Rigid analogues as probes of excitatory amino acid receptors.

The classes of compounds to be discussed are based on rigid analogues of glutamic and aspartic acids. The glutamate analogue 1-amino-1,3-cyclopentane dicarboxylic acid (1,3-ACPD) exists as two enantiomeric pairs of geometric isomers. The absolute configurations were assigned and the compounds were found to differentiate between the kainic acid (KA) and N-methyl-D-aspartic acid (NMDA) receptor subtypes when applied iontophoretically to hippocampal CA1 pyramidal neurones. The results indicate a high degree of specificity for the interaction of D-cis-1,3-ACPD with the NMDA receptor, while the remaining three isomers of 1,3-ACPD were KA-like in their action. The results are augmented with binding studies and patch clamp analysis. The second class of compound is the closely related aspartate analogue 1-amino-1,2-cyclopentane dicarboxylic acid (1,2-ACPD). The geometric isomers have been examined and found to be somewhat less active than their 1,3-ACPD counterparts; however, the cis isomer does have antagonistic properties against quisqualate (QA) evoked excitation. The results indicate that while the three-dimensional arrangement of functional groups is important for the activation of receptor subtypes, other considerations must be made, including stereochemistry and receptor affinity for sterically hindered analogues of excitatory amino acids.     

Glutamate excitatory effects on ampullar receptors of the frog

Summary The action of glutamate on frog ampullar receptors was investigated to assess the potential role of this excitatory amino acid as an afferent transmitter in the hair cell system. Intracellular recordings from single afferent units in the isolated labyrinth revealed that glutamate and the glutamate receptor agonists, N-methyl-D-aspartic acid, quisqualic acid and kainic acid increase dose-dependently the frequency of the resting afferent discharge of EPSPs and spikes and produce long lasting depolarizations. After blocking synaptic transmission by using 5 mM Co²⁺, the same compounds elicited only depolarizations of amplitude comparable to those observed in normal saline. Quisqualic acid and kainic acid were much more potent than N-methyl-D-aspartic acid in increasing the frequency of afferent discharge and in causing axonal depolarizations. The depolarization caused by glutamate was reduced dose-dependently by the competitive non-NMDA receptor antagonist 6-cyano-7-nitroquinaxoline-2,3 dione and disappeared almost completely in Na⁺-free Ringer solution. These results are consistent with the hypothesis that glutamate is the afferent transmitter in vestibular organs and indicate that receptors mainly of the non-NMDA type are present not only at postsynaptic level but also in hair cells. Presynaptic glutamate receptors may function as autoreceptors controlling by a positive feed-back mechanism the release of the afferent transmitter.     

The interaction of beta-N-methylamino-L-alanine with bicarbonate: an 1H-NMR study

The mode of action of the neurotoxic, non-protein amino acid beta-N-methylamino-L-alanine (L-BMAA) is unknown. We have shown, using 1H-NMR spectroscopy, that L-BMAA forms a stable adduct with bicarbonate (probably a carbamate). The properties of this adduct may explain the observation that L-BMAA and N-methyl-D-aspartic acid appear to act at the same central nervous system receptors.     

A Glycine Site Associated with N-Methyl-d-Aspartic Acid Receptors: Characterization and Identification of a New Class of Antagonists

Membranes from rat telencephalon contain a single class of strychnine-insensitive glycine sites. That these sites are associated with N-methyl-D-aspartic acid (NMDA) receptors is indicated by the observations that [3H]glycine binding is selectively modulated by NMDA receptor ligands and, conversely, that several amino acids interacting with the glycine sites increase [3H]N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding to the phencyclidine site of the NMDA receptor. The endogenous compound kynurenate and several related quinoline and quinoxaline derivatives inhibit glycine binding with affinities that are much higher than their affinities for glutamate binding sites. In contrast to glycine, kynurenate-type compounds inhibit [3H]TCP binding and thus are suggested to form a novel class of antagonists of the NMDA receptor acting through the glycine site. These results suggest the existence of a dual and opposite modulation of NMDA receptors by endogenous ligands.     

The effect of L-glutamate and related agents on adenylate cyclase in the cestode Hymenolepis diminuta.

