周边γ-氨基丁酸通过GABA_B受体调控骶髓后联合核神经元谷氨酸能突触

目的研究突触周边γ-氨基丁酸(ambient GABA)通过GABAB受体调控骶髓后联合核(SDCN)神经元谷氨酸能突触的机制。方法在急性切取的骶段脊髓薄片上,利用全细胞膜片钳法记录骶髓后联合核神经元谷氨酸能兴奋性突触后电流(EPSCs),将GABAB受体用其特异性受体拮抗剂CGP52432阻断,观察谷氨酸突触终末上的GABAB受体被周边GABA作用的影响。结果在突触后GABAB受体被从胞内阻断的条件下,再灌流CGP52432阻断谷氨酸能突触前GABAB受体,可增加刺激引发的EPSCs(eEPSCs)幅度;改变配对刺激的两个EPSC比率(paired-pulse ratio,PPR),并激发沉默突触(silent synapse)。但CGP52432对微小兴奋性突触后电流(mEPSCs)无影响。结论位于SDCN神经元谷氨酸能突触前的GABAB受体受周边GABA调控。这种影响参与调节谷氨酸释放并可能参与痛觉信息在脊髓水平的传递。     

GABA and glutamate release affected by GABAB receptor antagonists with similar potency: no evidence for pharmacologically different presynaptic receptors.

1. The effects of a series of nine GABAB receptor antagonists of widely varying potencies on electrically stimulated release from cortical slices of [3H]-GABA in the absence or presence of 10 microM of the GABAB agonist, (-)-baclofen and of endogenous glutamate in the presence of (-)-baclofen were compared. 2. The concentrations of the compounds half maximally increasing [3H]-GABA release (EC50's) at a stimulation frequency of 2 Hz correlated well with the IC50 values obtained from the inhibition of the binding of the agonist, [3H]-CGP 27492, to GABAB receptors in rat brain membranes (rank order of potency: CGP 56999 A > or = CGP 55845 A > CGP 52432 > or = CGP 56433 A > CGP 57034 A > CGP 57070 A > or = CGP 57976 > CGP 51176 > CGP 35348). 3. Likewise, the concentrations causing half-maximal increases of [3H]-GABA in the absence or presence of (-)-baclofen, and of endogenous glutamate in the presence of (-)-baclofen, correlated well with each other. Reports in the literature suggesting the CGP 35348 exhibits a 70 fold preference for inhibition of (-)-baclofen's effects on glutamate over [3H]-GABA release, and that CGP 52432 shows a 100 fold preference in the opposite sense, could not be confirmed in our model. 4. Therefore, our results suggest that, if there are pharmacological differences between GABAB autoreceptors and GABAB heteroreceptors on glutamatergic nerve endings in the rat cortex, they are not revealed by this series of compounds of widely different potencies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiological actions of GABAB agonists and antagonists in rat dorso-lateral septal neurones in vitro.

1. The actions of GABAB-receptor agonists and antagonists on rat dorso-lateral septal neurones in vitro were recorded with intracellular microelectrodes. 2. In the presence of 1 microM tetrodotoxin to prevent indirect neuronal effects caused by action potential-dependent neurotransmitter release, bath application of baclofen (0.1-30 microM) or SK&F 97541 (0.01-3 microM) evoked concentration-dependent hyperpolarizations which reversed close to the potassium equilibrium potential; the EC50S were 0.55 and 0.05 microM, respectively. No significant desensitization was observed during prolonged agonist exposure (< or = 10 min). 3. Hyperpolarizations induced by baclofen were antagonized in a competitive manner by the following GABAB-receptors antagonists (calculated pA2 values in parentheses): CGP 36742 (4.0), 2-OH saclofen (4.2), CGP 35348 (4.5), CGP 52432 (6.7) and CGP 55845A (8.3). Responses to SK&F 97541 were also antagonized by CGP 55845A (pA2 = 8.4). 4. The amplitude of the late, GABAB receptor-mediated inhibitory postsynaptic potential (i.p.s.p.) was reduced by the GABAB antagonists as follows (means +/- s.e.mean): CGP 55845A (1 microM) 91 +/- 5%, CGP 52432 (1 microM) 64 +/- 5%, CGP 35348 (100 microM) 82 +/- 5%, CGP 36742 (100 microM) 76 +/- 8%, and 2-OH saclofen (100 microM) 68 +/- 3%. 5. It is concluded that neurones in the rat dorso-lateral septal nucleus express conventional GABAB receptors, which are involved in the generation of slow inhibitory postsynaptic potentials. CGP 55845A is the most potent GABAB receptor antagonist described in this brain area.     

