Potentiation by sevoflurane of the gamma-aminobutyric acid-induced chloride current in acutely dissociated CA1 pyramidal neurones from rat hippocampus.

1. The effects of a new kind of volatile anaesthetic, sevoflurane (Sev), on gamma-aminobutyric acid (GABA)-gated chloride current (Icl) in single neurones dissociated from the rat hippocampal CA1 area were examined using the nystatin perforated patch recording configuration under the voltage-clamp condition. All drugs were applied with a rapid perfusion system, termed the "Y-tube' method. 2. When the concentrations were higher than 3 x 10(-4) M, Sev, itself, induced an inward current (ISev) at a holding potential (VH) of -40 mV. The concentration-response curve of ISev was bell-shaped, with a suppressed peak and plateau currents at high concentrations (above 2 x 10(-3) M). The reversal potential of ISev (ESev) was close to the theoretical Cl- equilibrium potential, indicating that ISev was carried mainly by Cl-. 3. ISev was reversibly blocked by bicuculline (Bic), an antagonist of the GABAA receptor, in a concentration-dependent manner with a half-inhibitory concentration (IC50) of 7.2 x 10(-7) M. But ISev was insensitive to strychnine (Str), an antagonist of the glycine receptor. 4. At low concentrations (between 3 x 10(-4) and 10(-3) M), Sev markedly enhanced the 10(-6) M GABA induced current (IGABA) but reduced the IGABA with accelerating desensitization accompanied by a "hump' current after washout at high concentrations (higher than 2 x 10(-3) M). 5. Sev, 10(-3) M potentiated the current induced by low concentrations of GABA (between 10(-7) and 3 x 10(-6) M) but reduced the current induced by high concentrations (higher than 10(-5) M) of GABA with a clear acceleration of IGABA desensitization. 6. Sev, like pentobarbitone (PB), pregnanolone (PGN) or diazepam (DZP), potentiated the 10(-6) M GABA-induced response without shifting the reversal potential of IGABA. 7. ISev was augmented by PB, PGN, or DZP at concentrations that maximally potentiated IGABA, suggesting that Sev enhanced IGABA at a binding site distinct from that for PB, PGN, or DZP. 8. It is concluded that Sev acts on the GABAA receptor complex mimicking the GABA-induced chloride current at high concentrations. At low concentrations, Sev enhances GABA-gated chloride current at a binding site independent of the allosteric modulator sites of barbiturates, benzodiazepines or neurosteroids. The reversible potentiation of the inhibitory GABAA receptor-mediated Cl- current may result in the depressing of postsynaptic excitability and may, at least in part, underlie the anaesthetic action of Sev.     

Kinetic and pharmacological properties of the GABA-induced chloride current in Aplysia neurones: a 'concentration clamp' study

1. gamma-Aminobutyric acid (GABA) was applied by the 'concentration clamp' technique to isolated neurones of Aplysia. GABA induced a chloride current (ICl) due to activation of a single class of chloride-channel. 2. The concentration-response curve for the peak ICl gave an apparent dissociation constant of 6.4 X 10(-5) M and a Hill coefficient of 0.88. The current-voltage relationship was linear in the voltage range examined (-40 to +10 mV). 3. The activation phase of the ICl could be fitted to a single exponential function and desensitization followed the sum of two exponential functions. The time constants of activation and desensitization decreased with increasing concentrations of GABA but were voltage-independent. The recovery process from desensitization also followed the sum of two exponential functions. 4. As for the rate-limiting step of the channel activation, the hyperbolic relationship between the activation rate and GABA concentration showed that the rapid binding assumption holds, suggesting that the isomerization step is rate-limiting. The apparent channel closing rate constant was estimated to be 10 s-1 from the ordinate intercept of the linear part of the above relationship at lower concentrations. 5. Muscimol and beta-alanine induced a ICl, which cross-desensitized with that evoked by GABA. The GABA-ICl was not enhanced by diazepam (10(-6) M) or alpha-chloralose (10(-3) M), in fact depressant effects were evident. 6. Pentobarbitone decreased the GABA-ICl non-competitively without altering activation or desensitization kinetics. The concentration-inhibition curve gave a KD value of 8.9 x 10(-5) M and a Hill coefficient of 1.0. 7. These results suggest that GABA activates a single class of Cl channel in Aplysia neurones, which have one binding site for the agonist. The GABA receptor-Cl channel complex in Aplysia is pharmacologically and perhaps structurally different from that in vertebrates.     

