Kainate-enhanced release of D-[3H]aspartate from cerebral cortex and striatum: reversal by baclofen and pentobarbital

A study was made of the actions of the excitant neurotoxin, kainic acid, on the uptake and the release of D-[2,3-3H]aspartate (D-ASP) in slices of guinea pig cerebral neocortex and striatum. The slices took up D-ASP, reaching concentrations of the amino acid in the tissue which were 14-23 times that in the medium. Subsequently, electrical stimulation of the slices evoked a Ca2+-dependent release of a portion of the D-ASP. Kainic acid (10(-5)-10(-3) M) produced a dose-dependent inhibition of D-ASP uptake. The electrically evoked release of D-ASP was increased 1.6-2.0 fold by 10(-5) and 10(-4)M kainic acid. The kainate-enlarged release was Ca2+-dependent. Dihydrokainic acid, an analogue of kainic acid with little excitatory or toxic action, did not increase D-ASP release but depressed D-ASP uptake. Attempts were made to block the action of kainic acid with baclofen and pentobarbital, compounds which depress the electrically evoked release of L-glutamate (L-GLU) and L-aspartate (L-ASP). Baclofen (4 X 10(-6)M), an antispastic drug, and pentobarbital (10(-4)M), an anesthetic agent, each inhibited the electrically evoked release of D-ASP and prevented the enhancement of the release above control levels usually produced by 10(-4)M kainic acid. It is proposed that 10(-5) and 10(-4)M kainic acid may enhance the synaptic release of L-GLU and L-ASP from neurons which use these amino acids as transmitters. This action is prevented by baclofen and pentobarbital. In view of the possibility that cell death in Huntington's disease could involve excessive depolarization of striatal and other cells by glutamate, baclofen might be effective in delaying the loss of neurons associated with this condition.     

Inhibition of GABAB Receptor Binding by Guanyl Nucleotides

Abstract: GTP and GDP decreased the saturable binding of [³H]baclofen or [³H]γ-aminobutyric acid ([³H]GABA) to GABA_B but not GABA_A receptors whereas GMP displayed negligible activity. This effect was specific to guanyl nucleotides and was not mimicked by high concentrations of ATP. The inhibition of ligand binding was the result of a diminished receptor affinity with no change in receptor number. The use of a complete physiological saline solution rather than Tris buffer plus Ca²⁺ or Mg²⁺ increased the potency of GTP at the GABA_B receptor. The results are discussed in relation to the effects of GABA and GTP on adenylate cyclase activity in the brain.     

Role of GABA<Subscript>B</Subscript> receptor in the regulation of excitatory synaptic transmission in trigeminal motoneurons

The aim of the present study was to determine if excitatory synaptic transmission onto trigeminal motoneurons is subject to a presynaptic modulation by gamma-aminobutyric acid (GABA) via GABA(B) receptor in this system. Whole cell recordings were made from trigeminal motoneurons in longitudinal brain stem slices taken from 8-day-old rats. Monosynaptic excitatory postsynaptic potential (EPSP) activity was evoked by placing bipolar stainless steel electrodes dorsal-caudal to the trigeminal motor nucleus. Bath application of the GABA(B) receptor agonist, baclofen, produced a marked reduction in the mean amplitude and variance of evoked EPSPs and also increased the portion of transmission failures. It also produced a decrease in the frequency, but not in the mean amplitude, of spontaneous miniature EPSPs. Bath application of GABA(B) receptor antagonists 6-hydroxy-saclofen and CGP35348 increased both the amplitude and frequency of miniature EPSP activity. Taken together the above results suggest that the excitatory synaptic inputs onto trigeminal motoneurons are controlled by tonic presynaptic modulation by GABA(B) receptor.     

Mechanisms of carbacholine and GABA action on resting membrane potential and Na+/K+-ATPase of Lumbricus terrestris body wall muscles

This work was aimed to identify the action of several ion channel and pump inhibitors as well as nicotinic, GABAergic, purinergic and serotoninergic drugs on the resting membrane potential (RMP) and assess the role of cholinergic and GABAergic sensitivity in earthworm muscle electrogenesis. The nicotinic agonists acetylcholine (ACh), carbacholine (CCh) and nicotine depolarize the RMP at concentrations of 5 μM and higher. The nicotinic antagonists (+)tubocurarine, α-bungarotoxin, muscarinic antagonists atropine and hexamethonium do not remove or prevent the CCh-induced depolarization. Verapamil, tetrodotoxin, removal of Cl(-) and Ca(2+) from the solution also cannot prevent the depolarization by CCh. In a Na(+)-free medium, however, CCh lost this depolarization ability and this indicates that the drug opens the sodium permeable pathway. Serotonin, glutamate, glycine, adenosine triphosphate (ATP) and cis-4-aminocrotonic acid (GABA(C) receptor antagonist) had no effect on the RMP. On the other hand, isoguvacin, γ-aminobutyric acid (GABA) and baclofen (GABA(B) receptor agonist) hyperpolarized the RMP. Ouabain, bicucullin (GABA(A) antagonist) and phaclofen (GABA(B) antagonist), as well as the removal of Cl(-), suppressed the effect of GABA and baclofen. CCh did not enhance the depolarization generated by ouabain but, on the other hand, hindered the hyperpolarizing activity of baclofen both in the absence and presence of atropine and (+)tubocurarine. The long-term application of CCh depolarizes the RMP primarily by inhibiting the Na(+)/K(+)-ATPase. The muscle membrane also contains A and B type GABA binding sites, the activation of which increases the RMP at the expense of increasing the action of ouabain- and Cl(-) -sensitive electrogenic pumps.     

