acids investigated, proline gave promise of being a practical antagonist to the action of glutamate on the retina.     

Modulation of transmission at a glutamate synapse.

The amino acid L-aspartate markedly potentiates the responses elicited by L-glutamate at excitatory neuromuscular synapses in lobster walking limbs. Results are consistent with the idea that aspartate increases the affinity between glutamate and its binding sites in the postsynaptic receptor. Although complications due to release from other amino acid sources are a serious qualification, studies of neurally induced release of glutamate and aspartate suggest that both amino acids are released from excitatory nerve terminals. Experiments comparing the potentiating action of a variety of amino acids with their ability to inhibit glutamate uptake are not supportive of the notion that inhibition of agonist removal is the primary mode of action in the potentiation process. However, this idea, as well as the suggestion that aspartate may induce release of glutamate from extrajunctional entrapment sites, are not ruled out. Indeed, it is likely that the modulatory process embodies a multiplicity of reactions with given ones dominating from preparation to preparation.     

Regulation of intracellular protein degradation in the isolated perfused liver of the chicken (Gallus domesticus).

1. The effects of insulin, glucagon and a supply of exogenous amino acids on protein degradation have been studied in isolated perfused livers from growing chickens by measuring the rate of net valine release in the presence of cycloheximide. 2. Insulin inhibited protein degradation as did a supply of exogenous amino acids. 3. Addition of glucagon increased uric acid release from the livers but had no significant effect on protein degradation. 4. When the effects of the hormones and amino acid mixture are compared with published data for the rat it is evident that the action of glucagon differs in the two species.     

Scope and limitations of in vivo brain dialysis: a comparison of its application to various neurotransmitter systems

Brain dialysis is rapidly becoming a routine research method with a wide range of applications. Since 1982 this sampling technique is frequently used as a method to study the in vivo release of endogenous neurotransmitters such as dopamine, noradrenaline, serotonin, acetylcholine and certain amino acids. In this review most of the studies that have appeared in this field, are evaluated. Special attention was given to the question whether the neurotransmitter content in the dialysate is related to neurotransmission. Criteria such as the presence of a high tissue/dialysate concentration ratio, the sensitivity of the transmitters to membrane active compounds and the occurrence of receptor-mediated effects, are discussed. It is concluded that dopamine, noradrenaline and acetylcholine found in the dialysate are directly derived from neurotransmission, whereas the overflow of excitatory amino acid neurotransmitters is related to neurogenic as well as to metabolic events.     

Synaptosomal release of newly-synthetized or recently accumulated amino acids. Differential effects of kainic acid on naturally occurring excitatory amino acids and on [d-3H]aspartate

Kainic acid, a powerful neuroexcitant and neurotoxin, stimulates the release of naturally occurring excitatory amino acids, l-glutamate and l-aspartate, from hippocampal synaptosomes. The release stimulation affects in a similar way both the general pool of the two amino acids and the fraction of l-glutamate and l-aspartate, newly-synthetized from precursors or recently accumulated through the high-affinity uptake mechanism. Kainic acid exerts its stimulatory action on the basal release of the two amino acids as well as on the high K⁺-stimulated release of l-glutamate. Kainic acid has, however, different effects on the release of exogenously accumulated [d-³H]aspartate. In particular, the high K⁺-stimulated release of this false transmitter is strongly inhibited by 1 mM kainic acid. The present data confirm the presynaptic action of kainic acid on the general as well as on the recently-formed pools of naturally occurring excitatory amino acids. At the same time, our results suggest that [d-³H]aspartate is not a reliable substitute for l-glutamate and l-aspartate, in release studies and that the radioactivity released after preloading with [d-³H]aspartate does not necessarily reflect the release of naturally occurring excitatory amino acids.     