Strychnine-induced potassium current in isolated dorsal root ganglion cells of the rat.

1. Effects of strychnine (Str) on the dissociated dorsal root ganglion (DRG) cells of the rat have been investigated in whole-cells configuration by a conventional patch-clamp technique. 2. gamma-Aminobutyric acid (GABA)-induced Cl- current (ICl) increased sigmoidally with increasing concentration. The half-maximal response (Ka) was 3 x 10(-5) M and the Hill coefficient was 1.5. Both Str and bicuculline inhibited the GABA-induced ICl in a concentration-dependent manner. 3. Str itself could elicit the current at concentrations over 10(-5) M, at which concentrations the GABA response was completely suppressed. The concentration-response curve for the Str-induced current was bell-shaped, and a nearly maximum response occurred at 3 x 10(-4) M. A transient 'hump' current appeared immediately after the wash-out of external solution containing high concentrations of Str over 3 x 10(-4) M. 4. The Str-induced outward current and a transient 'hump' current were augmented by the removal of extracellular K+ and were suppressed by the substitution of intracellular K+ for Cs+. But the current was not sensitive to extracellular Na+, Ca2+ and Cl-. 5. The reversal potential of Str-induced current (EStr) was -75 mV, which was close to the K+ equilibrium potential (EK = -76.3 mV). The change of EStr for a ten fold change in extracellular K+ concentration was 58 mV, indicating that the membrane behaves like a K+ electrode in the presence of Str. The reversal potential of the 'hump' current was also close to EK.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dual mechanisms of GABAA response inhibition by beta-lactam antibiotics in the pyramidal neurones of the rat cerebral cortex.

1. The effects of beta-lactam antibiotics on the gamma-aminobutyric acid (GABA)-induced Cl- current were investigated in pyramidal neurones freshly dissociated from the rat frontal cortex by the use of a nystatin-perforated patch recording mode under voltage-clamp conditions. 2. The GABA-induced inward current increased in a concentration-dependent manner with an EC50 of 6.7 x 10(-6) M at a holding potential of -40 mV. The GABA response was accompanied by an increase in the membrane conductance and reversed at near the Cl- equilibrium potential. 3. All beta-lactams (penicillin, imipenem, aztreonam and cefotiam) inhibited the 10(-5) M GABA-induced response in a concentration-dependent manner with an IC50 and Hill coefficient of 1.3 x 10(-3) M and 0.64 for penicillin, 9.6 x 10(-4) M and 0.83 for imipenem, 2.5 x 10(-3) M and 9.99 for aztreonam, and 2.9 x 10(-4) M and 1.03 for cefotiam. 4. Imipenem inhibited the GABA-response competitively while penicillin inhibited the same response in a noncompetitive fashion. 5. The inhibitory action of imipenem showed no voltage-dependency, whereas the effect of penicillin was voltage-dependent. 6. It is thus proposed that some classes of beta-lactams, including imipenem may have a mechanism that is different from penicillin and competitively affects the GABAA receptor.     

Effects of benzodiazepines and non-benzodiazepine compounds on the GABA-induced response in frog isolated sensory neurones

1. The effects of benzodiazepines and non-benzodiazepine compounds on the gamma-aminobutyric acid (GABA)-induced chloride current (ICl) were studied in frog isolated sensory neurones by use of a concentration-jump (termed 'concentration-clamp') technique, under single-electrode voltage-clamp conditions. The drugs used were classified into four categories as follows: full benzodiazepine receptor agonists (diazepam, clonazepam, nitrazepam, midazolam, clotiazepam and etizolam), partial agonists (CL 218,872, Ro 16-6028, Ro 17-1812 and Ro 23-0364), inverse agonists (Ro 15-3505, FG 7142 and beta-CCE) and a benzodiazepine receptor antagonist, Ro 15-1788 (flumazenil). 2. All full agonists at concentrations of 3 x 10(-6) M or less increased dose-dependently the peak amplitude of ICl elicited by 3 x 10(-6) M GABA to twice to three times larger than the control. However, no further augmentation of the GABA response was observed at concentrations of 1 x 10(-5) M or higher. Partial agonists also showed a dose-dependent augmentation of the GABA response at concentrations ranging from 3 x 10(-8) M to 3 x 10(-5) M, but their efficacies of augmentation of the GABA response were only about half or less of those of full agonists. Of the inverse agonists, beta-CCE had a unique dose-dependent effect on the GABA response. Beta-CCE reduced dose-dependently the GABA response at concentrations of less than 3 x 10(-6) M, but augmented it at concentrations of 3 x 10(-5) M and 6 x 10(-5) M. The inverse agonists reduced dose-dependently the GABA response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiological effects of piperazine and diethylcarbamazine on Ascaris suum somatic muscle.

