Differential desensitization of ionotropic non-NMDA receptors having distinct neuronal location and function

The release of tritium from rat hippocampal synaptosomes prelabeled with [³H]noradrenaline ([³H]NA) or [³H]5-hydroxytryptamine ([³H]5-HT) and from rat neocortex synaptosomes prelabeled with [³H]choline and the release of endogenous GABA and glutamate from rat neocortex synaptosomes were monitored during superfusion with media containing varying concentrations of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or kainic acid. Concentration-dependent release potentiations were elicited by both excitatory amino acids (EAAs) in all the transmitter systems investigated. The releases evoked by 100 μM AMPA were, in all cases, almost totally dependent on external Ca²⁺ and sensitive to 6,7-dinitroquinoxaline-2,3-dione (DNQX), indicating involvement of non-NMDA receptors. When cyclothiazide, a drug able to prevent desensitization of AMPA-preferring receptors, was added to the superfusion medium (at 1 or 10 μM) concomitantly with 100 μM AMPA or kainate, the EAA-evoked release of [³H]NA was significantly enhanced. Concanavalin A, a lectin thought to prevent desensitization of kainate-preferring receptors, had no effect (up to 10 μM) on the release of [³H]NA evoked by AMPA or kainate. The effect of cyclothiazide was lost if, after an 8-min pretreatment, the drug was removed just before the AMPA stimulus. When added concomitantly with the EAAs, cyclothiazide potentiated the release of [³H]5-HT elicited by AMPA and, less so, that evoked by kainate. Concanavalin A was ineffective. Neither cyclothiazide (1 or 10 μM) nor concanavalin A (3 or 10 μM) could affect the release of [³H]ACh or endogenous GABA provoked by 100 μM AMPA or kainate, suggesting that the receptors involved do not desensitize. Exposure of neocortex synaptosomes to AMPA or kainate concomitantly with cyclothiazide caused endogenous glutamate release that did not differ from that evoked by the EAAs alone. In contrast, the effects of AMPA and kainate were potentiated by concanavalin A. The activity of the lectin (3 μM) persisted when it was applied for 8 min and then removed before the AMPA or kainate (100 μM) pulse. When hippocampal synaptosomes prelabeled with [³H]NA were subjected to three subsequent AMPA (100 μM) stimuli (S₁, S₂ and S₃), the release of [³H]NA decreased dramatically from S₁ to S₃ (S₃/S₁ = 0.14 ± 0.04); a significant ‘protection’ of the AMPA effect was offered by 1 μM cyclothiazide (S₃/S₁ = 0.36 ± 0.06). This value did not differ from the S₃/S₁ ratio (0.38 ± 0.04) obtained in parallel experiments with 12 mM K⁺. The release evoked by high-K⁺ was insensitive to cyclothiazide. Finally, the effect of AMPA on the release of [³H]ACh did not respond to cyclothiazide also during three subsequent stimuli with 100 μM AMPA. To conclude: a) ionotropic non-NMDA receptors mediating enhancement of NA, 5-HT, ACh, GABA and glutamate release exist on the corresponding nerve terminals; b) the receptors present on noradrenergic and serotonergic neurons are AMPA-preferring receptors, whereas the glutamate autoreceptors resemble most the kainate-preferring subtype; the receptors mediating ACh and GABA release can not be subclassified at present; c) desensitization may not be a property of all non-NMDA ionotropic receptors. The receptors here characterized represent five models of native non-NMDA receptors suitable for pharmacological and molecular studies. Received: 28 January 1997 / Accepted: 14 April 1997     