Behavioral study of chemoreception in the sea starMarthasterias glacialis: Structure-activity relationships of lactic acid,amino acids,and acetylcholine

Behavioral responses of Marthasterias glacialis to low molecular compounds were studied under laboratory conditions. Feeding postures, stomach eversions and locomotion of initially inactive animals can be released with very dilute solutions of lactic acid, neutral 2 and 3 carbon amino acids, L isomers of 4 to 6 carbon neutral amino acids, L-arginine, acetylcholine iodide, and several of their analogues. Hunger was induced by temporary withdrawal of food. Responsiveness to feeding stimuli was controlled with L-cysteine and L-leucine. The lowest behavioral thresholds for the most effective feeding stimuli were 3 X 10(-11) mol/l for both enantiomers of lactic acid, 10(-8) mol/l for L-proline and both enantiomers of cysteine and 10(-7) mol/l for acetylcholine iodide and some of the effective neutral amino acids. The behavioral threshold values for chemical stimuli differed by a factor between 30 and 100 in different sea stars. The test concentration was 3 X 10(-7) mol/l, the level at which L-cysteine elicited a complete feeding response from all the animals. Structure-activity comparison of substances less effective than the control stimulus was thus possible. The behavioral threshold of fully effective substances was determined later. The independence of receptor mechanisms for different substances can be inferred as: L-cysteine controlled responsiveness is not always accompanied by responsiveness to neutral amino acids. Autotomized marthasterias arms crawled after stimulation with lactic acid, cysteine, and acetylcholine iodide but did not respond to the feeding stimuli betaine and L-proline. An animal became inactive if electric shocks were paired with L-proline or L-cysteine emanating from an 'electric' food model.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of leu-enkephalin and the delta-sleep-inducing peptide, M (DSIP), on endogenous noradrenaline release by brain synaptosomes in the rat

Fluorometry was employed to measure the noradrenaline (NA) content in rat brain synaptosomes depending on the duration of incubation, depolarization effects (40 mM KCl or 1.5 mM ouabain), composition of the synaptosomal fraction and concentration of the peptides. The 10-minute incubation in a potassium medium of a suspension of light synaptosomes was used as an optimal test-system for studying the peptide action. Leu-enkephalin inhibited the depolarization-induced NA release. The effect was abolished by naloxone. The delta-sleep-inducing peptide (DSIP) did not influence the neurotransmitter release at concentrations of 10(-8)-10(-5) M. A mixture of amino acids imitating the amino acid composition of the DSIP influenced spontaneous release of NA. This effect is discussed in connection with the physiological action of the peptide on its intraventricular injection.     

Development of spontaneous motility in chick embryos. Participation of the central cholinergic mechanism

Development of the effectivensess of the systemic administration of oxotremorin (200 micrograms/kg egg weight) on spontaneous motility in chick embryos was studied from the 11th to the 19th day of incubation. 1) Oxotremorin activated spontaneous motility from the 15th day of incubation; subsequently, its effect increased with the embryo's age. 2) Glycine (100 mg/kg e.w.) blocked the activating effect of oxotremorin, again in a direct correlation to the embryo's age. 3) The spontaneous motility of spinal embryos was activated significantly less, thereby demonstrating the importance of the supraspinal component of the effect of oxotremorin. 4) The results are evaluated primarily as evidence of the role of the cholinergic mechanism (Everett 1964) in embryonal development of supraspinal control of the spontaneous activity of spinal motoneurones.     

Modulation of amino acid neurotransmitter actions by other neurotransmitters: some examples.

Developments in the field of central neurotransmission indicate that amino acids serve as important and widespread transmitters throughout the central nervous system. There are increasing indications from recent experimental studies that several of the other central neurotransmitters may exert potent effects on central neurons by modulating the actions of amino acids. Noradrenaline and serotonin have received particular attention as potential modulators, and a wide variety of actions has been reported for them. Modulatory actions have been reported at both pre- and post-synaptic levels, including both short- and long-term effects and facilitation or inhibition of amino acid actions. Selectivity has been found both for specific receptor subtypes of the neuromodulator and for specific effects of amino acids. Examples of such selectivity are modification of actions of an amino acid with little effect on spontaneous activity or membrane properties of the target cell, or in comparison to the actions of other neurotransmitters, or even other selective amino acid analogs. Modulatory actions on amino acids have also been reported for several other neurotransmitters including acetylcholine and various peptides. Recent studies of angiotensin II demonstrate that when iontophoretically applied, it can potently and selectively block the depolarizing action of glutamate on locus coeruleus neurons. It is possible that physiological influences of these various transmitter substances are expressed through modification of amino acid actions, rather than through direct effects on central neurons.     

