Structure-function studies on N-oxalyl-diamino-dicarboxylic acids and excitatory amino acid receptors: evidence that beta-L-ODAP is a selective non-NMDA agonist

Excitatory amino acids and their receptors play an important role in both normal synaptic transmission and excitotoxic-mediated neuronal death. In the present investigation we have prepared a series of glutamate analogs and examined the pharmacological specificity with which they interact with excitatory amino acid receptors. Included within this group of compounds is a potent excitotoxic amino acid, beta-N-oxalyl-L-alpha, beta-diaminopropionic acid (beta-L-ODAP). This excitotoxin is of particular interest because it has been identified as a major causative agent of human neurolathyrism, a disease characterized by permanent spastic paralysis. The site of action of beta-L-ODAP was delineated with both electrophysiological recordings in hippocampal slices and radioligand binding assays in synaptic plasma membranes. We report that beta-L-ODAP is a potent agonist at the non-N-methyl-D-aspartate (NMDA) type of excitatory amino acid receptor. beta-L-ODAP interacts most potently with the quisqualate class of non-NMDA receptors (IC50 = 1.3 microM), less potently with the kainate receptor (IC50 = 17 microM), and very weakly with NMDA receptors. The specificity of this binding was consistent with physiological experiments that demonstrated that beta-L-ODAP-induced depolarizations were potently blocked by the newly identified non-NMDA receptor antagonist, CNQX, but were not affected by the NMDA antagonist D-AP5. These results extend recent studies that have focused on the contribution of NMDA receptors to excitotoxicity and highlight the potential involvement of non-NMDA receptors in excitotoxic-mediated cell death.     

Modulation of pentylenetetrazol-induced seizure activity by branched-chain amino acids and α-ketoisocaproate

Branched-chain amino acids, and mainly leucine act as nitrogen donors in the cerebral glutamate–glutamine cycle, thereby reducing brain excitability. Rats equipped with cortical electrodes received 300 mg/kg of leucine, isoleucine, valine or the ketoacid of leucine, α-ketoisocaproate at 2 h before the induction of seizures by 40 mg/kg pentylenetetrazol. Control groups received saline or a commercial mixture of amino acids, Vamine®. Leucine and isoleucine increased the latency to absence-like and tonic–clonic seizures but did not influence the duration of the tonic–clonic seizure. Vamine®, valine and α-ketoisocaproate had no effect. These data are consistent with the role of leucine in buffering brain glutamate concentration.     

Changes with aging in the levels of amino acids in rat CNS structural elements: IV. Methionine and basic amino acids

This paper reports the distribution of methionine, histidine, lysine, arginine, and ornithine in 53 discrete brain areas of 3- and 29-month-old male Fischer 344 rats microdissected by the punch technique. Like that of the other amino acids were reported in previous papers of this series, the distribution of methionine and the basic amino acids was regionally highly heterogeneous. The ratios of levels in the areas of highest concentrations to levels in the areas of lowest concentration varied from 10 to 15 for these amino acids, except that it was 23 for arginine. This heterogeneity is also illustrated by the finding that in some areas arginine was more than 5% of the total amino acid content and in others was less than 0.5%. The distribution of methionine differed from that of the basic amino acids, which were high in hypothalamic areas and low in the limbic system. With aging, methionine and basic amino acids, like the other amino acids studied in this series, mainly decreased in level, although in a few cases increases could be seen. Significant decreases were noted more than five times as often as increases.     

Regulation of nitric oxide synthase activity in cortical slices by excitatory amino acids and calcium