钙离子对4-氨基吡啶诱发肥大细胞释放组胺作用的影响

4-Aminopyridine(4-AP) has been shown to induce histamine release fromisolated peritoneal mast cells(PMC)in mice and rats. In the presence of extracellular calcium atnormal level(0.9 mmol·L^-1)histamine release induced by 4-AP(13. 6 mmol·L^-1) from mice PMCwas 33.0%±4.6%,while at low calcium concentration (0.5 mmol·L^-1) and in calcium-freemedium this parameter decreased to 25.5%±4.2%and 16.3%±3.7%respectively.Histaminerelease in response to 4-AP(10 mmol·L^-1)from rat PMC was 39.1%±6.7%(0.9 mmol·L^-1calcium),while at low calcium concentration(0. 5 mmol·L^-1) and in calcium-free medium thisparameter decreased to 29.3%±4.7%and 20.2%±2.9% respectively. Results of statisticalanalysis indicate that 4-AP induced histamine release is related to Ca²⁺ concentration. When rat PMCwere preincubated in calcium-free medium with EDTA 0.1 mmol·L^-1 for 180 min　4-AP inducedhistamine release was 13.8%±1.6%.This shows that 4-AP also elicited mobilization of endogenous calcium stores in mast cells.The mechanism of 4-AP　induced histamine release was discussed.     

In vitro studies on the mechanism by which (+)-norfenfluramine induces serotonin and dopamine release from the vesicular storage pool

(+)-Norfenfluramine is the main metabolite of the serotoninergic anorectic agent (+)-fenfluramine. Both compounds inhibit 5-HT reuptake and stimulate its release, although they induce release from different pools, with (+)-norfenfluramine acting primarily on the cytoplasmic pool. Moreover, (+)-norfenfluramine was more potent than the parent drug in inducing dopamine release. In order to investigate whether (+)-norfenfluramine induces a Ca²⁺-dependent vesicular release, like some amphetamine derivatives, in the present study we preloaded synaptosomes with the [³H]neurotransmitter ([³H]5-HT or [³H]dopamine), superfused (washed) them for 47 min in the absence of pargyline and then exposed them to the releasing stimulus. With this protocol, the cytoplasmic pool should be absent and the [³H]neurotransmitter should mainly be stored in synaptic vesicles, where (+)-norfenfluramine should act to induce release. This was confirmed by a significant decrease of (+)-norfenfluramine-induced [³H]5-HT and [³H]dopamine release after reserpine pretreatment. The dose-response curves of (+)-norfenfluramine-induced [³H]5-HT release were superimposable in hippocampus and hypothalamus, and also superimposable on the curve for (+)-fenfluramine-induced [³H]5-HT release; the dopamine releasing potency of (+)-norfenfluramine in the striatum was more than ten times lower. The [³H]5-HT release induced by (+)-norfenfluramine was partly (about 50%) but significantly Ca²⁺-dependent, and it was also markedly (68%) inhibited by Cd²⁺, a non-specific blocker of voltage-dependent Ca²⁺ channels, suggesting that the Ca²⁺-dependent release is mediated by entry of Ca²⁺ into the synaptosomes through these channels. The [³H]dopamine release induced by 5 μM (+)-norfenfluramine was completely Ca²⁺-independent whereas at higher concentrations (10 and 20 μM) it was only slightly (20%) Ca²⁺-dependent. We have no clear explanation why (+)-norfenfluramine has these different effects on serotoninergic and dopaminergic synaptosomes. Received: 6 April 1998 / Accepted: 9 June 1998     

Na+ channel modulation and force-frequency relationship in human myocardium

The enhancement of force of contraction (FOC) following increasing frequencies of stimulation is an important mechanism of positive inotropy in human myocardium. The present study aimed to investigate the influence of alterations in Na⁺ influx on FFR in human myocardium. Isometric FOC of electrically stimulated right auricular trabeculae (AUT, n=12) from human nonfailing hearts (n=8) was measured at different stimulation rates (0.5-3 Hz) under control conditions, after increasing Na⁺ influx by the addition of (±)BDF 9148 (BDF, 3 μmol l^-1) and after decreasing Na⁺ influx by the addition of lidocaine (LIDO, 10 μmol l^-1). Additionally, the rate dependent changes in diastolic tension (DT) were measured in all experiments. Under control conditions FOC increased with increasing frequencies of stimulation. The rate at which maximal FOC was observed (SFmax) was 2.0±0.2 Hz and maximal increase in FOC (PIEmax) by increasing frequency of stimulation was +1.5±0.5 mN. After increase of Na⁺ influx by BDF (3 μmol l^-1) SFmax was decreased to 0.8±0.1 Hz (p<0.05 versus control) and PIEmax was +0.1±0.3 mN (p<0.05). When Na⁺ influx was diminished by LIDO (10 μmol l^-1) SFmax and PIEmax were increased compared to control (2.4±0.1 Hz and +4.1±0.9 mN, p<0.05 versus control). The diastolic tension (DT) of AUT at 3 Hz was not changed at higher rates in the control group and after application of LIDO (10 μmol l^-1), whereas after enhancement of Na⁺ influx by BDF there was an increase in DT of +0.7±0.2 at 3Hz (p<0.05 versus control and LIDO). An enhanced Na⁺ influx leads to a decrease in the optimal frequency and to a smaller force potentiation by higher stimulation rates which could be at least partly due to incomplete relaxation at higher frequencies, whereas a reduced Na⁺ influx is followed by opposite alterations. It is concluded that besides Ca²⁺ handling also Na⁺ influx and Na⁺ homeostasis might determine the frequency-induced force potentiation in human myocardium. Thus, the negative FFR in diseased human myocardium might result from changes in cellular Ca²⁺ or Na⁺ regulatory sites. Received: 20 November 1996 / Accepted: 21 February 1997     