The effect of the putative amino acid transmitter, L-glutamate, on adenylate cyclase in crude membrane preparations of the rat tapeworm Hymenolepis diminuta was investigated to determine if glutamate effects the generation of the second messenger cAMP. Addition of glutamate at 10(-3) and 5.5 x 10(-9) M resulted in significant elevations in basal activity of adenylate cyclase, while concentrations in the 10(-5)-10(-7) M range caused significant depressions below basal activity. Assays with glutamate agonists and other acidic compounds showed glutamate to be the only amino acid, dicarboxylic acid, or acidic compound capable of this pattern of stimulation and inhibition. While the response of adenylate cyclase to glutamate agonists suggested that an N-methyl-D-aspartic acid (NMDA) type receptor may be present, glutamate agents acting as NMDA antagonists in vertebrate systems were agonists. Metabolic end products of glycolysis stimulated adenylate cyclase, suggesting that these, along with metabolic glutamate may regulate glycolytic enzymes. Only 10(-3) M L-glutamate significantly stimulated adenylate cyclase activity in tissue slices, and this response was restricted to those slices rich in nervous tissues. L-Glutamate eliminated the 5-hydroxytryptamine (5-HT) stimulated adenylate cyclase response suggesting that glutamate can modulate the 5-HT stimulated elevations in adenylate cyclase activity. The data support the hypothesis that L-glutamate is a neurotransmitter-modulator in the cestode.     

Control of Locomotion In Vitro: II. Chemical Stimulation

Previous studies have described the presence of alternating activity induced in left and right ventral roots of the neonate rat in vitro brainstem-spinal cord preparation, following application of certain neuroactive substances to the bathing solution. The present findings show the presence of chemically induced, adult-like coordinated airstepping demonstrated by electromyographic recordings in the hindlimb-attached in vitro brainstem-spinal cord preparation. Analysis of muscular activity demonstrated alternation between antagonists of one limb and between agonists of different limbs, as well as a proximodistal delay in agonists active at different joints of the same limb. Neuroactive agents were applied independently to either the brainstem or spinal cord bath. The substances surveyed in the present studies included some of those used previously, as well as additional compounds: bicuculline and picrotoxin (γ-aminobutyric acid-ergic antagonists), N-methyl-D-aspartic acid (excitatory amino acid agonist), substance P, acetylcholine, carbachol (cholinergic agonist), and serotonin. Application of these substances to the brainstem bath produced rhythmic airstepping. Application of dopamine, aspartate, glutamate, and N-methyl-D-aspartic acid to the spinal cord bath also produced rhythmic airstepping, while application of acetylcholine produced tonic, long-lasting co-contractions. These findings reveal the presence of several neurochemical systems in the central nervous system that can be activated at birth to induce coordinated airstepping in the neonate rat in vitro brainstem-spinal cord preparation.     

Control of locomotion in vitro: II. Chemical stimulation.

Previous studies have described the presence of alternating activity induced in left and right ventral roots of the neonate rat in vitro brainstem-spinal cord preparation, following application of certain neuroactive substances to the bathing solution. The present findings show the presence of chemically induced, adult-like coordinated airstepping demonstrated by electromyographic recordings in the hindlimb-attached in vitro brainstem-spinal cord preparation. Analysis of muscular activity demonstrated alternation between antagonists of one limb and between agonists of different limbs, as well as a proximodistal delay in agonists active at different joints of the same limb. Neuroactive agents were applied independently to either the brainstem or spinal cord bath. The substances surveyed in the present studies included some of those used previously, as well as additional compounds: bicuculline and picrotoxin (gamma-aminobutyric acid-ergic antagonists), N-methyl-D-aspartic acid (excitatory amino acid agonist), substance P, acetylcholine, carbachol (cholinergic agonist), and serotonin. Application of these substances to the brainstem bath produced rhythmic airstepping. Application of dopamine, aspartate, glutamate, and N-methyl-D-aspartic acid to the spinal cord bath also produced rhythmic airstepping, while application of acetylcholine produced tonic, long-lasting co-contractions. These findings reveal the presence of several neurochemical systems in the central nervous system that can be activated at birth to induce coordinated airstepping in the neonate rat in vitro brainstem-spinal cord preparation.     

The action of some analogues of the excitatory amino acids in the dentate gyrus of the rat

We have confirmed that gamma-D-glutamylglycine and the L-isomer of 2-amino-4-phosphonobutyric acid, and have shown also that L-2-amino-5-phosphonovaleric (L-APV) acid, are antagonists of synaptic excitations of dentate granule cells induced from both lateral and medial perforant paths. The N-methyl-D-aspartic acid (NMDA) antagonist D-APV is without effect. The synaptic antagonists reduce the presynaptic fibre volley particularly in the lateral path, suggesting that a reduced transmitter output contributes to their action. NMDA receptors exist upon the granule cells, but they are not involved with these synaptic process.     