Nitric oxide synthase (NOS) activity was determined in adult rat frontal cortex and hippocampus by measuring the conversion of L-[³H]arginine to L-[³H]citrulline. N-methyl-D-aspartate (NMDA), but not kainate or α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), stimulated NOS activity. This effect was concentration dependent (EC₅₀ ≈ 30μM) and was inhibited by tetrodotoxin, EGTA, N^ω-nitro-L-arginine (NOARG), Mg²⁺, phencyclidine, and (cis)-4-phosphonomethyl-2-piperidine carboxylate (CGS 19755), but not by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). NOS activity was increased to an even greater extent by the calcium ionophores ionomycin and A23187 and by depolarization with 50 mM K⁺. Interestingly, neither caffeine nor 1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), drugs that would be expected to increase intracellular Ca²⁺ concentration by release of Ca²⁺ from intracellular ryanodine- and inositol-1,4,5-trisphosphate-sensitive stores, respectively, had any significant effect on NOS activity. It is concluded that NOS can be activated by NMDA binding to a classic NMDA glutamate receptor subtype as well as by depolarization or other agents that increase the influx of extracellular Ca²⁺. The paradoxical lack of effect of caffeine, as well as the inhibitory effect of tetrodotoxin, are discussed. © 1994 Wiley-Liss, Inc.     

Selective potentiation of NMDA-induced activity and release of excitatory amino acids by dynorphin: possible roles in paralysis and neurotoxicity.

Selective antagonists of N-methyl-D-aspartate (NMDA) excitatory amino acid (EAA) receptors have been shown to protect against dynorphin-A (DYN)-induced paralysis and neurotoxicity in the spinal cord. To test the hypothesis that either DYN-induced paralysis or neurotoxicity involves an enhanced release of EAAs, we used microdialysis to monitor aspartate (Asp) and glutamate (Glu) release in both the lumbar spinal cord extracellular fluid (ECF) and the spinal cord cerebral spinal fluid (CSF) of conscious rats in response to DYN (1-13). Injection of 5 nmol of DYN produced temporary paralysis in 8 of 10 animals, but did not significantly change Asp or Glu release in either the ECF or the CSF. Injection of 20 nmol of DYN caused permanent paralysis and neuronal cell loss in all animals tested as well as a significant increase of Asp and Glu in both the ECF and the CSF, and a decrease in glutamine (Gln) release only in the ECF. Pretreatment with 1 mg/kg of the NMDA antagonist MK-801 blocked both paralysis and amino acid changes in the ECF. Pretreatment of animals with 5 mg/kg naloxone inhibited glutamate release in the ECF, but did not block paralysis, Asp release or inhibition of Gln release. As MK-801 sensitive paralysis by DYN was not mediated through enhanced EAA release, we examined whether DYN could act through postsynaptic facilitation of NMDA receptors by testing the ability of DYN to alter the magnitude of a behavioral response produced by intrathecal injection of NMDA in mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Biochemistry, Physiology and Pathological roles of PAI-1 and the requirements for PAI-1 inhibition in vivo

Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) and is consequently one the most important inhibitors of the plasminogen/plasmin system. PAI-1 attenuates fibrinolysis and increased levels of active PAI-1 have been associated with an increased risk for cardiovascular diseases. PAI-1 knock-out mice as well as PAI-1 overexpressing mice have been generated and characterized to study the role of PAI-1 in vivo. A number of PAI-1 inhibitors have been generated to study the pharmacological effect of PAI-1 inhibition in vitro and in vivo. The current review provides an overview of 1) the biochemical features of PAI-1, 2) the role of PAI-1 in diverse pathologies, 3) the in vitro and in vivo data obtained with PAI-1 inhibitors and 4) the vitronectin, glycosylation and species dependency of PAI-1 inhibition.     

Effects of kynurenate on root potentials evoked by synaptic activity and amino acids in the frog spinal cord