一叶萩碱对5种神经递质受体和蛙脊髓膜电位的影响

Using radioligand binding assay method, securinine was shown to be specifically bound to GABA receptors of rat brain with IC₅₀ of 10^(-4)～10^-5mol/L. However, securinine did not exhibit affinity for a₁-, a₂- adrenoceptors and M-cholinergic receptors of rat brain and the β-adrenoceptors of duck erythrocyte membrane. In isolated and perfused bullfrog spinal cord experiment, the depolarization induced by GABA was slightly depressed by securinine (1 mmol/L), but no antagonistic effect on glycine or taurine evoked depolarization was observed with securinine. These results indicate that securinine is a weak GABA antagonist.     

Endothelium-derived hyperpolarizing factor, but not nitric oxide or prostacyclin release, is resistant to menadione-induced oxidative stress in the bovine coronary artery

The interaction of superoxide anions (O₂ ^–) generated by menadione with the synthesis and/or action of nitric oxide (NO), prostacyclin (PGI₂) and endothelium-derived hyperpolarizing factor (EDHF) was investigated in segments of the left anterior descending coronary artery (LAD) isolated from bovine hearts. EDHF and NO release were monitored by superfusion bioassay in segments pre-constricted with the thromboxane mimetic, U46619, PGI₂ release in addition by enzyme immunoassay for 6-keto-prostaglandin F_1α, and generation of O₂ ^– by lucigenin-enhanced chemiluminescence. Bradykinin (1–1,000 pmol) elicited a prominent, endothelium-dependent, relaxant response, 50–60% of which was insensitive to combined blockade of cyclooxygenase with diclofenac (1 μM) and NO synthase with N ^G-nitro-l-arginine (50 μM). This diclofenac/N ^G-nitro-l-arginine-insensitive relaxant response was completely abrogated in the presence of tetrabutylammonium (3 mM), a non-selective inhibitor of Ca²⁺-dependent K⁺ channels, or when the segments were pre-constricted with potassium chloride (60 mM) instead of U46619, and thus most likely mediated by EDHF. Despite causing a two- to fourfold increase in the concentration of O₂ ^– in or at the vessel wall, menadione (30 μM) did not affect the diclofenac/N ^G-nitro-l-arginine-insensitive relaxant response to bradykinin. When administered in the absence of N ^G-nitro-l-arginine, however, menadione significantly inhibited the relaxant response to bradykinin, presumably by attenuating the NO-mediated relaxation. Menadione also abolished the bradykinin-stimulated release of PGI₂ from luminally perfused segments of the LAD. This effect was more pronounced in the absence of N ^G-nitro-l-arginine, indicating that PGI₂ release in this preparation may be more sensitive to inhibition by peroxynitrite, the reaction product of NO and O₂ ^–, than to O₂ ^– alone. These findings suggest that, in contrast to NO and PGI₂, the release, and presumably also the mechanism of action, of EDHF in the bovine LAD is resistant to an increase in the local concentration of O₂ ^– or peroxynitrite which is thought to play an important role in coronary heart disease. Received: 27 August 1998 / Accepted: 18 November 1998     