Release-regulating autoreceptors of the GABAB-type in human cerebral cortex.

1. The depolarization-evoked release of gamma-aminobutyric acid (GABA) and its modulation mediated by autoreceptors were investigated in superfused synaptosomes prepared from fresh human cerebral cortex. 2. The release of [3H]-GABA provoked by 15 mM K+ from human cortex nerve endings was almost totally (85%) calcium-dependent. 3. In the presence of the GABA uptake inhibitor SK&F 89976A (N-(4,4-diphenyl-3-butenyl)-nipecotic acid), added to prevent carrier-mediated homoexchange, GABA (1-10 microM) decreased in a concentration-dependent manner the K+-evoked release of [3H]-GABA. The effect of GABA was mimicked by the GABAB receptor agonist (-)-baclofen (1-100 microM) but not by the GABAA receptor agonist muscimol (1-100 microM). Moreover, the GABA-induced inhibition of [3H]-GABA release was not affected by two GABAA receptor antagonists, bicuculline or SR 95531 (2-(3'-carbethoxy-2'-propenyl)-3-amino-6-paramethoxy-phenyl-pyr idazinium bromide). 4. (-)-Baclofen also inhibited the depolarization-evoked release of endogenous GABA from human cortical synaptosomes. 5. It is concluded that GABA autoreceptors regulating the release of both newly taken up and endogenous GABA are present in human brain and appear to belong to the GABAB subtype.     

SKF97541抑制大鼠骶髓后联合核神经元

目的研究GABAB受体特异性激动剂SKF97541对骶髓后联合核(SDCN)神经元的作用。方法在大鼠骶段脊髓横切薄片上,利用全细胞膜片钳法记录骶髓后联合核神经元。电流钳记录模式下,观察SKF97541对神经元膜电位和动作电位发放的影响。电压钳模式下,观察谷氨酸能兴奋性突触后电流(EPSCs)对SKF97541处理的变化。结果SKF97541(0.5μmol.L-1)通过作用于GABAB受体,减少SDCN神经元动作电位发放,同时促进细胞膜超极化。SKF97541在电压钳模式下,减少谷氨酸介导的微小EPSCs的频率,但对振幅无影响,提示SKF97541通过作用于突触前GABAB受体抑制谷氨酸释放。突触前刺激引起的突触后电位,也被SKF97541抑制。结论在骶髓后联合核,SKF97541通过作用于突触后GABAB受体,直接抑制神经元的兴奋性和动作电位发放;并通过突触前GABAB受体,抑制谷氨酸的释放。以上结果提示SKF97541的抑制作用可能抑制骶髓后联合核神经元对伤害性信息的传递。     

Selective serotonin re-uptake inhibition attenuates evoked glutamate release in the dorsal horn of the anaesthetised rat in vivo.

