兴奋性和抑制性氨基酸以及机体应激激素表达与前庭性偏头痛的关系探究

目的探究兴奋性、抑制性氨基酸及机体应激激素表达与前庭性偏头痛的关系。方法选取2016年7月至2018年3月我院诊治的30例前庭性偏头痛患者为试验组,同时期的30例健康体检者为对照组。比较两组的血清兴奋性、抑制性氨基酸及机体应激激素表达情况,并比较试验组中不同疼痛程度患者的检测结果,采用Logistic分析处理兴奋性、抑制性氨基酸及机体应激激素与前庭性偏头痛的关系。结果试验组的血清兴奋性、抑制性氨基酸及机体应激激素表达水平均显著高于对照组,试验组中重度疼痛患者的血清兴奋性、抑制性氨基酸及机体应激激素表达水平均显著高于中度疼痛患者,差异均有统计学意义(P<0.05),且Logistic分析显示,血清兴奋性、抑制性氨基酸及机体应激激素均与前庭性偏头痛有密切的关系。结论前庭性偏头痛患者的兴奋性、抑制性氨基酸及机体应激激素均呈现异常表达,且不同疼痛程度患者的表达差异较大,上述指标与前庭性偏头痛之间有密切关系。     

A quantitative study of the actions of excitatory amino acids and antagonists in rat hippocampal slices.

1. A quantitative pharmacological investigation of the actions of excitatory amino acids on hippocampal CA1 neurones has been made using a new slice preparation developed for grease gap recording; d.c. potential was measured across a grease barrier placed between alvear fibres and the bathing medium. 2. In Mg2+-free perfusate, N-methyl-D-aspartate (NMDA, 1-100 microM), quisqualate (1-500 microM), kainate (1-200 microM) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 1-100 microM) caused dose-dependent depolarizations. 3. The dose-response relationships were fitted to logistic expressions. The maximum responses to AMPA, NMDA and kainate were similar; their respective EC50 values were 5, 13 and 23 microM. Quisqualate had a smaller maximum; its EC50 value was 10 microM. The slopes of the dose-response relationships were different for the 4 agonists; the order of steepness of the slopes was NMDA greater than AMPA greater than kainate greater than quisqualate. 4. Similar amino acid-induced depolarizations were observed in slices of just the CA1 region or in whole slices bathed in tetrodotoxin. Isolated alvear fibres, however, were insensitive to the excitatory amino acids. 5. D-2-Amino-5-phosphonovalerate (APV, 50 microM) selectively and reversibly antagonized responses induced by NMDA (apparent pA2 = 5.21). 6. Kynurenic acid (1 mM) reversibly depressed responses to the three agonists tested. The dose-ratios for antagonism of AMPA, kainate and quisqualate were 6.9, 5.6 and 4.6 respectively. 7. This preparation has a different sensitivity profile to agonists from those of previously reported preparations of spinal cord, neocortex and cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cocaine behavioral sensitization and the excitatory amino acids

Studies were conducted to identify neuroeffector systems involved in behavioral sensitization to cocaine-induced stereotypy in mice, and to compare the results with those from our previous amphetamine studies. The effects of eight relatively selective neuroeffector agonists and antagonists were measured in mice in order to identify specific functional changes associated with the sensitization. In contrast to amphetamine, the only neuroeffector response altered by cocaine sensitization was a decrease in convulsive threshold to kainate. The persistence of the change in convulsive threshold correlated with the persistence of behavioral sensitization. The induction of sensitization was blocked by pretreatment with four different classes of drugs, represented by haloperidol, dizocilpine, diltiazem and DNQX. These results suggest that the mechanism of induction to cocaine is similar to that of amphetamine; both the glutamate and dopaminergic systems appear to be involved in induction. The expression of the sensitized cocaine response was blocked by haloperidol, CPP and diltiazem. These results differed from those obtained previously insofar as CPP did not affect the expression of sensitization to amphetamine. Furthermore, DNQX, in contrast to its antagonism of the expression of amphetamine sensitization, did not affect the expression of cocaine sensitization. The pharmacological data suggest that the mechanism of induction differs from that of expression, and that the mechanism of expression for cocaine sensitization differs from that for amphetamine.     