LC-MS/MS法测定人血浆中西酞普兰及其在制剂生物等效性中的应用

A sensitive and selective LC-MS/MS method for determination of citalopram in human plasma was established to study the bioequivalence of different formulations containing citalopram. The samples were simply pretreated by protein precipitation using acetonitrile, and then analyzed on a Zorbax Extend C₈column. The mobile phase consisted of acetonitrile-water-formic acid (60∶40∶0.2), at a flow-rate of 0.5 mL·min^-1. A Thermo Finnigan TSQ Quantum Ultra tandem mass spectrometer equipped with electrospray ionization source was used as detector and was operated in the positive ion mode. Selected reaction monitoring using the precursor to product ion combinations of m/z325 → m/z109 and m/z265 → m/z167 was performed to quantify citalopram and the internal standard, respectively. The pharmacokinetic parameters of citalopram in different formulations were calculated by non-compartment model. The linear calibration curves were obtained in the concentration range of 0.10-100 μg·L^-1. The lower limit of quantification was 0.10 μg·L^-1. The intra- and inter-day relative standard deviation (RSD) over the entire concentration range was less than 5.2%. Accuracy determined at three concentrations (0.25, 8.00 and 90.0 μg·L^-1 for citalopram) ranged from -4.7% to 1.3%. Each plasma sample was chromatographed within 3.0 min. The method was successfully used in bioequivalence study of citalopram in human plasma after oral administration of 20 mg citalopram. Calculated with AUC_{1-120 h}, the bioavailability of two formulations was (102.1±10.9)%. The method is rapid, selective, robust and is proved to be suitable for bioequivalence evaluation of different formulations containing citalopram.     

甲基黄酮醇胺盐酸盐对β受体的阻断作用

The β-receptor blocking action of methylflavonolamine hydrochloride(MFA) was studied and compared with those of propranolol. The doseresponse curves of isoproterenol were shifted to the right by MFA on isolated rabbit atrium in this experiment. The pA₂ value and the slope of the regression line of MFA calculated from Schild plot were 5.53 and -0.84 respectively. The effects of MFA and propranolol on duck erythrocyte membranes were studied by the radioligand binding method. Both MFA and propranolol inhibited the binding of [³H] dihydroalprenolol to β-receptors. Their apparent equilibrium dissociation constants were 1.12×10^-5 mol/L and 5.50×10^-9 mol/L respectively. The affinity of propranolol to β-receptors of duck erythrocyte membranes was 2039-fold higher than that of MFA. These results demonstrates that MFA is a weak competitive β-receptor blocking agent.     

Desensitization of capsaicin-evoked neuropeptide release — Influence of Ca2+ and temperature

Summary Capsaicin-induced stimulation and desensitization of neuropeptide release from primary afferent neurons was investigated in the rat urinary bladder in-vitro. The capsaicin (5 min contact time)-evoked release of calcitonin gene-related peptide-like immunoreactivity (CGRP-IR) was dose-dependent; threshold to produce detectable release was 0.1 μmol/l, the EC₅₀ was 0.17 μmol/l. Pre-exposure of tissues to capsaicin (0.1–1.0 μmol/l, 5 min contact time) caused a dose-dependent reduction of the amount of CGRP-IR which was released by a second exposure to capsaicin. At 0.1 and 0.3 μmol/l, capsaicin was less effective to inhibit the subsequent K⁺-evoked release than that evoked by a second capsaicin exposure. Pre-exposure to 1 μmol/l capsaicin completely prevented subsequent K⁺- or capsaicin-evoked release of CGRP-IR. Exposure of the preparation to capsaicin (0.3μmol/l) in a Ca²⁺-free, EDTA-containing medium did not produce release of CGRP-IR. A subsequent stimulation with capsaicin in a 2.5 mmol/l Ca²⁺-containing superfusion solution was not less effective to release CGRP-IR than in tissues which had not been pre-exposed to capsaicin. At 18°C, the capsaicin-evoked release of CGRP-IR was reduced to 20% of the value obtained by the same dose (0.3 μmol/l for 5 min) of capsaicin at 37°C. Comparison of the desensitizing effect of 0.3 and 0.1 μmol/l capsaicin at 18°C and 37°C, respectively, showed significant inhibition of desensitization at 18°C. Inhibition of desensitization was also observed when the amount of CGRP-IR, which was released during preexposure to capsaicin (0.3 μmol/l for 10 min) at 18°C, was 3-fold higher than that produced by pre-exposure to capsaicin (0.1 μmol/l for 5 min) at 37°C. The present results show that in a narrow range of concentrations, capsaicin induces “selective” desensitization which is entirely dependent on the presence of external Ca²⁺ — and which is attenuated at low temperature.     