Augmentation of serotonergic neurotransmission at the level of the dorsal spinal cord is proposed to contribute to the analgesic activity of selective serotonin (5-HT) reuptake inhibitors (SSRIs). In this study we have utilised microdialysis perfusion to determine the effect of two structurally unrelated SSRIs on depolarisation-induced aspartate and glutamate release in the dorsal spinal cord of the anaesthetised rat. Perfusion with artificial extracellular fluid containing 45 mM potassium produced a significant increase in aspartate and glutamate efflux. Sensitivity, at least in part, to antagonism of calcium entry by high extracellular Mg2+ indicated a neuronal origin for a proportion of stimulated release. Reverse dialysis of paroxetine (1-30 microM) reduced the increase in glutamate in a concentration dependent manner, with a significant reduction evident following inclusion in the perfusate of 30 microM. Administration of an equi-potent dose of citalopram (300 micoM) also reduced depolarisation induced glutamate release. Aspartate levels tended to decrease in the presence of paroxetine and citalopram, but this trend did not reach significance. Co-perfusion of paroxetine (30 microM) with the selective 5-HT(1A) receptor antagonist WAY 100635 (100 microM) did not prevent the reduction in depolarisation induced glutamate efflux. These results demonstrate that local administration of SSRIs has an inhibitory influence on evoked release of glutamate in the dorsal horn. This could indicate regulation of excitatory neurotransmission mediated through augmented serotonergic neurotransmission and activation of a serotonergic receptor other than the 1A subtype. Alternatively, direct inhibition with voltage dependent calcium channels, potentially a property intrinsic to molecules with high selectivity for the 5-HT transporter, may underlie this effect.     

Phaclofen-insensitive presynaptic inhibitory action of (+/-)-baclofen in neonatal rat motoneurones in vitro.

1. Intracellular recordings were made from antidromically identified motoneurones in transverse spinal cord slices from neonatal (12-16 day) rats. 2. Superfusion of (+/-)-baclofen (0.5-50 microM) reduced the excitatory postsynaptic potentials (e.p.s.ps) and inhibitory postsynaptic potentials (i.p.s.ps) evoked by dorsal root or dorsal root entry zone stimulation in a concentration-dependent manner; the calculated EC50 was 2.4 microM. Baclofen in comparable concentrations also reversibly eliminated spontaneously occurring e.p.s.ps and i.p.s.ps. 3. (-)-Baclofen was more effective as compared to baclofen in reducing the synaptic responses, whereas (+)-baclofen at concentrations as high as 50 microM was ineffective. 4. Baclofen (less than 5 microM) attenuated the synaptic responses without causing a significant change of passive membrane properties and depolarizations induced by exogenously applied glutamate. In addition to synaptic depression, baclofen (greater than 5 microM) caused a hyperpolarization associated with decreased membrane resistance in some of the motoneurones; the glutamate responses were also attenuated. 5. Baclofen reversibly depressed the spike after-hyperpolarization of the motoneurones. 6. GABA (1-10 mM) depressed synaptic transmission and depolarized or hyperpolarized motoneurones. While potentiated by the uptake inhibitor nipecotic acid, the synaptic depressant effect of GABA was not antagonized by bicuculline. 7. The synaptic depressant effect of baclofen was neither blocked by GABAA antagonists bicuculline and picrotoxin (10-50 microM) nor by the GABAB antagonist phaclofen (0.1-1 mM).(ABSTRACT TRUNCATED AT 250 WORDS)     

GABA(B) receptor antagonists elevate both mRNA and protein levels of the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) but not neurotrophin-3 (NT-3) in brain and spinal cord of rats

In this study we show that single, physiologically-active and non-convulsive doses of the three GABA(B) receptor antagonists CGP 36742, CGP 56433A and CGP 56999A increase NGF and BDNF mRNA levels by 200-400% and protein levels by 200-250% in rat neocortex, hippocampus as well as spinal cord. In all areas examined the increase in NGF protein preceded that of BDNF. Peak levels of both neurotrophins are transient and occur between 24 and 72 h, depending on the region. In contrast, NT-3 protein concentrations in the neocortex and hippocampus were decreased significantly to 50% of control values within 48-96 h. The decrease in the spinal cord was less than 30% and did not reach significant levels. These data clearly demonstrate that GABA(B) receptor antagonists induce a specific neurotrophin expression in the central nervous system at physiologically relevant doses, as opposed to the extreme conditions of seizure paradigms. The results are in line with the concept that neuronal neurotrophin synthesis and release in brain are controlled by afferent nerve activity. GABA(B) receptor antagonists could therefore be a valuable new approach to selectively increase endogenous neurotrophin levels in the central nervous system.     