A structure-activity study of the neuropeptide PF1, SDPNFLRFamide, using the dorsal body wall muscle of the chicken nematode, Ascaridia galli

The action of a range of N terminally modified peptides structurally related to the nematode peptide PF1, SDPNFLRFamide, has been investigated using a dorsal muscle strip preparation from the chicken nematode, Ascaridia galli. Acetylcholine contracts this muscle preparation in a concentration-dependent manner when applied in the range 1-100 microM with an EC50 value of 9 microM. These contractions are reduced in the presence of PF1 and its analogues, with a threshold effect of PF1 of around 1 nM and an IC50 value of 470 nM against 10 microM acetylcholine. All the PF1 analogues tested were less potent than PF1 in reducing the acetylcholine contractions, indicating the importance of the N terminal amino acids in the action of PF1 in this preparation.     

Release of Endogenous Amino Acids from the Striatum from Developing and Adult Mice in Ischemia

In most other studies the release of amino acid neurotransmitters and modulators in vitro has been studied mostly using labeled preloaded compounds. For several reasons the estimated release may not reliably reflect the release of endogenous compounds. The magnitudes of the release cannot thus be quite correctly estimated using radioactive labels. The basal and K⁺-evoked release of the neuroactive endogenous amino acids γ-aminobutyrate (GABA), glycine, taurine, glutamate and aspartate was now studied in slices from the striatum from 7-day-old to 3-month-old mice under control (normoxic) and ischemic conditions. The release of alanine, threonine and serine was assessed as control. GABA and glutamate release was much greater in 3-month-old than in 7-day-old mice, whereas with taurine the situation was the opposite. Ischemia markedly enhanced the release of all these three amino acids. The release of aspartate and glycine was markedly enhanced as well whereas no effects were discernible in the release of glutamine, alanine, serine and threonine. K⁺ stimulation (50 mM) enhanced the release of GABA, glutamate, taurine, aspartate and glycine in most cases, except with taurine in 3-month-old mice under the ischemic conditions and with aspartate in 7-day-old mice under the control conditions. K⁺ stimulation did not affect the release of glutamine, alanine, serine or threonine. The results on endogenous amino acids are qualitatively similar to those obtained in our earlier experiments with labeled preloaded amino acids. In conclusion, in developing mice only inhibitory taurine is released in such amounts that may counteract the harmful effects of excitatory amino acids in ischemia.     

Further studies on the properties of bile acids. II. Effect of bile acids on the reactivity of adrenergic and cholinergic receptors in rat intestine

The effects of bile acids (deoxycholic, cholic and dehydrocholic) were studied on the action of certain autonomic system drugs (isoprenaline, adrenaline, noradrenaline and acetylcholine). It was also tried to explain the causes of these changes in the light of the results of experiments with the widely used antagonists of adrenergic and cholinergic receptors. The experiments were carried out on isolated rat intestine by the method of Magnus. It was found that bile acids decreased the relaxing effect of isoprenaline and caused inversion of the action of adrenaline and noradrenaline on the intestine. Changes in the action of catecholamines are caused by stimulation of alpha-adrenergic receptors enhanced by bile acids, with a simultaneous decreased stimulation of beta-receptors. Bile acids cause also an increase of the effect of acetylcholine on the intestine.     

Characterization of acetylcholine-induced insulin secretion in the isolated perfused dog pancreas.

In the presence of a nonstimulatory concentration of glucose, a 60-min perfusion with 50 muM acetylcholine was shown to elicit a monophasic release of insulin in the isolated dog pancreas preparation. A decline in secretory response, which may be due to desensitization of the beta-cell to acetylcholine, was noted during the latter part of the perfusion interval. The potent insulin secretory response elicited by acetylcholine during the 60-min period was abolished 0y 25 muM atropine. Inhibition of the insulinotropic action of acetylcholine was also noted with administration of the mitotic spindle inhibitor, colchicine. When compared to 20-min control perfusions, addition of 1 mM colchicine resulted in a 50% reduction in acetylcholine-induced insulin release. These results suggest that insulin secretion stimulated by acetylcholine can be considered to be due to a muscarinic action of this agent which is dependent, at least in part, upon the microtubular system of the beta-cell.