化学—酶法立体控制合成光学纯L-羟基苯丙氦酸的新方法

Optically pure L-3(2-hydroxyphenyl) alanine(L-o-tyrosine ,Ⅲa,),L-3-(3-hydroxyphenyl) alanine(L-m-tyrosine,Ⅲb )and L-3-(4-hydroxyphenyl )alanine(L-p-tyrosine,Ⅲc )were synthesized by the stereocontrolled amination of corresponding hydroxycinnamic acld(Ⅱ)catalyzed by L-phenylalanine ammonia-lyase(PAL,EC4.3.1.5 )contained in Rhodoterula rubramycelium. The amination of compound Ⅱ was completed in aqueous ammonia solution( 6.4mol·L^-1,pH10.5, 30℃) with the conversion of 74.9%(Ⅱa),21.1%(Ⅱb)and 20.6%(Ⅱc)respectively.The absolute configuration of the products Ⅲa～c were confirmed by circular dichroism(CD),and chiral high-performance ligand exchange chromatography(HPLEC)showed that productsⅢ were optically pure L-isomers.     

地佐环平对缺血突触体氨基酸释放和游离钙的影响(英文)

目的　初步探讨地佐环平 (MK 80 1)对脑缺血的保护作用与兴奋性氨基酸 (EAA)和抑制性氨基酸(IAA)释放的关系。方法　营养液中去除糖和氧 ,建立大鼠脑突触体缺血模型 ,检测静息及高钾去极化状态下 ,缺血突触体EAA和IAA释放量及游离钙浓度。结果　大鼠突触体缺血状态致天冬氨酸和甘氨酸、牛磺酸、γ 氨基丁酸释放量明显增加 ,而谷氨酸亦有增加趋势。缺血 + 5 0mmol·L- 1K+刺激后 ,上述EAA及IAA释放量均进一步增加。静息及去极化状态下的缺血突触体游离钙浓度显著增加。MK 80 10 .1mmol·L- 1可显著阻遏静息及去极化状态缺血突触体释放EAA及IAA ,但对EAA的抑制作用更明显 ;同时亦明显降低两种状态下缺血突触体内游离钙浓度。结论　MK 80 1的抗脑缺血致脑损伤作用 ,至少部分与其对抗缺血引起的突触体内游离钙浓度升高和EAA释放量增加的作用有关。     

丙泊酚对大鼠海马突触体释放谷氨酸和γ-氨基丁酸的影响

目的观察丙泊酚对大鼠海马突触体谷氨酸和γ氨基丁酸(GABA)Ca2+依赖性释放和非Ca2+依赖性释放的影响。方法制备突触体后用人工脑脊液(aCSF)孵育,分为7组,应用丙泊酚(propofol)的浓度分别为3(Pro3组)、10(Pro10组)、30(Pro30组)、100(Pro100组)和300μmol·L-1(Pro300组),脂肪乳剂对照组加入脂肪乳(Intralipid组),空白对照组(Control组)不加入任何药物。在观察Ca2+依赖性释放时,加入二氢海人藻酸和哌啶酸;在观察非Ca2+依赖性释放的时候,去除aCSF中的Ca2+。在37℃的条件下,应用20μmol·L-1藜芦定或30mmol·L-1KCl诱发递质释放。应用反相高效液相色谱法测定aCSF中谷氨酸和GABA的浓度。结果①Ca2+依赖性递质释放:应用藜芦定时,Pro30、Pro100和Pro300组的谷氨酸和GABA释放量低于Intralipid组(P<0.01或P<0.05);应用KCl时,各组的谷氨酸和GABA释放量无差异(P>0.05)。②非Ca2+依赖性释放:各组应用藜芦定和KCl诱发的谷氨酸和GABA释放量无差异(P>0.05)。结论丙泊酚可以抑制Ca2+依赖性谷氨酸和GABA的释放,而对非Ca2+依赖性谷氨酸和GABA释放无影响。     