GABAB receptors do not mediate the inhibitory actions of gabapentin on the spinal reflex in rats

The clinical effectiveness of gabapentin for the treatment of epilepsy, spasticity, and neuropathic pain has been established. The mechanisms responsible for those actions, however, are still not clearly understood. We have recently demonstrated that gabapentin reduces the spinal reflex in rats via mechanisms that do not involve gamma-aminobutyric acid (GABA)A receptors. In the study, we attempted to explore the involvement of GABAB receptors in gabapentin-induced inhibition of the spinal reflexes in spinalized rats. Stimulation of the dorsal root at L5 elicited the segmental mono-(MSR) and polysynaptic reflex (PSR) in the ipsilateral ventral root. The microinjection of gabapentin (1.5 and 5 nmol) into the ventral horn reduced both MSR and PSR, whereas the injection into the dorsal horn only inhibited the PSR, indicating that systemic gabapentin inhibits the MSR at the ventral horn and it inhibits the PSR at both the ventral and dorsal horns. The GABAB-receptor antagonist CGP35348 (0.5 nmol) injected into the ventral horn antagonized the inhibition of the spinal reflexes by the GABAB-receptor agonist baclofen (i.v.) but not by gabapentin (i.v.). Thus, GABAB receptors do not appear to contribute to the gabapentin-induced inhibition of the spinal reflex.     

GABAB autoreceptors in rat cortex synaptosomes: response under different depolarizing and ionic conditions

Rat cerebral cortex synaptosomes prelabeled with [3H]gamma-aminobutyric acid [( 3H]GABA) were exposed in superfusion to various concentrations of KCl (9-50 mM). The evoked release of [3H]GABA reached a plateau at about 35 mM KCl. The K+-induced release was Ca2+-dependent, particularly at the lowest K+ concentrations. The GABAB agonist (-)-baclofen concentration dependently inhibited the release of [3H]GABA evoked by K+; this effect decreased with increasing K+ concentration and disappeared at 35 mM KCl. The GABAA agonist muscimol (1-100 microM) was totally ineffective to inhibit the release of [3H]GABA. Veratrine (1-30 microM) induced the release of [3H]GABA and the effect was tetrodotoxin-sensitive. (-)-Baclofen, but not muscimol, decreased the veratrine-induced [3H]GABA release; the GABAB agonist was particularly effective in presence of low concentrations of veratrine (1-3 microM) but the effect disappeared when 30 microM of the alkaloid was used. The inhibitory effect of (-)-baclofen on the release of [3H]GABA evoked by 15 mM KCl was dependent on the concentration of Ca2+: the effect increased as the concentration of Ca2+ was raised, reaching a plateau at 0.6 mM Ca2+. Exogenous GABA, in presence of the GABA uptake blocker SK & F 89976A, inhibited the release of [3H]GABA evoked by K+; this effect was antagonized by phaclofen. The data support the idea that terminal GABA autoreceptors in the rat cerebral cortex are of the GABAB type.     

Interaction of group I mGlu and NMDA receptor agonists within the dorsal horn of the spinal cord of the juvenile rat

1. The modulatory effects of mGlu receptors on NMDA-induced potential changes in spinal motoneurones were studied in vitro. 2. Selective activation of mGlu5 receptors by 10 microM (RS)-2-Chloro-5-hydroxyphenylglycine (CHPG; EC(50)=280 +/- 24 microM) did not produce any change in the ventral root potential. However, the same concentration of CHPG (10 min perfusion) significantly attenuated the NMDA-induced ventral root depolarization (VRD). The effect persisted for 10 min after washout. NMDA-induced responses returned to control in 30 min. Brief co-application of CHPG and NMDA did not alter the NMDA-induced response indicating lack of direct receptor interaction. 3. The attenuating effect of CHPG on the NMDA-induced VRD was inhibited by the mGluR5 receptor antagonist, 2-methyl-6-phenyl-ethynylpyridine (MPEP). 4. In the presence of CGP56433A, a GABA(B) receptor antagonist, the NMDA-induced VRD was unchanged. However, NMDA-induced responses were potentiated after 10 min co-application of CHPG and CGP56433A. 5. (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate ((2R,4R)-APDC), a group II mGlu receptor agonist did not attenuate the NMDA-induced response. 6. Under normal physiological conditions group I mGlu receptor agonists activate at least two populations of neurones: (1) GABA-ergic cells, which could release GABA and inhibit dorsal horn neurones, and (2) deep dorsal horn neurones/motoneurones which express NMDA receptors. Therefore, activation of mGlu5 receptors located on GABA-ergic interneurones could influence any direct potentiating interaction between mGlu5 and NMDA receptors in spinal cord and result in depression of the VRD. In the presence of a GABA(B) receptor antagonist, the direct synergistic interaction is unmasked. These data suggest that group I mGlu receptors provide a complex modulation of spinal synaptic processes.     