The effects of kynurenate (kyn) on synaptic- and excitatory amino acid-mediated responses in isolated, hemisected spinal cords of frog were examined. Kyn (0.5 mM) rapidly and reversibly blocked >90% of the synaptically mediated ventral root potential (VRP) produced by stimulation of the dorsal root. Spontaneous activities recorded from both ventral and dorsal roots were also reversibly blocked by Kyn. However, Kyn had no effect on action potentials or excitability per se, nor was it a general inhinitor of synaptic transmission since Kyn concentrations as high as 2.5 mM had no effect on synaptically mediated dorsal root potentials produced by stimulation of the ventral root. In addition, Kyn had no effect on synaptic transmission in sympathetic ganglia of frog. Although Kyn (2.5 mM) by itself produced no ventral root response in spinal cords treated with tetrodotoxin, it antagonized those induced by the excitatory amino acids N-methyl-d,l-aspartate, quisqualite, kainate, aspartate, and glutamate. The ventral responsed to all concentrations of quisqualate tested were depressed by 2.5 mM Kyn. In addition, when Kyn was washed out, the rate of recovery from Kyn block was accelerated by the presence of quisqualate. These results indicate that quisqualate and Kyn compete for common binding sites. However, low concentrations of Kyn (e.g. 0.1 mM) potentiated the peak of the response to saturating concentrations of quisqualate by as much as 30%. The durations of the potentiated quisqualate responses were significantly shorter than the control responses. Thus, Kyn does not act simply as a competitive inhibitor of quisqualate. Incontrast, Kyn appears to simply block N-methyl-d,l-aspartate responses with no signs of poptentiation or charges in kinetics.     

Antagonism of morphine analgesia by nonopioid cold-water swim analgesia: direct evidence for collateral inhibition

The demonstrated existence of opioid and nonopioid forms of pain control has raised questions as to how they interact. Previous indirect evidence suggests that activation of one system inhibited the activation of the other. The present study assessed this directly using morphine as an opiate form of analgesia and continuous cold-water swims (CCWS, 4 degrees C, 2 min) as the nonopioid form. A significant reduction in morphine (8 mg/kg, SC) analgesia on the tail-flick test was observed if rats were acutely exposed to CCWS immediately prior to morphine administration. The inability of naloxone (10 mg/kg, SC) to reduce CCWS analgesia verified its nonopioid nature. The antagonism of morphine (3 mg/kg, SC) analgesia was greater following preexposure to 2 min of CCWS than 1 min of CCWS. CCWS was also more effective in antagonizing analgesia induced by the 3 mg/kg than the 8 mg/kg dose of morphine. The antagonism of morphine analgesia by CCWS was dependent upon the temporal patterning of stimulus presentation: exposure to CCWS 20 or 60 min prior to morphine failed to alter subsequent morphine analgesia. A significant reduction in analgesia induced by intraperitoneal administration of morphine (10 mg/kg) was also observed when CCWS was presented immediately prior to injection, suggesting that pharmacokinetic factors such as altered drug absorbance by CCWS-induced vasoconstriction do not appear to explain these effects. These data provide direct support for the existence of collateral inhibitory mechanisms activated by CCWS and morphine, and suggests that these opioid and nonopioid forms of analgesia do not function synergistically, but instead involve some form of hierarchical order.     

The antinociceptive activity of excitatory amino acids in the rat brainstem: an anatomical and pharmacological analysis.

Rats were stereotaxically implanted with microinjection cannulae aimed at sites ranging caudally from the lower medulla and rostrally to the diencephalon and received microinjections of the excitatory amino acid: L-glutamate 30 nmol/0.5 microliters. The subsequent spontaneous behavioral response and the effect on the thermal noxious-evoked tail flick (TF) and hot plate (HP) responses was recorded. From 331 brain sites mapped with glutamate, an elevation of tail flick and hot plate response latencies was observed in 59 cases and in 34 of these sites the antinociceptive activity was preceded by a shortlasting aversion characterized by vocalization and running. The glutamate-sensitive sites at which TF and HP response latencies were elevated were exclusively distributed in the medullary reticular formation (MRF) and the mesencephalic periaqueductal gray matter (PAG). The aversive and antinociceptive activity of glutamate was dose-dependent and mimicked by the excitatory amino acid (EAA) receptor agonists N-methyl-D-aspartate + (NMDA) kainate and less so quisqualate. The EAA receptor antagonists MK-801 and AP-5, but not glutamyl-amino-methyl-sulfonic acid, antagonized in a dose-dependent fashion both the aversive and antinociceptive responses evoked from the PAG. It is suggested that NMDA receptor-linked neurons in the PAG activate both nociceptive and antinociceptive systems.     