1 Electrophysiological recordings were made from the bag region of Ascaris suum muscle. Membrane potential and input conductance or membrane current under voltage clamp were measured. 2 In high-Cl- Ringer, bath-applied piperazine, at concentrations greater than 10(-4)M, produced a dose-dependent and reversible increase in input conductance associated with a hyperpolarizing potential. The increase in input conductance was reduced when the preparations were bathed in low-Cl- Ringer. Gamma-Aminobutyric acid (GABA) and piperazine reversal potentials were measured with a voltage clamp on the same cells using iontophoretic application of the agonists. The reversal potentials were the same and close to the predicted Nernst Cl- potential (-65 mV). When GABA and piperazine were applied simultaneously piperazine reversibly reduced the amplitude of the control outward GABA current response. It was concluded that piperazine acts as a GABA agonist of low potency on the extra-synaptic GABA receptors of the bag, mediating an increase in Cl- conductance. 3 Acetylcholine was applied iontophoretically within 100 micron of the bag region while the preparation was bathed in a low-Ca2+, low-Cl- Ringer. The response under voltage clamp was a dose-dependent inward current associated with an increase in input conductance. This response was reversibly antagonized by 3 X 10(-5)M tubocurarine, high concentrations of diethylcarbamazine (10(-3) to 10(-2)M) but not high concentrations of piperazine (10(-3) to 10(-2)M). It was concluded that there are extra-synaptic acetylcholine receptors on the bag region of Ascaris muscle and that diethylcarbamazine but not piperazine acts as an antagonist. 4 Bath-applied diethylcarbamazine (10(-4) to 2 X 10(-3)M) produced a reversible dose-dependent depolarization of the membrane potential which was associated with an increase in the amplitude and frequency of spontaneous depolarizing potentials in active preparations at 32 degrees C to 35 degrees C in high-Cl- Ringer. The excitatory action of diethylcarbamazine was not blocked by 3 X 10(-5)M tubocurarine. Diethylcarbamazine (10(-4) to 10(-3)M) had no effect on the outward current response to GABA iontophoresis. Diethylcarbamazine (10(-4) to 10(-2)M) reversibly antagonized in a dose-dependent manner the delayed rectification of the bag membrane. In a low-Ca2+, low-Cl- Ringer, diethylcarbamazine (10(-4) to 2 X 10(-3)M) reversibly antagonized the voltage-sensitive outward current of the bag. This effect was mimicked by high-K+ Ringer or perfusion with 4-aminopyridine (10(-3) to 2 X 10(-3)M). It was concluded that diethylcarbamazine did not react with the GABA receptor but antagonized a voltage-sensitive K+ conductance.     

Caffeine and related compounds block inhibitory amino acid-gated Cl- currents in freshly dissociated rat hippocampal neurones.

1. The effects of caffeine and related compounds on responses mediated by inhibitory amino acids were investigated in freshly dissociated rat hippocampal pyramidal neurones by conventional and nystatin perforated patch-clamp techniques. 2. Glycine and gamma-aminobutyric acid (GABA) evoked Cl- currents in hippocampal neurones. The half-maximum effective concentrations (EC50) of glycine and GABA were 8.5 x 10(-5) and 5 x 10(-6) M, respectively. 3. Caffeine reversibly inhibited both 10(-4) M glycine- and 10(-5) M GABA-induced Cl-currents in a concentration-dependent manner. The half-maximum inhibitory concentrations (IC50) of caffeine were 4.5 x 10(-4) M for the glycine response and 3.6 x 10(-3) M for the GABA response. 4. Caffeine shifted the concentration-response curve of IGly to the right without affecting the maximum response. 5. The inhibitory action of caffeine did not show voltage-dependency. 6. The blocking action of caffeine was not affected by intracellular perfusion with 5 mM BAPTA or by pretreatment with the protein kinase A inhibitor, H-8. This excludes the participation of Ca2+ or cyclic AMP in the inhibitory action of caffeine. 7. Caffeine failed to inhibit the augmentations of aspartate- and N-methyl-D-aspartate (NMDA) -gated current by glycine, suggesting that caffeine has no effect on the allosteric glycine binding site on the NMDA receptor. 8. The inhibitory effects of some xanthine derivatives on IGly were compared. The inhibitory potency of those compounds on IGly was in the order of pentoxifylline > theophylline > or = caffeine > paraxanthine > IBMX > or = theobromine > dyphylline. Xanthine had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Non-competitive inhibition of GABAA responses by a new class of quinolones and non-steroidal anti-inflammatories in dissociated frog sensory neurones.