氯苯氨丁酸抑制脊髓背角神经元谷氨酸量子释放的机制

目的　研究GABAB 受体特异性激动剂氯苯氨丁酸(baclofen)在脊髓背角神经元抑制谷氨酸量子释放的机制。方法　在脊髓薄片标本上 ,采用全细胞电压钳法记录脊髓背角神经元谷氨酸能的微兴奋性突触后电流 (miniatureexcita torypostsynapticcurrents;mEPSCs) ,通过分析这些电流的变化来研究baclofen影响谷氨酸量子释放的机制。结果　ba clofen抑制mEPSCs的发放频率 ,但对平均幅度无明显影响 ,表明baclofen抑制谷氨酸释放的作用部位在突触前。在无钙溶液或者K+ 通道阻滞剂 4 AP存在的条件下 ,baclofen对mEPSCs发放频率的抑制作用不受影响 ,但腺苷酸环化酶激动剂foskolin (可使cAMP保持在较高水平 )能降低其抑制作用。而蛋白激酶C (PKC)激动剂PDBu对baclofen的抑制作用无影响。用NEM破坏G蛋白 ,则可取消baclofen的抑制效果。结论　baclofen不是通过影响突触前Ca2 + 通道或K+通道 ,或PKC途径 ,而是通过作用于G蛋白和 (或 )cAMP途径抑制谷氨酸的释放 ;这种抑制作用可能参与baclofen在脊髓水平的镇痛     

Gabapentin may inhibit synaptic transmission in the mouse spinal cord dorsal horn through a preferential block of P/Q-type Ca2+ channels

Gabapentin is a lipophilic analog of gamma-amino butyric acid (GABA) with therapeutic activity against certain forms of epilepsy and neuropathic pain. Despite its structural similarity to GABA, it does not bind GABAA or GABAB receptors and the mechanism, especially of its analgesic action, has remained elusive. Here, we have studied its effects on synaptic transmission mediated by the major spinal fast excitatory and inhibitory neurotransmitters, L-glutamate and glycine, in the superficial layers of the spinal cord dorsal horn, a CNS area, which is critically involved in nociception. Gabapentin reversibly reduced evoked excitatory postsynaptic currents mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA-EPSCs) and inhibitory postsynaptic currents mediated by glycine (gly-IPSCs). Inhibition of AMPA-EPSCs and gly-IPSCs occurred with similar potencies (approximately 10-50 nM) and by about the same degree (approximately 40% at 1 microM). Gabapentin did not affect membrane currents elicited by exogenously applied glutamate or glycine arguing against a postsynaptic site of action. Selective blockade of N-type Ca2+ channels with omega-conotoxin GVIA dramatically increased and blockade of P/Q-type channels with omega-agatoxin IVA strongly attenuated inhibition of evoked synaptic transmission by gabapentin. These results show that gabapentin affects both excitatory and inhibitory spinal neurotransmission via a presynaptic mechanism which preferentially involves P/Q-type Ca2+ channels.     

Human brain somatostatin release from isolated cortical nerve endings and its modulation through GABAB receptors.