Stimulation of [3H]norepinephrine release from hippocampal slices by excitatory amino acids

[3H]norepinephrine efflux from preloaded rat hippocampal slices was increased in a dose-dependent manner by excitatory amino acids (EAA) in the following potency order: N-methyl-D-aspartic acid (NMDA) greater than kainic acid greater than L-glutamic acid greater than or equal to D,L-homocysteic acid greater than L-aspartic acid greater than quinolinic acid greater than quisqualic acid. The effect of EAA was blocked by physiological concentration of Mg2+, with the exception of kainic acid. D,L-2-amino-7-phosphonoheptanoic acid (APH) dose-dependently inhibited NMDA effect (IC50 = 69 mumol/L), whereas at 1 mmol/L it was ineffective versus kainic acid. The release of [3H]norepinephrine induced by quinolinic acid was blocked by APH 0.1 mmol/L. gamma-D-glutamylglycine dose-dependently inhibited kainic acid effect with an IC50 = 1.15 mmol/L. Tetrodotoxin 2 mumol/L reduced NMDA and kainic acid effects by 40 and 20%, respectively. The data indicate a possible involvement of central noradrenergic system in the modulation of excitotoxic action of EAA and offer a reliable system for testing new compounds acting at EAA-receptors by measuring norepinephrine release in vitro.     

Ethanol inhibits release of excitatory amino acids from slices of hippocampal area CA1.

Slices of hippocampal area CA1 were employed to test the hypothesis that release of excitatory amino acids glutamate and aspartate is regulated by ethanol. K(+)-evoked release of glutamate and aspartate was inhibited by ethanol (25-100 mM) in a dose-dependent manner. Ethanol (100 mM) also inhibited K(+)-evoked gamma-aminobutyric acid release. This selective inhibition of excitatory amino acid release by ethanol may contribute to some of the learning and memory deficits associated with ethanol abuse, since excitatory amino acid receptors play an important role in synaptic plasticity processes.     

皮质酮对原代培养大鼠海马神经细胞的毒性作用及其与兴奋性氨基酸的关系

探讨了皮质酮对原代培养海马神经细胞的毒性作用及作用机理. 结果显示,无血清培养基中加入皮质酮（10 nmol·L^-1-0.1 mmol·L^-1）可浓度依赖地损伤原代培养的海马和皮层神经细胞,其EC₅₀分别为3.2 和85 μmol·L^-1. 预先加入N-甲基-D-天冬氨酸(NMDA)受体拮抗剂地佐环平(MK 801,终浓度为50 μmol·L^-1）可显著拮抗皮质酮（10 μmol·L^-1）对海马神经细胞的毒性作用. 皮质酮同海马脑薄片共同孵育10和20 min可明显增加海马脑薄片谷氨酸和谷氨酰胺的释放,对天冬氨酸的释放则无明显影响. 实验结果提示,皮质酮可选择性地损伤原代培养海马神经细胞,该毒性作用可能与其增加兴奋性氨基酸释放有关.     

Stimulatory and inhibitory actions of excitatory amino acids on inositol phospholipid metabolism in rat cerebral cortex.