1. The interaction of a new class of quinolone antimicrobials (new quinolones) and non-steroidal anti-inflammatory agents (NSAIDs) with the GABAA receptor-Cl- channel complex was investigated in frog sensory neurones by use of the internal perfusion and 'concentration clamp' techniques. 2. The new quinolones and the NSAIDs (both 10(-6)-10(-5) M) had little effect on the GABA-induced chloride current (ICI) when applied separately. At a concentration of 10(-4) M the new quinolones, and to a lesser degree the NSAIDs, produced some suppression of the GABA response. 3. The co-administration of new quinolones and some NSAIDs (10(-6)-10(-14) M) resulted in a marked suppression of the GABA response. The size of this inhibition was dependent on the concentration of either the new quinolone or the NSAID tested. The inhibitory potency of new quinolones in combination with 4-biphenylacetic acid (BPAA) was in rank order norfloxacin (NFLX) much greater than enoxacin (ENX) greater than ciprofloxancin (CPFX) much greater than ofloxacin (OFLX), and that of NSAIDs in combination with ENX was BPAA much greater than indomethacin = ketoprofen greater than naproxen greater than ibuprofen greater than pranoprofen. Diclofenac, piroxicam and acetaminophen did not affect GABA responses in the presence of ENX. 4. In the presence of ENX or BPAA, there was a small shift to the right of the concentration-response curve for GABA without any effect on the maximum response. However, the co-administration of these drugs suppressed the maximum of the GABA concentration-response curve, indicating a non-competitive inhibition, for which no voltage-dependency was observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

5-HT2受体介导大鼠DRG神经元膜GABA-激活电流的增强作用

目的　探讨 5 HT对大鼠DRG神经元膜GABA 激活电流的调节作用及其机制。方法　在新鲜分离的大鼠DRG神经元标本上,以全细胞膜片钳技术记录膜电流,用排管快速换液装置行胞外给药,以胞内透析技术分析信号转导途径。结果　给予GABA可使多数受检细胞产生浓度依赖性内向电流 (IGABA)。预加 5 HT,可使IGABA增加。此效应可被 5 HT2受体特异性激动剂α methyl 5 HT( 1×10-6mol·L-1 )所模拟,被 5 HT2受体选择性拮抗剂cyproheptadine所阻断。在部分细胞, 5 HT本身可引起由 5 HT3受体介导的快速内向电流,但并未发现该电流与 5 HT对IGABA的增强作用有必然的联系。从GABA激活电流的量效曲线可见,预加 5 HT后和对照曲线相比,阈浓度不变、EC50值相近,IGABA最大值增加 33. 6%。胞内透析GDP β S或H 7可取消 5 HT增强IGABA的效应,而透析H 9无效。结论　5 HT可增强GABA 激活电流,其机制为 5 HT2受体激活后通过PKC引起GABAA受体胞内磷酸化所致。     

36Cl- flux measurements on GABAA receptor-activated chloride exchange. Multiple mechanisms of the chloride channel inactivation

The technique of radiotracer 36Cl- influx in primary culture of rat cerebellar granule cells was applied to study the mechanism of inactivation of the GABAA receptor-activated chloride channel. During sustained application of GABA, muscimol and THIP the specific bicuculline-sensitive 36Cl- influx tends to decline with time. The sequence in decay half-time is GABA less than muscimol less than THIP. Diazepam accelerates the rate of decay of the peak response to GABA. (-)-Baclofen enhances the rate of decline of the response to muscimol in a dose-dependent manner. Treatment of the cells with pertussis toxin antagonized the effect of (-)-baclofen. It is concluded that rat neonatal cerebellar neurons maintained in tissue culture exhibit complex inactivation of the GABAA channel, indicating some interaction with the GABAB receptor system.     

Two actions of gamma-aminobutyric acid on the responses of the isolated basilar artery from the rabbit.