1. The release of somatostatin-like immunoreactivity (SRIF-LI) in the human brain was studied in synaptosomal preparations from fresh neocortical specimens obtained from patients undergoing neurosurgery to remove deeply sited tumours. 2. The basal outflow of SRIF-LI from superfused synaptosomes was increased about 3 fold during exposure to a depolarizing medium containing 15 mM KCl. The K(+)-evoked overflow of SRIF-LI was almost totally dependent on the presence of Ca2+ in the superfusion medium. 3. The GABAB receptor agonist, (-)-baclofen (0.3 - 100 microM), inhibited the overflow of SRIF-LI in a concentration-dependent manner (EC50 = 1.84 +/- 0.20 microM; maximal effect: about 50%). The novel GABAB receptor ligand, 3-aminopropyl(difluoromethyl)phosphinic acid (CGP 47656) mimicked (-)-baclofen in inhibiting the SRIF-LI overflow (EC50 = 3.06 +/- 0.52 microM; maximal effect: about 50%), whereas the GABAA receptor agonist, muscimol, was ineffective up to 100 microM. 4. The inhibition by 10 microM (-)-baclofen of the K(+)-evoked SRIF-LI overflow was concentration-dependently prevented by two selective GABAB receptor antagonists, 3-amino-propyl (diethoxymethyl)-phosphinic acid (CGP 35348) (IC50 = 24.40 +/- 2.52 microM) and [3-[[(3,4-dichlorophenyl) methyl]amino]propyl] (diethoxymethyl) phosphinic acid (CGP 52432) (IC50 = 0.06 +/- 0.005 microM). 5. The inhibition of SRIF-LI overflow caused by 10 microM CGP 47656 was abolished by 1 microM CGP 52432. 6. When human synaptosomes were labelled with [3H]-GABA and depolarized in superfusion with 15 mM KCl, the inhibition by 10 microM (-)-baclofen of the depolarization-evoked [3H]-GABA overflow was largely prevented by 10 microM CGP 47656 which therefore behaved as an autoreceptor antagonist. 7. In conclusion: (a) the characteristics of SRIF-LI release from synaptosomal preparations of human neocortex are compatible with a neuronal origin; (b) the nerve terminals releasing the neuropeptide possess inhibitory receptors of the GABAB type; (c) these receptors differ pharmacologically from the GABAB autoreceptors present on human neocortex nerve terminals since the latter have been shown to be CGP 35348-insensitive but can be blocked by CGP 47656.     

Gabapentin activates presynaptic GABAB heteroreceptors in rat cortical slices

In electrically stimulated rat neocortical brain slices preloaded with [3H]gamma-aminobutyric acid (GABA) or [3H]glutamic acid, the pharmacological actions of 1-(aminomethyl)-cyclohexaneacetic acid (gabapentin, Gp) were compared with the GABAB receptor agonists baclofen (Bac) and (3-amino-2-(S)-hydroxypropyl)-methylphosphinic acid (CGP 44532). Gabapentin, baclofen and CGP 44532 all reduced the electrically stimulated release of [3H]glutamic acid (IC50=20 microM, 0.8 microM and 2 microM, respectively). These effects were sensitive to the GABAB receptor antagonists (+)-(S)-5,5 dimethylmorpholinyl-2-acetic acid (Sch 50911) or N-3-[[1-(S)-(3,4-dichlorophenyl)ethyl]amino]-2-(S)-hydroxypropyl-P-(cyclo-hexylmethyl)-phosphinic acid (CGP 54626). By contrast, gabapentin was without effect on the release of [3H]GABA, whilst baclofen (IC50=8 microM) and CGP 44532 (IC50=1 microM) inhibited [3H]GABA release. It is concluded that gabapentin selectively activates presynaptic GABAB heteroreceptors, but not GABAB autoreceptors, and may be a useful ligand to discriminate between presynaptic GABAB receptor subtypes.     

Actions of the GABAB agonist, (-)-baclofen, on neurones in deep dorsal horn of the rat spinal cord in vitro.