Effects of γ-acetylenic GABA and γ-vinyl GABA on synaptosomal release and uptake of GABA

The effects of the irreversible inhibitors of GABA-transaminase (E.C.2.6.1.19), γ-acetylenic GABA (4-amino-hex-5-ynoic acid, RMI 71645) and γ-vinyl GABA (4-amino-hex-5-enoic acid, RMI 71754) on the release and uptake of endogenous and exogenous GABA by rat cerebral cortical synaptosomes were studied. γ-Acetylenic GABA increased the release of preloaded [U¹⁴C]-GABA by 12 to 46% over the concentration range 0.5 to 5 mM. With γ-vinyl GABA, the concentration range 0.25–5 mM caused an increase of 22–95% above controls. All concentrations of γ-vinyl GABA were more effective than equivalent levels of γ-acetylenic GABA. The two agents had similar effects on the release of endogenous GABA, no change was observed in any other amino acid. The effect of both drugs on [U¹⁴C]-GABA release was inhibited significantly by tetrodotoxin and blocked by Verapamil.When animals were pretreated with γ-acetylenic GABA (100 mg/kg) for 8 hr or γ-vinyl GABA (1000 mg/kg) for 14 hr, the control release of GABA from synaptosomes prepared subsequently was increased, but other amino acids showed no change. The GABA content of the tissue was also greatly increased. The veratrine-stimulated release of GABA, glutamate and aspartate were increased by this pretreatment, but no change occurred in the rates of release of other amino acids. All veratrine-stimulated changes in the release of endogenous GABA, glutamate and aspartate were prevented by tetrodotoxin.     

克仑特罗对大鼠肝细胞氮代谢及6-磷酸葡萄糖脱氢酶活性的影响

目的探讨克仑特罗(Cl)调节大鼠肝细胞物质代谢及药理学机制。方法测定Cl(1×10^-6 mol·L^-1)对大鼠原代肝细胞培养液尿素氮水平以及肝细胞³H-亮氨酸参入,胰岛素样生长因子I(IGF-I)水平和6-磷酸葡萄糖脱氢酶(G6PDH)活性的影响。结果Cl使培养液尿素氮浓度下降25.51%(P<0.05);使肝细胞³H-亮氨酸参入量增加23.35%(P<0.05);增加肝细胞产生IGF-I的趋势(P>0.05);使肝细胞G6PDH活性下降45.22%(P<0.05)。β受体阻滞剂普萘洛尔(Pro)对Cl的上述作用均有阻断作用。结论Cl可增强大鼠肝细胞氮素保留和蛋白质合成,以及抑制肝细胞G6PDH活性而具有调节机体物质代谢的药理学效应。     

褪黑激素对大脑皮层谷氨酸释放及其神经毒的拮抗作用

AIM: To observe the effects of melatonin (Mel) on glutamate (Glu) release from the cortical synaptosomes in old mice and on neurotoxicity induced by KCl, Glu in cultured cortical cells of fetal rat and to explore the antiaging mechanism of Mel. METHODS: Glu release by the synaptosomes in old mouse cerebral cortex was detected in a spectrofluorophotometer. The neuronal viability in primary cultures from rat cerebral cortex was assessed using MTT stain and lactate dehydrogenase (LDH) efflux in the bathing medium. RESULTS: Mel inhibited the K+ (30 mmol.L-1)-induced Glu release from synaptosomes either in calcium dependent or independent conditions [control (10.6 +/- 1.1), (9.2 +/- 0.7) mumol.g-1 (protein); Mel 0.1 mumol.L-1 (6.5 +/- 0.9), (7.5 +/- 0.6) mumol.g-1 (protein), respectively, P < 0.01 vs control group), increased MTT activity (control 0.67 +/- 0.04, 0.81 +/- 0.03; Mel 0.1 mumol.L-1 0.715 +/- 0.023, 0.925 +/- 0.027, P < 0.01 vs control group] and decreased LDH efflux (control 0.400 +/- 0.016, 0.379 +/- 0.016; Mel 0.1 mumol.L-1 0.345 +/- 0.021, 0.340 +/- 0.012, respectively, P < 0.01 vs control group), therefore, protected the neuronal viability against KCl and Glu-induced injury. CONCLUSION: The inhibitory effect of Mel on Glu release from cortical synaptosome and the protective effect of Mel on cortical neurons against neurotoxicity are its antiaging mechanisms.     