海马NMDA受体与神经肽Y在慢性应激性抑郁发生中的作用及其关系

本文旨在探讨N-甲基-D-天冬氨酸(N-methyl-D-aspartic acid,NMDA)受体与神经肽Y(neuropeptide Y,NPY)在慢性应激抑郁发生中的作用与关系。建立慢性不可预见性温和应激(chronic unpredictable mild stress,CUMS)抑郁模型,海马单侧分别微量注射非竞争性NMDA受体拮抗剂MK-801、NPY-Y1受体阻断剂GR231118和NMDA后,利用体重测量及糖水偏爱测试、强迫游泳及敞箱实验等方法观察动物行为变化,运用免疫组织化学方法检测海马CA3区和齿状回(dentate gyrus,DG)内NPY的表达。结果显示,CUMS组大鼠表现出抑郁样行为变化,海马NPY表达显著降低;海马微量注射NMDA或NPY-Y1受体阻断剂GR231118,动物行为学表现均与CUMS组相同,注射NMDA可使NPY表达显著降低;海马微量注射MK-801能明显改善应激引起的抑郁样行为表现,并使海马NPY表达增加。联合注射GR231118与MK-801后,GR231118可以显著减弱MK-801的抗抑郁样行为的效应。以上结果表明,CUMS可能使谷氨酸(glutamic acid,Glu)过量释放,NMDA受体过度激活,抑制NPY表达,导致抑郁发生。NPY抗抑郁作用主要是通过NPY-Y1受体实现。     

Enantiomer separation of imidazo-quinazoline-dione derivatives on quinine carbamate-based chiral stationary phase in normal phase mode

The normal phase mode liquid chromatographic enantiomer separation capability of a quinine tert-butyl-carbamate-type chiral stationary phase (CSP) has been investigated for a set of polar [1,5-b]-quinazoline-1,5-dione derivatives. This class of chiral heterocycles is currently under development as potential alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and/or N-methyl-D-aspartic acid (NMDA) receptor antagonists. The effect of the nature and concentration of polar modifier, i.e., ethanol and isopropanol, in n-hexane-based mobile phases, as well as the substituent pattern of the phenyl ring attached to the quinazolone framework on retention factor, enantioselectivity, and resolution was investigated. The Soczewiński competitive adsorption model was used to describe the relationship between the retention and the binary mobile phase compositions. According to this model, linear plots of the logarithms of retention factor versus molar fractions of the polar modifiers were obtained over a wide concentration range (X(B) between 0.15 and 0.35). Addition of equimolar ethanol yields higher resolution than isopropanol, R(S) values ranging between 1.54 and 2.75, whereas the latter allows to achieve moderately increased enatioselectivity. The resolution was further improved by using a ternary mixture of n-hexane:methanol:isopropanol/85:5:10 (v/v). The most pronounced selectivity factor alpha and resolution R(S) values were obtained for the para-hydroxy substituted compound, indicating that chiral recognition is sensitive to steric and stereoelectronic factors. In the course of optimization, the temperature-dependence on the chiral separation was also investigated. It turned out that the enantiomer separation is predominantly enthalpically driven in normal phase mode.     

A specific enzymatic high-performance liquid chromatography method to determine N-methyl-D-aspartic acid in biological tissues

Recently we demonstrated that N-methyl-D-aspartic acid (NMDA) is present as an endogenous compound in the nervous tissues and endocrine glands of the rat where it plays a role in the regulation of the luteinizing hormone, growth hormone, and prolactin (FASEB J. 14 (2000) 699; Endocrinology 141 (2000) 3861). Based on the prediction that NMDA could have future importance in neuroendocrinology, we have devised an improved method for the specific and routine determination of NMDA in biological tissue. This method is based on the detection by HPLC of methylamine (CH(3)NH(2)) which comes from the oxidation of NMDA by D-aspartate oxidase, an enzyme which specifically oxidizes NMDA, yielding CH(3)NH(2) as one of the oxidative products of the reaction. The sensitivity of the method permits the accurate determination of NMDA in the supernatant of a tissue homogenate at levels of about 5-10 picomol/assay. However, for those tissues in which the concentration of NMDA is less than 1nmol/g, the sample must be further purified by treatment with o-phthaldialdehyde in order to separate the NMDA from the other amino acids and amino compounds and then concentrated and analyzed by HPLC. Using this method we have conducted a comparative study in order to measure the amount of NMDA in neuroendocrine and other tissues of various animal phyla from mollusks to mammals.     