1. The electrophysiological actions of the GABAB agonist, (-)-baclofen, on deep dorsal horn neurones were studied using an in vitro preparation of the spinal cord of 9-16 day old rat. 2. On all neurones tested, (-)-baclofen (100 nM-30 microM) had a hyperpolarizing action which was associated with a reduction in apparent membrane input resistance. The increase in membrane conductance was dose-dependent and had a Hill coefficient of 1.0. 3. The (-)-baclofen-activated hyperpolarization persisted in the presence of bicuculline (50 microM) and Mg2+ (20 mM). 4. The reversal potential of the hyperpolarizing event was estimated at 102 mV and was made less negative by increasing the external concentration of potassium ions. 5. Over the same concentration range, (-)-baclofen also depressed the polysynaptic composite excitatory postsynaptic potentials (e.p.s.ps) evoked in these neurones by electrical stimulation of the dorsal root entry zone. 6. The potassium channel blockers caesium, applied intracellularly, and barium, applied extracellularly, depressed the postsynaptic response to baclofen but not its effect on e.p.s.ps. 7. We propose that (-)-baclofen has more than one mechanism of action in spinal dorsal horn: a postsynaptic action mediated via an increase in potassium conductance and a presynaptic action that is not associated with potassium channels and may be mediated via calcium channels. Since previous studies have demonstrated little effect of (-)-baclofen on transmitter release in spinal cord, it is possible that the postsynaptic hyperpolarizing action of (-)-baclofen may account for its clinical potency as an anti-spastic agent.     

胍丁胺对炎性疼痛的镇痛作用及对吗啡镇痛作用的影响

目的观察胍丁胺对炎性疼痛的镇痛作用及其对吗啡镇痛作用的影响,研究胍丁胺的镇痛作用是否与激动咪唑啉受体或影响受体前谷氨酸和γ-氨基丁酸(gamma-aminobutyr-icacid,GABA)释放有关。方法应用福尔马林致大鼠炎性疼痛模型,观察胍丁胺镇痛和增强吗啡镇痛的作用。应用高效液相色谱技术测定胍丁胺对脊髓切片孵育液中谷氨酸和GABA基础释放量及对高钾诱发神经元去极化引起神经递质释放的影响。结果单侧足底注射5%福尔马林使大鼠出现明显的双相伤害性行为反应。胍丁胺抑制福尔马林引起的第二相疼痛行为反应及痛觉过敏,并增强吗啡对第二相疼痛的镇痛作用,但在第一相疼痛过程中,无明显镇痛和增强吗啡镇痛的作用。咪唑啉受体拮抗剂咪唑克生不能拮抗胍丁胺镇痛及增强吗啡镇痛的作用。1~1000μmol.L-1胍丁胺对脊髓谷氨酸和GABA的基础释放量和高钾诱发谷氨酸和GABA释放量的升高均没有影响。结论胍丁胺对炎性疼痛具有明确的镇痛作用,并明显增强吗啡的镇痛效果,其镇痛机制可能与咪唑啉受体无关,也不是通过在受体前水平抑制谷氨酸或促进GABA释放来实现的。     

Differential effects of wortmannin on the release of substance P and amino acids from the isolated spinal cord of the neonatal rat

1. Effects of wortmannin, an inhibitor of myosin light chain kinase, on the release of substance P and amino acids, GABA and glutamate, were investigated in the isolated spinal cord preparation of the neonatal rat.
2. Wortmannin at 0.5–10 μM depressed the release of substance P evoked by high-K⁺ (90 mM) medium from the spinal cord (IC₅₀=1.1 μM). Wortmannin also depressed the high-K⁺ (70 mM)-evoked release of substance P from cultured dorsal root ganglion neurons of neonatal rats. In contrast, the high-K⁺ (90 mM)-evoked release of GABA and glutamate from the spinal cord was not affected by wortmannin (0.1–10 μM).
3. Upon stimulation of a dorsal root, a monosynaptic reflex and a subsequent slow ventral root depolarization were evoked in the ipsilateral ventral root of the same segment in the isolated spinal cord preparation. The magnitude of the slow ventral root depolarization was depressed gradually to about 70% of the control during the course of 30 min under wortmannin (1 μM). In contrast, the monosynaptic reflex was unaffected by wortmannin.
4. Immunofluorescent staining revealed that immunoreactivities of substance P and myosin II were colocalized at presynaptic terminals in the dorsal horn of the neonatal rat spinal cord.
5. The present results suggest that myosin phosphorylation by myosin light chain kinase may play a crucial role in the release of substance P, but not in the release of GABA and glutamate in the neonatal rat spinal cord. This may reflect a difference in the exocytic mechanisms of substance P-containing large dense core vesicles and amino acid-containing small clear vesicles.