巴氯芬对慢性吗啡依赖后条件化位置偏好和戒断症状的抑制

小鼠连续7天腹腔注射吗啡(40mg/kg)建立条件化位置偏好模型,连续皮下递增注射吗啡(25、50、75、100、125、150mg/kg),成瘾后腹腔注射纳络酮(6mg/kg)诱导戒断症状(跳跃行为)建立戒断模型。腹腔注射GABAB受体激动剂巴氯芬(2mg/kg)可以有效地抑制吗啡诱导的条件化位置偏好和减轻纳络酮诱导的戒断症状,结果表明GABA系统参与动物成瘾后渴求和戒断过程,激动GABAB受体可以在一定程度上抑制成瘾的心理和生理戒断症状。     

In Vivo Evidence that GABAB Receptors Are Negatively Coupled to Adenylate Cyclase in Rat Striatum

Abstract: The characteristics of the cerebral GABA_B receptor/cyclic AMP (cAMP)-generating system were investigated using the in vivo microdialysis technique in freely moving rats. Addition of forskolin, an activator of adenylate cyclase, to perfusate for 20 min resulted in a dose-dependent increase of cAMP efflux from the striatum. Pre- and coinfusions of baclofen for 80 min had no effect on the basal efflux of cAMP from the striatum but induced a significant decrease of forskolin (10 µ M )-stimulated cAMP efflux from the striatum in a dose-dependent manner. SKF 97541 (100 µ M ), a GABA_B receptor agonist, and GABA (50 µ M ) also decreased forskolin-induced cAMP efflux from the striatum. Coinfusion of CGP 54626A (100 µ M ), a GABA_B receptor antagonist, counteracted the effect of baclofen on the forskolin-stimulated cAMP efflux. In contrast, the isoproterenol (5 m M )-induced increase of cAMP efflux from the striatum was significantly enhanced by pre- and coinfusions with baclofen. These results suggest that this test system using in vivo microdialysis may be useful for examining the effect of drugs on the GABA_B receptor-linked cAMP-generating system in vivo.     

巴氯芬注射液Beagle犬腰椎穿刺置管多次鞘内注射局部刺激性研究

目的模拟临床给药途径,Beagle犬腰椎穿刺置管多次鞘内注射巴氯芬注射液,观察其局部刺激性,同时进行犬行为学观察,为巴氯芬注射液安全性评价提供依据。方法12只Beagle犬分为假手术组,生理盐水对照组,巴氯芬给药组。行腰椎穿刺置管,使用单通道微量注射泵泵入给药,给药剂量为1000μg/d,生理盐水对照组给予生理盐水0.5mL,连续给药7d,恢复期7d。每日进行行为学观察,给药结束及恢复期结束时每组麻醉2只动物,取给药部位脊髓进行组织病理学检查。结果给药及恢复期期间动物行为未见异常,给药结束时各组均有部分动物观察到置管处表皮感染现象,进行局部消毒处理后在恢复期第3天恢复正常。组织病理学检查发现给药及恢复期结束时各组动物脊髓均可见血管周围炎细胞浸润或脊髓内钙盐沉积,各组无差别。给药结束时巴氯芬组1例动物脊膜处有肉芽组织形成,判定与置管操作有关。结论巴氯芬注射液Beagle犬腰椎穿刺置管连续7d鞘内注射,给药剂量为1000μg/d对脊髓无局部刺激性作用,动物行为也未见异常。     

Regulation of Glutamate and Aspartate Release from Slices of the Hippocampal CA1 Area: Effects of Adenosine and Baclofen