海马NMDA受体和NOS在慢性应激性抑郁发生中的作用及其相互关系

运用慢性不可预见性温和应激（chronic unpredicted mild stress, CUMS）建立抑郁动物模型,通过海马内微量注射、动物行为学观察及免疫组织化学方法检测海马内一氧化氮合酶（nitric oxide synthase,NOS）表达的变化,探讨CUMS诱发抑郁与海马谷氨酸N-甲基-D-天冬氨酸（N-methyl-D-aspartic acid,NMDA）受体、一氧化氮合酶（nitric oxide synthase,NOS)的关系。结果发现：CUMS组大鼠表现出抑郁样行为变化,海马NOS表达显著升高;海马微量注射NMDA受体激动剂,动物行为学表现与CUMS组相同,NOS表达升高;海马微量注射非竞争性NMDA受体拮抗剂MK-801能明显改善应激引起的抑郁样行为表现,并降低海马NOS表达。这些结果表明慢性不可预见性应激可能使谷氨酸（glutamic acid,Glu）过量释放,NMDA受体过度激活,NOS高表达,NO过量产生,损伤海马神经元,导致抑郁发生。     

A new class of N-methyl-D-aspartic acid receptor agonists and antagonists--derivatives of imidazole-4,5- and pyrazole-3,4-dicarboxylic acids]

New agonists and antagonists of the N-methyl-D-aspartic acid (NMDA) receptors were found among the derivatives of 1- or 2-alkyl-substituted imidazole-4,5- and pyrazole-3,4-dicarboxylic acids. Lipophilic surrounding of the nitrogen atom in these compounds was found to determine their ability to be either agonists or antagonists, while the distance between the terminal acidic functions was the same. An increase in the lipophilicity can also cause loss of selective action upon the NMDA receptors and occurrence of non- NMDA antagonistic activity.     

LY226936 Administered Orally and Centrally to Obese Zucker Rats Suppresses Food Intake and Body Weight Gain

LY226936, methylcarbamothoic acid-S-(4,5-dihydro-2-thiazolyl) ester, is a new compound that, when administered to obese Zucker rats, caused reduced food intake. LY226936 reduced the food consumption after a single oral dose of 50 and 100 mg/kg. On chronic oral administration to meal-fed obese (5 to 35 mg/kg. once daily) and to fed obese and lean (15 mg/kg. twice daily) Zucker rats, LY226936 reduced food intake and body weight gain for periods ranging from 40 to 48 days. The effect on both parameters was statistically significant. There is no evidence in our studies that tolerance to the actions of LY226936 developed. LY226936 decreased the consumption of both high carbohydrate and high fat diets. Food consumption of meal-fed obese Zucker rats was reduced significantly each time a single dose of 10 ugm LY226936 per rat was infused intracerebroventricularly. None of the receptors studied (mu and kappa opioid, CCK, serotonin, neuropeptide Y, galinin, N-methyl-D-aspartic acid) appeared to bind LY226936 and therefore, appear not to be involved in the depression of food intake by the obese Zucker rat.     

Strychnine-Sensitive, Glycine-Induced Release of [3H]Norepinephrine from Rat Hippocampal Slices

The amino acid glycine is the primary inhibitory neurotransmitter of the mammalian spinal cord. Glycine has also been shown to facilitate the excitatory actions of glutamate at the N-methyl-D-aspartic acid receptor subtype. In this article, glycine is shown to increase the Ca2(+)-dependent release of [3H]norepinephrine from preloaded slices of the rat hippocampus. This effect was inhibited noncompetitively by nanomolar concentrations of strychnine, which differentiates it from the glycine site associated with the N-methyl-D-aspartate receptor. Glycine also released [3H]acetylcholine, but was without effect on the efflux of [3H]serotonin or gamma-[3H]aminobutyric acid from the same tissue preparation. The release of [3H]norepinephrine was reversibly blocked by tetrodotoxin, indicating the effect is not initiated at the noradrenergic terminals, but requires propagation of an action potential. The results suggest that a glycine site that is pharmacologically similar to that found in the spinal cord exists in the rat hippocampus. We suggest that this site may participate in modulating the release of specific neurotransmitters in the brain.     