1. The effects of excitatory amino acids on [3H]-inositol phosphate levels have been examined in rat cortical slices under basal conditions or following agonist stimulation. 2. Ibotenate and quisqualate provoked a substantial dose-dependent (EC50, 30 microM and 20 microM respectively) increase in inositol phosphates; these responses were not additive suggesting a common site of action for the two amino acids. The responses to maximally effective concentrations of ibotenate and quisqualate were not blocked by verapamil, tetrodotoxin or Cd2+, indicating that these effects are not indirect. Small, but significant, increases in inositol phosphates were also seen with glutamate and N-methyl-DL-aspartate (NMDLA); kainate and aspartate were ineffective. 3. Each excitatory amino acid tested reduced carbachol (1 mM) stimulated inositol phosphate formation. Kainate (IC50, 20 microM) and NMDLA (IC50, 20 microM) were the most effective inhibitors. Kainate also reduced the responses to noradrenaline, 5-hydroxytryptamine and 20 mM K+. 4. The inhibitory action of NMDLA, but not kainate, could be reversed with the NMDA antagonists, DL-2-amino-5-phosphonovalerate (APV) and MK-801; DL-2-amino-4-phosphonobutyrate (APB) was without effect. Since MK-801 blocks the ion channels associated with the NMDA receptor, it appears that inhibition requires the entry of ions into the cell. 5. APV and MK-801 potentiated the stimulatory response to ibotenate but had no effect on the response to quisqualate. Potentiation was presumably the result of blocking the inhibition by ibotenate mediated through NMDA receptors. 6. In conclusion, excitatory amino acids appear to reduce agonist-mediated inositol phosphate formation in rat cerebral cortex by a non-specific action, possibly including the influx of Na+ ions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of inescapable shock and conditioned fear on the release of excitatory and inhibitory amino acids in the locus coeruleus

We investigated the importance of endogenous amino acids in the locus coeruleus in inescapable electric shock and conditioned fear. In naive rats and in rats exposed to noise (N), light (L) and electric shock (S) or to N + L only, the locus coeruleus was superfused with artificial cerebrospinal fluid through a push-pull cannula and the release of GABA, taurine, glutamate, aspartate, serine and glutamine was determined in the superfusate by HPLC after derivatization with o-phthaldialdehyde. Locomotor activity, arterial blood pressure and heart rate were telemetrically monitored. The placement of naive rats or conditioned rats from their home cage to a chamber provided with a grid-floor for shock virtually did not change the release rates of the amino acids in the locus coeruleus. Motility was enhanced in naive and conditioned rats to a similar extent. Blood pressure and heart rate were enhanced in conditioned rats only. Exposure to N + L + S for 5 min greatly enhanced the release rates of all determined amino acids in the locus coeruleus. In conditioned rats the increase in release of most amino acids lasted longer than in naive rats. Electric shock also enhanced motility, blood pressure and heart rate. In conditioned rats, motility and cardiovascular changes were more pronounced and/or lasted longer than in naive rats. Exposure of conditioned rats to the conditioned stimuli N + L for 5 min led to an increased release of taurine and aspartate. The enhanced release of taurine lasted 30 min. Exposure to N + L did not affect the release rates of amino acids in naive rats. N + L did not influence motility but arterial blood pressure and heart rate were elevated in conditioned rats. The findings show that inescapable electric shock enhances the release of several amino acids in the locus coeruleus, while conditioned fear selectively increases the outflow of taurine and aspartate. Moreover, conditioned fear prolongs the response of excitatory and inhibitory amino acids to electric shock. The results suggest that an excitatory amino acid (aspartate) and an inhibitory amino acid (taurine) of the locus coeruleus are implicated in conditioned fear.     

The release and uptake of excitatory amino acids

In this article, David Nicholls and David Attwell describe recent advances in our understanding of the mechanisms by which excitatory amino acids are released from cells, and of the way in which a low extracellular glutamate concentration is maintained. Glutamate can be released from cells by two mechanism: either by Ca2(+)-dependent vesicular release or, in pathological conditions, by reversal of the plasma membrane uptake carrier. The contrasting pharmacology and ionic dependence of the glutamate uptake carriers in the vesicle membrane and in the plasma membrane explain how glutamate (but probably not aspartate) can function as a neurotransmitter, and why the extracellular glutamate concentration rises to neurotoxic levels in brain anoxia.     

Actions of excitatory amino acids and their antagonism

Possible sites and mechanisms of interaction of glutamic acid diethylester with glutamate are considered, in relation to the thesis that the amino acid acts as an excitatory transmitter of certain central synapses. Differences in action of other substances which have been described as antagonists of glutamate are also discussed.     