Cholecystokinin-8S increases dynorphin B, aspartate and glutamate release in the fronto-parietal cortex of the rat via different receptor subtypes

The effect of sulphated cholecystokinin-8 (CCK-8S) on extracellular dynorphin B, aspartate, glutamate and GABA levels in the rat fronto-parietal cortex was investigated with in vivo microdialysis. The peptide was infused through the microdialysis probe trying to mimic local CCK-8S release. Basal levels of dynorphin B were around 20pM, aspartate 100nM, glutamate 600nM and GABA 30nM. CCK-8S (10μM) induced a ≈3-fold increase in extracellular dynorphin B, aspartate and glutamate levels, while GABA levels were only slightly increased. The effect of CCK-8S was restricted to the stimulated neocortex. Systemic pretreatment with the CCK_B antagonist, L-365, 260, but not with the CCK_A antagonist, L-364, 718, significantly antagonised the effect of CCK-8S on cortical dynorphin B and aspartate release. However, both CCK_A and CCK_B antagonists inhibited the increase in cortical glutamate levels. Thus, the present results indicate that cortical CCK release exerts a stimulatory modulation on cortical dynorphin B and aspartate release via the CCK_B receptor subtype, and on glutamate release via both CCK_A and CCK_B receptor subtypes. Considering electrophysiological evidence that CCK increases neuronal firing rates in many brain regions, it may be suggested that CCK represents a stimulatory system modulating the function of the neocortex. Received: 25 September 1996 / Accepted: 25 January 1997     

A novel type of GABA receptor in rat spinal cord?

Summary The depolarization-evoked release of -aminobutyric acid (GABA) and its possible modulation mediated by autoreceptors were studied in nerve endings isolated from rat spinal cord and prelabeled with the radioactive aminoacid. In the presence of the GABA uptake inhibitor SK&F 89976A [N-(4,4-diphenyl-3-butenyl)-nipecotic acid], used to minimize carrier-mediated homoexchange, exogenous GABA (1–10 µmol/l) decreased in a concentration-dependent way the release of³H-GABA evoked by 15 mmol/l KCl. The GABA_A receptor agonist muscimol (10–100 µmol/l) did not affect the K⁺(15 mmol/l)-evoked³H-GABA release. Similarly ineffective was the GABA_B receptor agonist (–)-baclofen (3–100 µmol/l). The effect of GABA was not counteracted by the GABA_A receptor antagonists bicuculline,picrotoxinorSR95531 [2-(3-carbethoxy-2-propenyl)-3-amino-6-paramethoxy-phenyl-pyridazinium bromide]. In a set of experiments performed in parallel with spinal cord or cerebral cortex synaptosomes depolarized with 9 mmol/l KCI it was found that: (a) GABA (1–10 µmol/l) decreased in a concentration-dependent way the release of³H-GABA in both brain regions and its effects were practically doubled when compared with those in synaptosomes depolarized with 15 mmol/l KCI; - (b) muscimol (1–100 µmol/l) was ineffective both in spinal cord and in cerebral cortex; - (c) (–)-baclofen (1-100 µmol/l) concentration-dependently inhibited³H-GABA release in cerebral cortex synaptosomes but was almost inactive in the spinal cord; the (+)-enantiomer of baclofen (100 µmol/l) did not affect significantly the release of³H-GABA; - (d) the GABA_B receptor antagonist phaclofen [3-amino-2-(4chlorophenyl)-propyl phosphonic acid] antagonized the effect of GABA in cerebral cortex but not in spinal cord synaptosomes. It is concluded that the release of GABA in rat spinal cord may be modulated by an autoreceptor which does not belong to the known GABA receptor subtypes. Send offprint requests to M. Raiteri at the above address     