Kinetic studies on the inhibition of creatine kinase activity by branched-chain α-amino acids in the brain cortex of rats

Maple syrup urine disease (MSUD) is a metabolic disorder biochemically characterized by the accumulation of branched-chain alpha-amino acids (BCAA) and their branched-chain alpha-keto acids (BCKA) in blood and tissues. Neurological dysfunction is usually present in the patients, but the mechanisms of brain damage in this disease are far from be understood. The main objective of this study was to investigate the mechanisms by which BCAA inhibit creatine kinase activity, a key enzyme of energy homeostasis, in the brain cortex of 21-day-old Wistar rats. For the kinetic studies, Lineweaver-Burk and a modification of the Chevillard et al. plots were used to characterize the mechanisms of enzyme inhibition. The results indicated that BCAA inhibit creatine kinase by competition with the substrates phosphocreatine and ADP at the active site. Considering the crucial role creatine kinase plays in energy homeostasis in brain, if these effects also occur in the brain of MSUD patients, it is possible that inhibition of this enzyme activity may contribute to the brain damage found in this disease. In this case, it is possible that creatine supplementation to the diet might benefit MSUD patients.     

Mood effects of the amino acids tryptophan and tyrosine: 'Food for Thought' III

Parker G, Brotchie H. Mood effects of the amino acids tryptophan and tyrosine. Objective: Reflecting increased scientific interest in any nutritional contribution to the onset and treatment of mood disorders, we overview research into two neurotransmitter precursors – the amino acids tryptophan and tyrosine – particularly examining whether any deficiency increases risk to depression and whether those amino acids have any antidepressant properties. Method: The theoretical relevance of the two amino acids was overviewed by considering published risk and intervention studies, technical papers and reviews. Results: There is some limited evidence, suggesting that depressed patients, especially those with a melancholic depression, have decreased tryptophan levels. Whether such findings reflect a causal contribution or are a consequence of a depressed state remains an open question. There is a small database supporting tryptophan preparations as benefitting depressed mood states. There is no clear evidence as to whether tyrosine deficiency contributes to depression, while the only randomized double‐blind study examining tyrosine supplementation did not show antidepressant benefit. Conclusion: Acute tryptophan depletion continues to provide a research tool for investigating the relevance of serotonin to depression onset. There is limited evidence that tryptophan loading is effective as a treatment for depression through its action of increasing serotonin production. Most clinical studies are dated, involve small sample sizes and/or were not placebo controlled. The development of the new serotonin reuptake inhibitor drugs seemingly signalled an end to pursuing such means of promoting increased serotonin as a treatment for depression. The evidence for tyrosine loading promoting catecholamine production as a possible treatment for depression appears even less promising, and depletion studies less informative.     

Cortical control of inhibition of return: Causal evidence for task-dependent modulations by dorsal and ventral parietal regions

Inhibition of return (IOR) reflects a bias to preferentially attend to non-previously attended or inspected spatial locations. IOR is paramount to efficiently explore our environment, by avoiding repeated scanning of already visited locations. Patients with left visual neglect after right parietal damage or fronto-parietal disconnection demonstrated impaired manual, but not saccadic, IOR for right-sided targets (Bourgeois et al., 2012). Here we aimed at investigating in healthy participants the causal role of distinct cortical sites within the right hemisphere in manual and saccadic IOR, by evaluating the offline effects of repetitive Transcranial Magnetic Stimulation (rTMS) on the right intra-parietal sulcus (IPS) and the right temporo-parietal junction (TPJ). Our results show that rTMS over both sites lastingly interfered with manual but not saccadic IOR for right-sided targets. This behavioral pattern closely mimicked the performance of neglect patients evaluated with the same paradigm. In contrast, for left-sided targets, rTMS over the right IPS impaired both manual and saccadic IOR, while rTMS over the right TPJ produced no modulation in either task. We concluded that distinct parietal nodes of the dorsal and ventral spatial attention networks of the right hemisphere make different contributions to exogenous orienting processes implicated in IOR, and that such effects are hemifield- and task-dependent.     