Neurotoxic effect of corticosterone on primary cultured hippocampal neurons and its relationship with excitatory amino acids 1

探讨了皮质酮对原代培养海马神经细胞的毒性作用及作用机理．结果显示,无血清培养基中加入皮质酮（１０ｎｍｏｌ·Ｌ－１－０．１ｍｍｏｌ·Ｌ－１）可浓度依赖地损伤原代培养的海马和皮层神经细胞,其ＥＣ５０分别为３．２和８５μｍｏｌ·Ｌ－１．预先加入Ｎ－甲基－Ｄ－天冬氨酸（ＮＭＤＡ）受体拮抗剂地佐环平（ＭＫ－８０１,终浓度为５０μｍｏｌ·Ｌ－１）可显著拮抗皮质酮（１０μｍｏｌ·Ｌ－１）对海马神经细胞的毒性作用．皮质酮同海马脑薄片共同孵育１０和２０ｍｉｎ可明显增加海马脑薄片谷氨酸和谷氨酰胺的释放,对天冬氨酸的释放则无明显影响．实验结果提示,皮质酮可选择性地损伤原代培养海马神经细胞,该毒性作用可能与其增加兴奋性氨基酸释放有关．     

On the configuration of the receptors for excitatory amino acids

Separate receptors are recognized for the excitation of mammalian neurones by (a) L-glutamic and quisqualic acids and (b) N-methyl-D-aspartic (NMDA), and other amino acids which have conformationally restricted molecules. Several other compounds, both agonists and antagonists, have been examined, and it is concluded that (i) the NMDA receptor reacts preferentially with substances in a relatively extended configuration, (ii) the glutamate/quisqualate receptor prefers folded molecules and (iii) the distance separating the amino group from the distal anionic function is the critical one determining receptor preference.     

Imbalance between excitatory and inhibitory amino acids at spinal level is associated with maintenance of persistent pain-related behaviors

Although the postsynaptic events responsible for development of pathological pain have been intensively studied, the relative contribution of presynaptic neurotransmitters to the whole process remains less elucidated. In the present investigation, we sought to measure temporal changes in spinal release of both excitatory amino acids (EAAs, glutamate and aspartate) and inhibitory amino acids (IAAs, glycine, γ-aminobutyric acid and taurine) in response to peripheral inflammatory pain state. The results showed that following peripheral chemical insult induced by subcutaneous bee venom (BV) injection, there was an initial, parallel increase in spinal release of both EAAs and IAAs, however, the balance between them was gradually disrupted when pain persisted longer, with EAAs remaining at higher level but IAAs at a level below the baseline. Moreover, the EAAs–IAAs imbalance at the spinal level was dependent upon the ongoing activity from the peripheral injury site. Intrathecal blockade of ionotropic (NMDA and non-NMDA) and metabotropic (mGluRI, II, III) glutamate receptors, respectively, resulted in a differential inhibition of BV-induced different types of pain (persistent nociception vs. hyperalgesia, or thermal vs. mechanical hyperalgesia), implicating that spinal antagonism of any specific glutamate receptor subtype fails to block all types of pain-related behaviors. This result provides a new line of evidence emphasizing an importance of restoration of EAAs–IAAs balance at the spinal level to prevent persistence or chronicity of pain.     

Ontogeny of amino acids related to excitatory amino acidergic transmission in the rat forebrain

Developmental changes in the concentration of putative amino acid neurotransmitters were investigated in the forebrain of prenatal and postnatal rats. Glutamate and aspartate, which interact with three-typed agonist sites of excitatory amino acid receptors, decreased until birth and then increased with maturation. Agonists for strychnine-insensitive glycine sites, i.e., glycine, serine and alanine, showed progressive decreases in the postnatal period. Although alanine rapidly decreased in preterm, there was no change in glycine and serine contents in this period. These developmental patterns of the compounds related to EAA transmission differed from those of inhibitory amino acid transmitters. GABA showed a steady increase during ontogeny. Taurine increased until birth and then decreased to the adult level. These results suggested that the ontogenic profile for EAA is different from that for the glycine receptor agonists and that other inhibitory amino acid transmitters develop with patterns different from those of amino acids related to EAAergic transmission.     