     

Disinhibition of spinal responses to primary afferent input by antagonism at GABA receptors in urethane-anaesthetised rats is dependent on NMDA and metabotropic glutamate receptors

Disruption of spinal GABAergic circuits, which regulate the conveyance of sensory information to spinal cord neurones from the primary afferent system, leads to miscoding of afferent input and often results in hyperresponsiveness states. In the present work, extracellular field potentials elicited by electrical peripheral nerve activation were recorded in the urethane-anaesthetised rat following spinal administration of GABA(A) or GABA(B) receptor-antagonists, and the involvement of glutamate receptors of the NMDA and metabotropic types in changes induced by altered GABAergic function was examined by pre-treating the spinal dorsal horn with appropriate antagonist drugs. Spinal administration of the GABA(A) receptor antagonist bicuculline (BIC) dose-dependently augmented poly- but not monosynaptic field potentials elicited by activation of A fibres or potentials elicited by activation of C fibres, whereas application of the GABA(B) receptor antagonist CGP35348 significantly increased the amplitudes of C- but not A fibre-evoked potentials. BIC-induced augmentation was blocked by pre-treatment with the NMDA receptor antagonist D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5) or the group I or II metabotropic glutamate receptor (mGluR)-antagonists (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) or (2S)-alpha-ethylglutamic acid (EGLU), respectively, but not by the group III mGluR-antagonist (RS)-alpha-methylserine-O-phosphate (MSOP). Augmentation of spinal field potentials induced by CGP35348 was prevented by pre-treatment with D-AP5 but not with mGluR-antagonists. The present findings provide novel evidence that disparate synaptic mechanisms subserved by metabotropic and NMDA glutamate receptors may be involved in spinal hyperresponsiveness states secondary to decreased GABA(A) or GABA(B) receptor activity.     

GABAB receptor modulation of the release of substance P from capsaicin-sensitive neurones in the rat trachea in vitro.

1. The role of gamma-aminobutyric acid (GABA) as an inhibitory transmitter in the central nervous system is well documented. Recently, GABAA and GABAB receptors have been identified in the peripheral nervous system, notably on primary afferent neurones (PAN). We have utilised a multi-superfusion system to investigate the effect of selective GABA receptor agonists and antagonists on the release of substance P (SP) from the rat trachea in vitro. 2. GABA (1-100 microM) did not affect spontaneous release of SP-like immunoreactivity (LI) but caused dose-related inhibition of calcium-dependent potassium (60 mM)-stimulated SP-LI release. The greatest inhibition of 77.7 +/- 18.8% was observed at 100 microM. 3. The inhibitory effect of GABA was mimicked by the GABAB receptor agonist, (+/-)-baclofen (1-100 microM), but not the GABAA receptor agonist, 3-amino-1-propane-sulphonic acid (3-APS, 1-100 microM). Baclofen (100 microM) had no effect on SP-LI release stimulated by capsaicin (1 microM). 4. The inhibitory effect of baclofen (30 microM) was significantly reduced by prior and concomitant exposure to the GABAB receptor antagonist, phacolofen (100 microM) but not the GABAA receptor antagonist, bicuculline (10 microM). Neither antagonist, alone, affected spontaneous or potassium-stimulated SP-LI release. 5. We conclude that activation of pre-synaptic GABAB receptors on the peripheral termini of PANs in the rat trachea inhibits SP-LI release and suggest that GABAB receptor agonists may be of value in the therapeutic treatment of asthma.