In vivo regulation of extracellular dopamine in the neostriatum: influence of impulse activity and local excitatory amino acids

Summary It has been suggested that dopamine release can be evoked by excitatory amino acids acting on dopaminergic terminals, as well as by the classical process of impulse-evoked exocytosis. We used in vivo microdialysis to examine whether endogenous excitatory amino acids locally evoke dopamine efflux under basal conditions. Infusion of N-methyl-d-aspartate (NMDA) or kainate into the neostriatum increased extracellular dopamine, and this effect was blocked by co-infusion of 2-amino-5-phosphonovalerate (APV) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively. However, neither these antagonists nor kynurenate decreased extracellular dopamine when administered alone. In contrast, infusion of tetrodotoxin into the medial forebrain bundle reduced extracellular dopamine to below the limit of detection of our assay. These and other findings reviewed in this report suggest to us that extracellular dopamine in the neostriatum is not stimulated locally by endogenous excitatory amino acids.     

Antihemostatic activity of heparin disaccharides and oligosaccharides obtained by chemical and enzymatic fragmentation: reversal of the hemorrhagic activity by ATP and myosin

Heparin and its fragments, namely, trisulfated disaccharide, pentasulfated tetrasaccharide, octasulfated hexasaccharide and an oligosaccharide (M.W. 6,300) prepared by enzymatic fragmentation and an oligosaccharide (M.W. 4,500) prepared by chemical fragmentation are potent inhibitors of skin hemostasis when applied topically. All the heparin fragments tested are 10 to 20 times more active than heparin itself on a weight basis in disrupting the normal hemostatic mechanism. As heparin, the fragments produce a residual antihemostatic effect which persists after extensive washing of the preparation with isotonic solutions. This residual effect could be removed either by ATP or myosin ATPase.     

Feedforward and feedback inhibition of hippocampal principal cell activity evoked by perforant path stimulation: GABA-mediated mechanisms that regulate excitability in vivo.

Hippocampal field potentials evoked by paired-pulse perforant path stimulation were used to identify normal feedforward and feedback inhibitory influences on hippocampal principal cells. Three distinct aspects of inhibitory function were identified in the dentate gyrus. They are: (1) first spike amplitude-dependent inhibition of the second spike, which at low stimulus frequency is primarily feedback in nature; (2) frequency-dependent inhibition of a single spike or the first spike of a pair, which occurs as stimulus frequency is increased from 0.1 to 1.0 Hz and which is primarily a reflection of feedforward inhibition; and (3) frequency-dependent inhibition of the second spike that is relatively independent of first spike amplitude and probably due to a combination of feedforward and feedback mechanisms. The results indicate that granule cell recurrent inhibition alone, evoked at low stimulus frequency, is relatively brief and weak. At higher frequencies, probably more relevant to physiological activity, feedforward inhibitory activity is recruited. The combination of feedforward and feedback mechanisms results in strong, maximal duration, granule cell inhibition. Similar frequency dependence of inhibition was not seen in area CA1 in response to ipsilateral perforant path stimulation since low frequency stimulation did not evoke CA1 spikes. CA3 stimulation did evoke large contralateral CA1 population spikes, but paired-pulse inhibition was weaker than that evoked by ipsilateral perforant path stimulation in terms of the duration of inhibition and the ability to suppress the development of epileptiform behavior. The identification of simple tests that reflect distinct inhibitory processes in vivo permits similar studies to be conducted in vitro to determine how to preserve inhibitory processes for cellular studies of normal and human epileptic tissue in which the state of excitatory--inhibitory balance is the subject. These results also form the basis for the interpretation of the following study (Sloviter, 1991), which addresses the relationship between selective dentate interneuron loss and the pathophysiology that accompanies it.     