1 In the isolated basilar artery of the rabbit, gamma-aminobutyrate acid (GABA) (ED50 +/- s.e. mean, 2.4 +/- 1.1 x 10(-5) M) produced a relaxation, if the tone had been increased with 5-hydroxytryptamine (5-HT). 2 3-Aminoproprane sulphonic acid (3-APS) produced a similar, but smaller relaxation, while baclofen had no effect. The relaxation produced by GABA was inhibited by bicuculline. 3 Transmural electrical stimulation produced a reproducible contraction of the isolated basilar artery. In 9 out 14 preparations GABA (ED50 +/- s.e. mean, 5.6 +/- 2.1 x 10(-7) M) caused a reduction of the response, with a maximum of 49.2 +/- 4.3%. Bicuculline did not inhibit these responses to GABA. 4 Baclofen (ED50 +/- s.e. mean, 6.8 +/- 1.4 x 10(-7) M) produced a similar inhibition (47.4 +/- 3.2% maximum) but 3-APS had no effect. 5 GABA (10(-4) M) had no effect on the tone of isolated mesenteric or internal carotid arteries from the rabbit, whether or not the tone was increased with 5-HT. Similarly, GABA (10(-4) M) did not produce any change in the responses to transmural stimulation in isolated mesenteric or internal carotid arteries. 6 These findings are consistent with the presence of two types of GABA receptor on the rabbit basilar artery.     

GABAB receptor-mediated mechanisms in human intestine in vitro.

The spontaneous motility of longitudinal muscle of human jejunum was recorded and the effect of gamma-aminobutyric acid-ergic (GABAergic) drugs was tested. GABA and (-)-baclofen (10(-6)-10(-4) M) dose dependently reduced the amplitude and frequency of the spontaneous contractions; muscimol and 3-aminopropanesulfonic acid (3 x 10(-5) M) were ineffective. The effect of 3 x 10(-5) M GABA was reduced by 3 x 10(-3) M 5-aminovaleric acid but not by 3 x 10(-5) M picrotoxin. The dose-response curve for GABA was shifted to the right by 3 x 10(-3) M 3-aminopropanesulfonic acid. Tetrodotoxin 3 x 10(-7) M prevented the GABAergic action, whereas various receptor antagonists tested did not affect it. GABAergic drugs did not influence the spontaneous motility of either circular or longitudinal muscles of human colon. It is suggested that GABAB receptor activation induces the inhibition of human jejunum longitudinal muscle motility by a neurogenic mechanism. The possible involvement of postganglionic cholinergic neurons is to be evaluated by other techniques.     

Evidence for a glutamate receptor of the AMPA subtype which mediates insulin release from rat perfused pancreas.

1. The effect of L-glutamate has been studied on insulin secretion by the isolated perfused pancreas of the rat. The glutamate receptor subtype involved has been characterized. 2. In the presence of a slightly stimulating glucose concentration (8.3 mM), L-glutamate (5 x 10(-5)-4 x 10(-3) M) induced an immediate, transient and concentration-dependent insulin response. On the other hand, in the presence of a non stimulating glucose concentration (2.8 mM), L-glutamate (10(-3) M) did not modify the basal insulin secretion. 3. The three non-NMDA receptor agonists, kainate (10(-4)-10(-3) M), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 5 x 10(-5)-10(-4) M) and quisqualate (5 x 10(-6)-5 x 10(-5) M) all provoked a transient and concentration-dependent insulin response from pancreas perfused with 8.3 mM glucose. Compared with glutamate, kainate exhibited a similar efficacy, whereas AMPA and quisqualate elicited only a 3 fold lower maximal insulin response. In contrast, NMDA (10(-4)-10(-3) M) was ineffective. 4. An antagonist of non-NMDA receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 5 x 10(-5) M) totally prevented the stimulatory effect of L-glutamate (4 x 10(-4) M) and kainate (2 x 10(-4) M). In contrast, the NMDA receptor antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ((+) MK801) was without effect. 5. The insulin secretory effect of glutamate (4 x 10(-4) M) was not affected by atropine (3 x 10(-7) M) or tetrodotoxin (3 x 10(-6) M). 6. Quisqualate at a high maximally effective concentration (4 x 10(-4) M) inhibited glutamate (10(-3) M) or kainate (4 x 10(-4) M)-induced insulin release. 7. This study shows that L-glutamate stimulates insulin secretion in rat pancreas, by acting on an excitatory amino acid receptor of the AMPA subtype.     

The effect of temperature on the GABA-induced chloride current in isolated sensory neurones of the frog.