N-methyl-D-aspartate-sensitive [3H]glutamate binding sites in brain synaptic membranes treated with Triton X-100

Specific binding activity of radiolabeled L-glutamic acid, a putative central excitatory neutrotransmitter, was drastically increased with increasing concentrations of Triton X-100 used for pretreatment of rat brain synaptic membranes. The binding in these Triton-treated membranes was a protein dependent, inversely temperature-dependent, stereospecific, structure-selective and saturable process with a high affinity for the amino acid. The binding activity was invariably inhibited by agonists and antagonists for the N-methyl-D-aspartic acid (NMDA)-sensitive subclass, but not by agonists for the other subclasses of excitatory amino acid neurotransmitter receptors in the brain. Scatchard analysis revealed that the binding sites consisted of a single component with a Kd of 24.4 +/- 2.5 nM and a Bmax of 0.94 +/- 0.09 pmol/mg protein. Some endogenous tryptophan metabolites such as kynurenic acid and quinolinic acid also inhibited the binding. These results suggest that synaptic membranes may indeed contain the NMDA-sensitive receptors which are disclosed by Triton X-100 treatment.     

MK-801 Increases Extracellular 5-Hydroxytryptamine in Rat Hippocampus and Striatum In Vivo

The effect of MK-801 (0.25 or 0.5 mg/kg) on the extracellular concentration of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in rat hippocampus and striatum was studied using intracerebral dialysis. The dialysate 5-HT concentration was dose-dependently increased by MK-801 in both regions. In the hippocampus, at the higher drug dose a slow increase in the 5-HIAA level was observed, and this became significant 3 h after treatment. In contrast to this, the extracellular 5-HIAA content in the striatum was significantly decreased 150 min after administration of both doses of MK-801. The data are discussed in the light of the known behavioural effects of MK-801 and possible N-methyl-D-aspartic acid receptor regulation of 5-HT release.     

Protection of neuronal cells against reactive oxygen species by carnosine and related compounds

Carnosine and related compounds were compared in terms of their abilities to decrease the levels of reactive oxygen species (ROS) in suspensions of isolated neurons activated by N-methyl-D-aspartic acid (NMDA) using both stationary fluorescence measurements and flow cytometry. Carnosine was found to suppress the fluorescent signal induced by ROS production and decreased the proportion of highly fluorescent neurons, while histidine showed opposite effects. N-Acetylated derivatives of both carnosine and histidine demonstrated weak (statistically indistinguishable) suppressive effects on the ROS signal. N-Methylated derivatives of carnosine suppressed intracellular ROS generation to the same extent as carnosine. This rank of effectiveness is distinct from that previously obtained for the anti-radical ability of CRCs (anserine>carnosine>ophidine). These differences suggest that the similar ability of carnosine and its N-methylated derivatives to protect neuronal cells against the excitotoxic effect of NMDA is not solely related to the antioxidant properties of these compounds.     

丝氨酸消旋酶在中枢神经系统中的研究进展

中枢神经系统中,丝氨酸消旋酶是5'吡哆醛依赖性酶,通过合成调控D型丝氨酸,参与N-甲基-D-天冬氨酸受体介导的神经发生、突触可塑性及学习记忆的调节。丝氨酸消旋酶表达与活性可以通过转录、翻译、翻译后修饰,小分子配基与蛋白相互作用,亚细胞分布多种方式调节。丝氨酸消旋酶失调影响了精神分裂症、脑损伤及神经退行性疾病等多种中枢神经系统疾病。本文简要介绍丝氨酸消旋酶的结构、分布、调节因素和在中枢神经系统中的生理病理功能,为神经及精神疾病的治疗和药物开发提供了新的思路。     

Disclosure by triton X-100 of NMDA-sensitive [3H] glutamate binding sites in brain synaptic membranes

Pretreatment of brain synaptic membrane homogenates with Triton X-100 resulted in a drastic disclosure of [3H] glutamate (Glu) binding activity which was sensitive to one of the central Glu receptor agonists, N-methyl-D-aspartic acid (NMDA). The NMDA-sensitive binding was inversely dependent on the incubation temperature, and was a reversible and saturable process. Scatchard analysis revealed that Triton X-100 treatment yielded in a significant enhancement of the affinity with a concomitant increment of the density of binding sites. Electrophysiologically identified agonists and antagonists for the NMDA receptors all significantly inhibited the binding to Triton-treated membranes. These results suggest that Triton-treatment may disclose NMDA-sensitive [3H] Glu binding sites in brain synaptic membranes.