Domoic acid induced spinal cord lesions in adult mice: evidence for the possible molecular pathways of excitatory amino acids in spinal cord lesions

Domoic acid (DA) is an excitatory amino acids (EAAs) analog which induced excitotoxicity lesion to central nervous system, but whether induced adult animal spinal cord is not known, furthermore, previous studies have shown that EAAs play an important role in spinal cord lesion, however, the molecular pathways in spinal cord lesion are not fully known. Therefore, a motor neuron-like cell culture system and a DA-induced spinal cord lesioned mice model were used to study the effect of DA on spinal cord in adult mice and the possible molecular pathways of EAAs in spinal cord lesions. Exposure of motor neuron-like cells NSC34 to DA dramatically increased reactive oxygen species (ROS) production by the DCF fluorescent oxidation assay, reduced mitochondrial function by MTT assay, cell viability by trypan blue exclusion assay, and was accompanied by an increase of cell apoptosis by histone protein release assay. In DA-induced spinal cord lesioned mice model, we showed that the decrease of proteasome activity, increase of UCP4 expression by immunohistochemistry and neural cell apoptosis by TUNEL staining, and was accompanied by an decrease of motor disturbance grade during the different stages of DA treatment. Taken together, the in vitro and in vivo data presented in the current report demonstrated that DA induces spinal cord lesions in adult mice, and the multiple molecular pathways promoted by EAAs in spinal cord lesions, at least partially was associated with ROS generation increase, mitochondrial dysfunction, proteasome activity decrease and UCP4 expression increase.     

Branched-chain amino acid-induced hippocampal norepinephrine release is antagonized by picrotoxin: evidence for a central mode of action

Previous studies indicated that administration of a 1:1:1 mixture of the branched-chain amino acids leucine, isoleucine, and valine (BCAA) decreased the response to pain. The present study investigates the effects of BCAA on release of norepinephrine (NE) from isolated hippocampal brain slices. BCAA evoked 3H-NE release in a concentration-dependent manner. This effect was antagonized by the gamma aminobutyric acid (GABA) receptor antagonist picrotoxin, again in a concentration-dependent manner, suggesting that the effect may be mediated via a GABA receptor. Given the role of NE and of GABA receptors in the central response to pain, it is possible that the BCAA may exert their antinociceptive properties through activation of GABA receptors.     

Response inhibition triggered by the briefly viewed image of a hand: Behavioural and electrophysiological evidence

Previous research has shown that subliminally presented arrows produce negative priming effect in which responses are performed slower when primes and targets are calling for the same response than different response. This phenomenon has been attributed to self-inhibitory mechanisms of response processes. Similar negative priming was recently observed when participants responded to the direction of the target arrow and the prime was a briefly displayed image of a left or right hand. Responses were made slower when the left-right identity of the viewed hand was compatible with the responding hand. This was suggested to demonstrate that the proposed motor self-inhibition is a general and basic functional principle in manual control processes. However, the behavioural evidence observed in that study was not capable of showing whether the negative priming associated with a briefly displayed hand could reflect other inhibitory processes than the motor self-inhibition. The present study uses an electrophysiological indicator of automatic response priming, the lateralized readiness potential (LRP), to investigate whether the negative priming triggered by the identity of the viewed hand does indeed reflect motor self-inhibition processes. The LRP revealed a pattern of motor activation that was in line with the motor self-inhibition hypothesis. Thus, the finding supports the view that the self-inhibition mechanisms are not restricted to arrow stimuli that are presented subliminally. Rather, they are general sensorimotor mechanisms that operate in planning and control of manual actions.     

Self-stimulation and noradrenaline: evidence that inhibition of synthesis abolishes responding only if the "reserve" pool is dispersed first.

Although noradrenaline (NA) is thought to play a critical role in electrical self-stimulation, suppression of NA synthesis by injection of the dopamine-beta-hydroxylase inhibitor, FLA-63 (H?ssle) (25 mg/kg), had little or no effect on response rates. But 3 or 5 days after prior reserpinisation, FLA-63 in the same dosage suppressed or profoundly depressed self-stimulation without eliciting signs of general incapacity. Suppression of self-stimulation could be reversed by intraventricular injection of NA, indicating that the depressant effect depended specifically on NA depletion. These findings support the view that NA may play a necessary role in self-stimulation and indicate that the NA available for this purpose included intraneuronal NA in a reserpine-sensitive reserve pool.