1. The effect of temperature on the kinetics of the activation and inactivation phases of gamma-aminobutyric acid (GABA)-induced Cl- current (ICl) was examined in frog isolated sensory neurones. 2. The peak ICl was reversibly reduced on changing the temperature and temperature-dependent coefficients were shown to exist, with the highest Q10 (1.58) occurring between 5-15 degrees C. 3. At both room temperature (20 degrees C) and 10 degrees C, the GABA dose-response curve was sigmoidal with a Hill coefficient of 2 and half-maximal responses to GABA, Kd, of 1.3 x 10(-5)M and 1.1 x 10(-5)M, respectively. Thus, indicating no change in the binding affinity of GABA when the temperature was decreased. 4. At GABA concentrations greater than 10(-5)M, both the activation and inactivation phases of the GABA-induced ICl consisted of double exponentials, fast and slow components respectively, in the temperature range of 10 to 30 degrees C. 5. The fast (tau af) and slow (tau as) activation time constants decreased with an increase in temperature and increased with a reduction in temperature. With an increased temperature, the reduction in peak ICl was due to a reduction in the slow time constant with no significant change in the fast time constant. 6. Both the fast (tau if) and slow (tau is) inactivation time constants were also increased by cooling to 10 degrees C; heating to 30 degrees C had little effect. 7. The concentration-dependence (10(-5) to 10(-3)M) of the slow activation (tau as) and inactivation (tau is) time constants was unaltered by the change in temperature. Similarly, the lack of concentration-dependence shown by the fast activation (tau af) and inactivation (tau if) time constants was unaltered by the temperature change. 8. From recordings made with 'inside-out' patches, the probability of opening of the GABA-induced Cl- channels showed a marked increase with cooling to 10 degrees C compared to room temperature (20 degrees C), with no change in channel conductance. 9. The change in the GABA-induced ICl at different temperatures is, therefore, not due to changes in binding but to subsequent channel activation. Possible mechanisms whereby this occurs are discussed.     

Pharmacological characteristics of two different types of inhibitory GABA receptors on Achatina fulica neurones

GABA (gamma-aminobutyric acid) receptors of Achatina fulica neurones have been classified into two types associated with neuronal inhibition and one type with excitation. The pharmacological features of muscimol I and baclofen types associated with inhibition were investigated in this study. Activation of muscimol I type receptors on TAN (tonically autoactive neurone) by GABA, muscimol and trans-4-aminocrotonic acid (TACA) produced a transient outward current (Iout) with an increase in membrane conductance (g). Their relative potencies at GABA ED50 (approximately 10(-4) M) were: GABA: muscimol: TACA = 1:0.6:0.3. The relation between Iout and g increase (delta g) induced by various concentrations of these compounds was linear. The Hill coefficients for GABA were close to 1.0. The GABA effects were potentiated by pentobarbitone, antagonized competitively by pitrazepin and non-competitively by picrotoxin and diazepam, and unaffected by bicuculline. The reversal potentials of the effects of GABA, muscimol and TACA on TAN changed under various [Cl-]0 according to the Nernst equation for Ec1, but not under various [K+]0 and [Na+]0. Activation of baclofen type GABA receptors on RPeNLN (right pedal nerve large neurone) by GABA and (+/-)-baclofen produced a slow Iout with an increase in g. The two compounds were almost equipotent (ED50: approximately 3 x 10(-4) M). The relation between Iout and delta g produced by various concentrations was linear. The Hill coefficients for GABA were also close to 1.0. The reversal potentials of GABA and (+/-)-baclofen on RPeNLN changed under various [K+]0 according to the Nernst equation for EK, but not under various [Cl-]0 and [Na+]0. The two compounds hardly affected the voltage-gated and slowly inactivating calcium current. The Iout produced by GABA and (+/-)-baclofen was reduced by tetraethylammonium chloride, but was unaffected by 4-aminopyridine, bicuculline, pitrazepin and picrotoxin. In conclusion, the pharmacological features of muscimol I type GABA receptors are partly comparable to those of mammalian GABAA receptors, except for the influences of bicuculline and diazepam: the features of the baclofen type GABA receptor, which did not occur with muscimol I type receptors in the same neurone, were similar to those of GABAB.     

Functional properties of ionotropic glutamate receptor channels in rat sacral dorsal commissural neurons.