Glutamate and/or aspartate is the probable transmitter released from synaptic terminals of the CA3-derived Schaffer collateral, commissural, and ipsilateral associational fibers in area CA1 of the rat hippocampal formation. Slices of the CA1 area were employed to test the effects of adenosine- and gamma-aminobutyrate (GABA)-related compounds on the release of glutamate and aspartate from this projection. Under the conditions of these experiments, the release of glutamate and aspartate evoked by 50 mM K+ was more than 90% Ca2+-dependent and originated predominantly from the CA3-derived pathways. Adenosine reduced the K+-evoked release of glutamate and aspartate by a maximum of about 60%, but did not affect the release of GABA. This action was reversed by 1 microM 8-phenyltheophylline. The order of potency for adenosine analogues was as follows: L-N6-phenylisopropyladenosine greater than N6-cyclohexyladenosine greater than D-N6-phenylisopropyladenosine approximately equal to 2-chloroadenosine greater than adenosine much greater than 5'-N-ethylcarboxamidoadenosine. 8-Phenyltheophylline (10 microM) by itself enhanced glutamate/aspartate release, whereas dipyridamole alone depressed release. These results support the view that adenosine inhibits transmission at Schaffer collateral-commissural-ipsilateral associational synapses mainly by reducing transmitter release and that these effects involve the activation of an A1 receptor. Neither adenosine, L-N6-phenylisopropyladenosine, nor 8-phenyltheophylline affected the release of glutamate or aspartate evoked by 10 microM veratridine. The differing effects of adenosine compounds on release evoked by K+ and veratridine suggest that A1 receptor activation either inhibits Ca2+ influx through the voltage-sensitive channels or interferes with a step subsequent to Ca2+ entry that is coupled to the voltage-sensitive Ca2+ channels in an obligatory fashion. Neither baclofen nor any other agent active at GABAB or GABAA receptors affected glutamate or aspartate release evoked by elevated K+ or veratridine. Therefore, either baclofen does not inhibit transmission at these synapses by depressing transmitter release or else it does so in a way that cannot be detected when a chemical depolarizing agent is employed.     

Different GABAB-Mediated Effects on Protein Kinase C Activity and Immunoreactivity in Neonatal and Adult Rat Hippocampal Slices

Abstract: The effects of GABA on protein kinase C (PKC) were investigated in rat hippocampal slices at various postnatal ages [postnatal day (P) 1-P60]. At P4, GABA (300 µ M ) induced a rapid (in 1–2 min) 40–50% increase of PKC activity in the membrane fraction and a decrease in the cytosol. These effects were mediated by GABA_B receptors because (a) they were neither blocked by 10 µ M bicuculline nor reproduced by 10 µ M isoguvacine and (b) they were mimicked by the GABA_B agonist baclofen (3–30 µ M ), an effect fully antagonized by the GABA_B antagonist 2-hydroxysaclofen (10 µ M ). A baclofen-induced increased PKC activity in the membrane fraction was only present during the early postnatal period (P1–P14); it was associated with a translocation from the cytosol to the membrane of the immunoreactivity of some PKC isoforms (α-, β-, and ε-PKCs). In contrast, after P21, PKC activity and α-, β-, ε-, and γ-PKC immunoreactivities were decreased by baclofen in the membrane fraction and increased in the cytosol. These results suggest that the stimulation of GABA_B receptors differentially modulates PKC activity via distinct second messenger pathways in developing and mature hippocampi.     

Comparative Stereostructure-Activity Studies on GABAA and GABAB Receptor Sites and GABA Uptake Using Rat Brain Membrane Preparations

The affinities of a number of analogues of gamma-aminobutyric acid (GABA) for GABAA and GABAB receptor sites and GABA uptake were studied using rat brain membrane preparations. Studies on the (S)-(+)- and (R)-(-)-isomers of baclofen, 3-hydroxy-4-aminobutyric acid (3-OH-GABA), and 4,5-dihydromuscimol (DHM) revealed different stereoselectivities of these synaptic mechanisms in vitro. Although (S)-3-OH-GABA and, in particular, (S)-DHM were more potent than the corresponding (R)-isomers as inhibitors of GABAA binding, the opposite stereoselectivity was demonstrated for the GABAB binding sites. Thus, (R)-3-OH-GABA and (R)-baclofen were more potent than the (S)-isomers as inhibitors of GABAB binding, (R)-baclofen being some five times more potent than (R)-3-OH-GABA. These two (R)-isomers actually have opposite orientation of the substituents on the GABA backbones, suggesting that the lipophilic substituent of (R)-baclofen interacts with a structural element of the GABAB receptor site different from that that binds the very polar hydroxy group of (R)-3-OH-GABA. The O-methylated analogue of 3-OH-GABA, 3-methoxy-4-aminobutyric acid (3-OCH3-GABA), did not interact significantly with GABAB sites. The homologues of GABA, trans-4-aminocrotonic acid (trans-ACA), muscimol, and 3-OH-GABA, that is, 5-aminovaleric acid (DAVA), trans-5-aminopent-2-enoic acid, homomuscimol, and 3-hydroxy-5-aminovaleric acid (3-OH-DAVA), respectively, were generally much weaker than the parent compounds, whereas 2-hydroxy-5-aminovaleric acid (2-OH-DAVA) showed a significantly higher affinity for GABAB sites than the corresponding GABA analogue.(ABSTRACT TRUNCATED AT 250 WORDS)