硫喷妥钠对大鼠前额皮层突触体谷氨酸和γ-氨基丁酸释放的影响

目的　观察硫喷妥钠对大鼠前额皮层突触体谷氨酸和γ 氨基丁酸 (GABA)Ca2 + 依赖性和Ca2 + 非依赖性释放的影响。方法　制备大鼠前额皮层粗突触体。用人工脑脊液(aCSF)孵育 ,分为 5组 :基础释放组 (Base)、硫喷妥钠 10μmol·L-1组 (THS10 )、硫喷妥钠 30 μmol·L-1组 (THS30 )、硫喷妥钠 10 0 μmol·L-1组 (THS10 0 )、硫喷妥钠 30 0 μmol·L-1组 (THS30 0 ) ,每组含 8份突触体。向每组各等份突触体反应液中加入不同浓度的硫喷妥钠 (Base组不加入 ) ,于 37℃条件下应用 30mmol·L-1KCl或 2 0 μmol·L-1veratridine诱发谷氨酸和GABA释放。应用反相高效液相色谱法 (RP HPLC)测定各反应液中的谷氨酸和GABA浓度。观察谷氨酸和GABACa2 + 非依赖性释放时 ,aCSF中不含Ca2 + 。结果　硫喷妥钠 30、10 0、30 0 μmol·L-1可显著抑制KCl或vera tridine诱发的Ca2 + 依赖性谷氨酸释放 (P <0 0 1,P <0 0 1,P <0 0 1) ,IC50 分别为 2 5 6 μmol·L-1和 2 6 3μmol·L-1;THS10与THS30组 ,THS30与THS10 0组之间差异有显著性 (P <0 0 1,P <0 0 1)。硫喷妥钠各浓度组中 ,Ca2 + 依赖性GABA释放及Ca2 + 非依赖性谷氨酸和GABA释放水平与Base组比较差异无显著性 (P >0 0 5 )。结论　硫喷妥钠可剂量依赖性地抑制大     

Cannabinoid CB1 receptor-mediated inhibition of glutamate release from rat hippocampal synaptosomes

Cannabinoid receptors are widely expressed in the brain and have been shown to regulate synaptic transmission through a presynaptic mechanism. Using synaptosomal preparation, I show here that 2,3-dihydro-5-methyl-3-(4-morpholinyl-methyl)-pyrrolo-1,4-benzoxazin-6-yl-1-naphthalenylmethanone (WIN 55212-2) strongly depressed 4-aminopyridine-evoked glutamate release in a concentration-dependent manner, and this effect was reversed by the selective cannabinoid CB(1) receptor antagonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-morpholinyl-1H-pyrazole-3-carboxamide (AM 281). The inhibitory modulation by WIN 55212-2 was not due to a decrease in synaptosomal excitability or a direct effect on the release machinery because WIN 55212-2 did not alter 4-aminopyridine-mediated depolarization and ionomycin-induced glutamate release. In addition, the WIN 55212-2-mediated inhibition of glutamate release was blocked by the G(i)/G(o) protein inhibitor pertussis toxin, but not by the protein kinase A inhibitor 2,3,9,10,11,12-Hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo-benzodiazocine-10-carboxylic acid, hexyl ester (KT 5720). Furthermore, this inhibitory effect was associated with a decrease in 4-aminopyridine-evoked Ca(2+) influx, which could be completely prevented in synaptosomes pretreated with the N- and P/Q-type Ca(2+) channel blockers. Together, these observations indicate that activation of cannabinoid CB(1) receptors inhibit 4-aminopyridie-evoked glutamate release from hippocampal synaptosomes through a inhibitory G protein to suppress N- and P/Q-type Ca(2+) channel activity.     

The effect of delta-aminolaevulinic acid on the high affinity uptake of aspartic acid by rat brain synaptosomes

delta-Aminolaevulinic (delta-ALA) acid is an analogue of GABA, glutamate and aspartate. High affinity GABA uptake by synaptosomes is inhibited by delta-ALA in a competitive fashion at high concentrations (greater than 0.5 mM). Glutamate uptake is inhibited in a non-competitive fashion at all delta-ALA concentrations (0.05-2.0 mM). High affinity aspartate uptake is inhibited in a non-competitive fashion at high concentration (greater than 1.0 mM) but exhibits a curve which resembles that for the inhibition of GABA uptake. The different interactions between delta-ALA, glutamate and aspartate may be evidence for a separate transport system for aspartate.