Pharmacological separation of cannabinoid sensitive receptors on hippocampal excitatory and inhibitory fibers

Our earlier studies demonstrated that in the hippocampus, cannabinoids suppress inhibitory synaptic transmission via CB(1) cannabinoid receptors, whereas a novel cannabinoid-sensitive receptor modulates excitatory synapses (Katona, I. et al., Journal of Neuroscience 19 (1999) 4544; Hájos, N. et al., European Journal of Neuroscience 12 (2000) 3239; Hájos, N. et al., Neuroscience 106 (2001) 1). The novel receptor does not correspond to CB(2), since this receptor type is not expressed in the brain (Munro, S. et al., Nature 365 (1993) 61). Recent binding experiments revealed that the synthetic cannabinoid WIN 55,212-2 binds with lower affinity to brain membranes of CB(1) receptor-knockout mice indicating that pharmacological differences exist between these two types of cannabinoid receptors in the hippocampus (Breivogel et al., Molecular Pharmacology 60 (2001) 155). To analyze this difference in detail, we first determined the EC(50) values of WIN 55,212-2 for excitatory and inhibitory transmission in rat hippocampal slices using whole-cell patch-clamp recordings. The estimated EC(50) value for inhibitory postsynaptic currents (IPSC) evoked by electrical stimulation in CA1 pyramidal cells was 0.24 microM, whereas for excitatory postsynaptic currents (EPSC) it was 2.01 microM, respectively. The cannabinoid antagonist, AM251, blocked the WIN 55,212-2-induced inhibition of evoked IPSCs, but not of EPSCs, providing evidence for its selectivity for CB(1). We then tested the hypothesis of whether the cannabinoid effect on hippocampal excitatory neurotransmission is mediated via receptors with an affinity for vanilloid ligands. Co-application of the vanilloid receptor antagonist capsazepine (10 microM) with cannabinoids (WIN55,212-2 or CP55,940) prevented the reduction of EPSCs, but not of IPSCs. The amplitude of evoked EPSCs was also suppressed by superfusion of the vanilloid receptor agonist capsaicin (10 microM), an effect which could also be antagonized by capsazepine. In contrast, capsaicin did not change the amplitude of evoked IPSCs.These results demonstrate that WIN 55,212-2 is an order of magnitude more potent in reducing GABAergic currents via CB(1) than in inhibiting glutamatergic transmission via the new CB receptor. The sensitivity of the new CB receptor (and EPSCs) to vanilloid ligands, but not to the cannabinoid antagonist AM251, represents another pharmacological tool to distinguish the two receptors, since CB(1) (and its effect on IPSCs) is not modulated by vanilloids, but is antagonized by AM251.     

Antagonists of excitatory amino acids and cyclic guanosine monophosphate in cerebellum

The excitatory amino acid analogues kainate, quisqualate, domoic acid, 4-fluoroglutamate, homocysteic acid and N-methylaspartate as well as the tremor-inducing drugs harmaline and oxotremorine all induced significant elevations in cyclic guanosine monophosphate (cGMP) levels in the cerebellum in vivo. The putative antagonists of excitatory amino acids, 2-amino-5-phosphonovalerate (APV) and piperidine dicarboxylate (PDA) both blocked the actions of the tremorogens. Piperidine dicarboxylate also blocked the in vivo activity of all the amino acid analogues except homocysteic acid and N-methylaspartate. 2-Amino-5-phosphonovalerate (APV) was inactive against kainate, quisqualate and homocysteic acid. It therefore appears that PDA and APV are useful tools for the further study of the function of glutamate and asparatate receptors.     