Nystatin perforated patch and conventional whole-cell recording configurations were used to characterize the properties of ionotropic glutamate receptor (GluR) channels in neurons freshly dissociated from the rat sacral dorsal commissural nucleus (SDCN). L-Glutamate (Glu), N-methyl-D-aspartate (NMDA), quisqualate (QA), alpha-amino-3-hydroxy-5-methyl-4-isoxazoleprop ionate (AMPA) and kainate (KA) applied via a Y-tube produced inward currents at -44 mV which increased in a concentration-dependent manner; they desensitized when induced at higher concentrations except for the KA-induced current (IKA). (1S-3R)1-amino-cyclopentane-1,3-dicarboxylate (1S-3R-ACPD) evoked no response. The EC50 and Hill coefficient (nH) values of the GluR responses were 3.3 x 10(-5) M, 0.74 for Glu; 9.0 x 10(-5) M, 0.83 for NMDA; 6.4 x 10(-7) M, 1.30 for QA; 1.3 x 10(-4) M, 1.10 for AMPA and 9.6 x 10(-5) M, 1.30 for KA, respectively. The reversal potentials of the GluR responses were all near 0 mV. The 6-Cyano-7-nitroquinoxaline-2-3-dione (CNQX) and D-2-amino-5-phosphonovalerate (D-APV) suppressed the non-NMDA and NMDA responses in a concentration-dependent manner, respectively. Cyclothiazide strongly potentiated both KA- and AMPA-induced responses while concanavalin A potentiated both the responses to a much lesser degree. NS-102 produced no significant effect on either KA- or AMPA-activated currents, while GYKI 52466 reversibly blocked both the currents. The Ca2+ permeabilities (PCa/PCs) of the NMDA and AMPA receptor channels were 8.33 and 1.23, respectively. In addition, the current-voltage (I-V) relationship of IKA showed little rectification. There was a poor correlation between the Ca2+ permeability and the shape of the I-V curves of IKA. These results suggest that rat SDCN neurons possess NMDA and non-NMDA receptor channels, and express AMPA type receptors with unique properties (slow desensitization to AMPA, high Ca2+ permeability but lack of inward rectification). These ionotropic receptor channels may play important roles in mediating and regulating pelvic visceral information including nociception.     

gamma-Aminobutyric acid (GABA)- and barbiturate-mediated 36Cl- uptake in rat brain synaptoneurosomes: evidence for rapid desensitization of the GABA receptor-coupled chloride ion channel

"Desensitization" of the gamma-aminobutyric acid (GABA) receptor-coupled chloride ion channel was studied using an in vitro method for measuring chloride (Cl-) permeability in brain vesicles (synaptoneurosomes). Muscimol, a GABA agonist, stimulated 36Cl- uptake in rat cerebral cortical synaptoneurosomes in a concentration-dependent manner (EC50 7.3 +/- 0.5 microM), whereas pentobarbital stimulated 36Cl- uptake in a biphasic manner, indicated by a bell-shaped concentration-response relationship, with a maximal response at 500 microM (EC50 271 +/- 17 microM). Higher concentrations of pentobarbital led to progressively smaller stimulation of 36Cl- uptake and blocked muscimol-stimulated 36Cl- uptake. Lower concentrations of pentobarbital (100-200 microM), when added with muscimol, produced an additive effect in stimulating 36Cl- uptake, whereas even lower (subthreshold) concentrations of pentobarbital (50 microM) potentiated muscimol-stimulated 36Cl- uptake. Following continuous exposure of synaptoneurosomes (up to 20 min) to muscimol (50 microM) or pentobarbital (500 microM), the 36Cl- uptake response diminished to a new steady state level with a t1/2 of approximately 6 sec and 30 sec, respectively. The decrement in response to these agonists was dependent on both concentration and length of exposure. No decrement was observed in the ability of subthreshold concentrations of pentobarbital to enhance muscimol-stimulated 36Cl- uptake following prolonged (20 min) incubation. "Heterologous desensitization" between muscimol and pentobarbital was observed in experiments where either muscimol or pentobarbital was added to the vesicles following pretreatment with the other. These findings suggest that "desensitization" of the GABA receptor/Cl- ion channel may involve both the GABA and barbiturate recognition sites or a common effector component such as the ionophore itself.     

Cholinergic-mediated secretion in the rat colon: neuronal and epithelial muscarinic responses

The acetylcholine receptor agonists, acetylcholine (10(-5)-10(-4 M), carbachol (5 x 10(-6)-5 x 10(-5) M), bethanechol (5 x 10(-5)-5 x 10(-4) M) and dimethylphenylpiperazinium (DMPP, 10(-5) M) increased the short-circuit current (Isc) in the rat colon descendens by a tetrodotoxin (TTX)-sensitive mechanism. Blockade by TTX was still observed after removal of the submucosa, indicating the involvement of neurons of the mucosal plexus. Hexamethonium (10(-5) M) and atropine (10(-6) M) were used to distinguish between nicotinic and muscarinic neuronally mediated effects. The inhibitor of choline uptake, hemicholinium-3 (1 mM), reversibly inhibited the effect of repeated electric field stimulation (EFS). The EFS response was only inhibited by high concentrations of atropine (greater than or equal to 10(-5) M). In mucosa-submucosa preparations 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) was more effective than telenzepine whereas pirenzepine was ineffective. Pirenzepine inhibited the EFS response in mucosa preparations as did telenzepine and 4-DAMP. It was not possible to differentiate between the muscarinic receptors involved in the different parts of the enteric nervous system on the basis of our results.     

A study on benzodiazepine receptor binding in audiogenic seizure-susceptible rats

Benzodiazepine receptors were investigated in the cerebral cortex, the hippocampus, the brainstem, and the cerebellum of audiogenic seizure (AGS)-susceptible and seizure-resistant (ER) control rats. In AGS-susceptible rats of Sprague-Dawley descent, muscimol (10-6 M and 3 x 10-5 M) activated the binding of 3H-diazepam (0.4 nM) significantly less than in ER-rats. This finding may be strain selective, since it was not observed in AGS-susceptible rats of Wistar descent. Specific binding of the convulsant benzodiazepine receptor ligand methyl 6,7-dimethoxy-4-ethyl carboline-3-carboxylate (3H-DMCM), the benzodiazepine receptor ligand 3H-diazepam and the chloride channel directed cage convulsant t-butylbicyclophosphorothionate 35S-TBPS were not significantly changed in AGS-susceptible as compared to control rats. Our findings indicate that a disturbance at the level of the benzodiazepine receptor/GABA receptor/chloride channel complex is not a likely general aetiological factor for audigenic seizures in rats.     

An electrophysiological investigation of the properties of 5-HT3 receptors of rabbit nodose ganglion neurones in culture.

1. The biophysical and pharmacological properties of 5-hydroxytryptamine (5-HT)-evoked currents in rabbit nodose ganglion neurones in culture have been determined by use of the whole-cell and outside-out membrane patch recording modes of the patch-clamp technique. 2. In 49% of cells investigated the bath application of 10(-5) M 5-HT at negative holding potentials elicited an inward current. The whole-cell response to 5-HT reversed in sign (E5-HT) at approximately -2 mV and exhibited inward rectification. 3. The influence of various ion substitutions upon E5-HT established that the 5-HT-evoked current is mainly mediated by a mixed Na+, K+ cation conductance with little or no contribution from Cl- ions. The omission of Ca2+ and Mg2+ from the extracellular solution enhanced the amplitude of the 5-HT-induced current. 4. On isolated outside-out membrane patches, the bath application of 10(-6) M 5-HT induced single channel currents with a chord conductance of approximately 17 pS at -70 mV and an average slope conductance of 19 pS over the range -100 to -40 mV. The 5-HT-induced single channels exhibited modest inward rectification and were reduced in frequency, but not amplitude, by the 5-HT3 receptor antagonist metoclopramide (10(-6) M). 5. The bath application of 5-HT (3 x 10(-7)-3 x 10(-5) M) to whole cells voltage clamped at -60 mV produced dose-dependent inward currents which were mimicked by 2-methyl-5-HT and 1-phenylbiguanide with equipotent molar ratios, relative to 5-HT, of 2.5 and 32 respectively. 6. Whole-cell inward currents produced by the local pressure application of 5-HT (10(-5) M) were unaffected by 10(-6) M methysergide, 10(-6) M ketanserin or 10(-6) M citalopram, but were concentration-dependently antagonized by the selective 5-HT3 receptor antagonists tropisetron (IC50 = 4.6 x 10(-11) M) ondansetron (IC50 = 5.7 x 10(-11) M), and bemesetron (IC50 = 3.3 x 10(-10) M). The response to 5-HT was also blocked by the non-selective antagonists metoclopramide (IC50 = 1.2 x 10(-8) M), cocaine (IC50 = 8.3 x 10(-8) M) and (+)-tubocurarine (IC50 = 1.6 x 10(-7) M).