The time course of NMDA-and kainate-induced cGMP elevation and glutamate release in cultured neuron

The levels of extracellular glutamate, intracellular Ca²⁺ ([Ca2+]_i) and cGMP were determined for 1 h with the excitatory amino acids, N-methyl-D-aspartate (NMDA) or kainate in cultured cerebellar granule cells. Both NMDA and kainate produced a time-dependent release of glutamate, and kainate was more potent than NMDA in glutamate elevation. The elevation of extracellular glutamate was not purely governed by intracellular Ca²⁺ concentration. However, in opposite to the time-dependent elevation of glutamate, the elevation of cGMP by NMDA and kainate were at maximum level in short-time (1 min) incubation then remarkably decreased with longer incubation times. Post-applications (30 min after agonist) of EAA antagonst did not block EAAs-induced glutamate elevation. However, NMDA antagonist, phencyclidine (PCP), blocked NMDA-induced cGMP elevation at pre- or post-application, but kainate antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX), paradoxically augmented kainate-induced cGMP elevation for 1 h incubation. These results show that NMDA or kainate induces time-dependent elevations of extracellular glutamate, while the elevations of cGMP by these EAAs are remarkably decreased with longer incubation times. However, NMDA- and kainate-induced glutamate release was blocked by pre-application of each receptor antagonist but not by post-application while EAA-induced [Ca²⁺]_i was blocked by post-application of antagonist. These observations suggest that EAA-induced elevation of [Ca²⁺]_i is not parallel with elevation of glutamate release or cGMP.     

gamma-Hydroxybutyrate depresses monosynaptic excitatory and inhibitory postsynaptic potentials in rat hippocampal slices.

The action of gamma-hydroxybutyrate was studied on pre- and postsynaptic GABA(B) receptors in rat hippocampal neurones in vitro using intracellular recording. gamma-Hydroxybutyrate (1-10 mM) caused a 4-8 mV hyperpolarization of CA1 cells and a 20-80% decrease in monosynaptic excitatory and inhibitory postsynaptic potentials in a concentration-dependent manner. These actions were reversibly inhibited by a novel and selective GABA(B) antagonist, CGP 36742 (20-500 microM) suggesting that gamma-hydroxybutyrate can activate presynaptic as well as postsynaptic GABA(B) receptors.     

Regulation of serotonin release by GABA and excitatory amino acids

Regulation of serotonin release by gamma-aminobutyric acid (GABA) and glutamate was examined by microdialysis in unanaesthetized rats. The GABA(A) receptor agonist muscimol, or the glutamate receptor agonists kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolaproprionate or N-methyl-D-aspartate were infused into the dorsal raphe nucleus (DRN) while extracellular serotonin was measured in the DRN and nucleus accumbens. Muscimol produced decreases, and the glutamate receptor agonists produced increases in serotonin. To determine if these receptors have a tonic influence on serotonergic neurons, glutamate or GABA(A) receptor antagonists were infused into the DRN. Kynurenate, a nonselective glutamate receptor blocker, produced a small, 30% decrease in serotonin. A similar decrease was obtained with combined infusion of AP-5 and DNQX into the DRN. The GABAA receptor blocker bicuculline produced an approximately three-fold increase in DRN serotonin. In conclusion, glutamate neurotransmitters have a weak tonic excitatory influence on serotonergic neurons in the rat DRN. However, the predominate influence is mediated by GABA(A) receptors.     

Effects of excitatory amino acids on dopamine synthesis in the rat retina

Intraocular administration of quisqualate but not of N-methyl-D-aspartate or kainate augments dopamine synthesis in the retinas of dark-maintained rats. The effect of quisqualate is dosage-dependent but not blocked by the glutamate antagonist, glutamate diethyl ester. These results are the first to show that the quiescent dopamine neurons of the dark-adapted rat retina can be activated by an excitatory amino acid analogue of the putative endogenous neurotransmitter, glutamate.