GABAA and GABAB receptor-mediated effects in guinea-pig ileum

1 The effects of γ-aminobutyric acid (GABA) and related substances were examined in guinea-pig ileum longitudinal muscle.

2 GABA at doses ranging from 10^-7 M to 3 × 10^-6 M elicited a relaxation while at higher doses (3 × 10^-6 M — 10^-4 M), as previously described, it caused a contraction followed by relaxation.

3 GABA-induced relaxation was bicuculline-insensitive, was mimicked by (-)-baclofen but not by homotaurine and muscimol. The effect of baclofen was stereospecific. GABA- and (-)-baclofen-induced relaxations were dose-dependent and their ED₅₀ values were similar. A specific cross-desensitization occurred between GABA and (-)-baclofen.

4 The bicuculline-insensitive relaxation induced by GABA and (-)-baclofen was prevented by tetrodotoxin and hyoscine but not by phentolamine plus propranolol, naloxone or theophylline.

5 In preparations in which the muscle tone was raised by histamine or prostaglandin F_2α, GABA and (-)-baclofen induced relaxation to the same extent as before increasing the tone. If the tone was raised by DMPP, a greater bicuculline-insensitive relaxation occurred.

6 Contraction caused by GABA was bicuculline-sensitive and was mimicked by homotaurine and muscimol. Contraction was dose-dependent and muscimol was about three times more potent than GABA or homotaurine. A specific cross-desensitization occurred between the contractile effects of GABA and those of homotaurine or muscimol.

7 Bicuculline competitively antagonized the contractile effects of GABA, homotaurine and muscimol and gave closely similar pA₂ values. The slope of the Schild plot for the above drugs was near 1, confirming the competitive nature of the antagonism.

8 The bicuculline-sensitive contraction induced by GABA, homotaurine and muscimol was abolished by tetrodotoxin and was non-competitively antagonized by hyoscine, while it was unaffected by hexamethonium, mepyramine and methysergide.

9 It is concluded that two receptors mediate the GABA effects in guinea-pig ileum: a bicuculline-sensitive GABA_A receptor, which elicits contraction through an excitatory action on cholinergic post-ganglionic neurones; and a bicuculline-insensitive GABA_B receptor which causes relaxation through an inhibitory presynaptic action on cholinergic post-ganglionic neurones. We confirm that GABA, homotaurine and muscimol are GABA_A agonists, while GABA and (-)-baclofen are GABA_B agonists.

     

Interactions of ginsenosides with ligand-bindings of GABAA and GABAB receptors

• 1.1. Total saponin fraction decreased the affinity of specific [³H]muscimol binding without changes in B_max. Ginsenoside Rb₁ Rb₂, Rc, Re, Rf and Rg₁ inhibited the specific [³H]muscimol binding to the high-affinity site.
• 2.2. Total saponin fraction increased the affinity of specific [³H]flunitrazepam binding. Ginsenoside Re and Rf enhanced specific [³H]flunitrazepam binding.
• 3.3. Total saponin fraction decreased the affinity of specific [³⁵S]TBPS binding without changes in B_max. Ginsenosides did not affect specific or non-specific [³⁵S]TBPS binding.
• 4.4. Total saponin fraction decreased the affinity of specific [³H]baclofen binding without changes in B_max. Ginsenoside Rc inhibited specific [³H]baclofen binding.

     

Differential involvement of GABAA and GABAB receptors in propofol self-administration in rats

Aim:

Propofol has shown abuse potential. The aim of the present study is to investigate the effects of GABA_A antagonist and GABA_B agonist on propofol reinforcement.

Methods:

Sprague-Dawley rats were trained to self-administer propofol at a dose of 1.7 mg/kg per infusion under a fixed ratio (FR1) schedule of reinforcement for 14 d. In a separate set of experiments, food-maintained self-administration under a fixed ratio (FR5) schedule and locomotor activities of Sprague-Dawley rats were examined.

Results:

GABA_A receptor antagonist bicuculline (0.25 mg/kg, ip) significantly increased the number of injections and active responses. Pretreatment with GABA_B receptor agonist baclofen (3 mg/kg, ip) significantly decreased the number of active responses and total infusions of propofol during the training session. Moreover, microinjection of baclofen (50 and 100 ng/side) into the ventral tegmental area (VTA) significantly decreased the number of active responses and total infusions of propofol. Neither baclofen (1-3 mg/kg, ip) nor bicuculline (0.25-1 mg/kg, ip) affected food-maintained responses or motor activities.

Conclusion:

Propofol maintains its reward properties partially through GABA_A receptor activation. Stimulation of GABA_B receptors in VTA may counteract the reinforcing properties of propofol.     

Effects of pantoyl-GABA on GABAA and GABAB receptors in the rat brain

The interactions of the "antidementia drug" pantoyl-gamma-aminobutyric acid (pantoyl-GABA) with gamma-aminobutyric acid (GABA) receptors were investigated by studies on bindings of radiolabelled ligands in rat brain. Pantoyl-GABA inhibited the binding of [3H]GABA to GABAA receptors and those of [3H]baclofen and [3H]GABA to GABAB receptors in the rat cerebral cortex. These data suggest that pantoyl-GABA interacts with both types of GABA-receptors in the rat brain.     

Opposing effects of activation of central GABAA and GABAB receptors on brown fat thermogenesis in the rat

Baclofen (a GABAB agonist) stimulates body temperature, metabolic rate and brown adipose tissue (BAT) in the rat through a central action, but no effects of gamma-aminobutyric acid (GABA) itself on these parameters were observed. In the present study, it was found that the central effects of (+/-)baclofen (0.5-2.0 micrograms injection i.c.v.) on the temperature (1.2 degrees C increase) and metabolic rate (44-76% increase) of brown adipose tissue were inhibited by previous treatment with the GABAA agonist, muscimol (0.05 micrograms). Injection of GABA alone (12 micrograms) did not significantly affect these parameters, but in the presence of the GABAA antagonist bicuculline (2.5 micrograms), GABA significantly increased the temperature (0.3 degrees C) and oxygen consumption (22%) of brown fat. (-)Baclofen was found to be approximately 50-times more effective in stimulating the temperature of brown adipose tissue than (+/-)baclofen. The results indicate that activation of central GABAB receptors stimulates the activity and hence metabolic rate of brown adipose tissue. However, activation of the GABAA receptors opposes the effects of GABAB stimulation on the thermogenesis of brown fat.     

Excitatory amino acid receptors in rat locus coeruleus

Summary The goal of this study was to investigate whether locus coeruleus neurons of the rat are sensitive to agonists of the different excitatory amino acid receptors. All experiments were performed on a midpontine rat slice preparation. Bath-applied l-glutamate, kainate, N-methyl-d-aspartate (NMDA) and quisqualate induced concentration-dependent activations of all neurons which were reflected in an increase of the neurons' mean discharge rate. The rank order of cell activation was kainate quisqualate > NMDA > l-glutamate. None of the agonists induced a bursting-type of discharge. The NMDA-receptor blocker dl-2-amino-5-phosphonovaleric acid (APV, 30 M) selectively antagonized the NMDA-induced increase in cell firing. Kynurenic acid (100 M) non-selectively attenuated the response to NMDA, kainate and quisqualate. Neither APV nor kynurenic acid per se had any effect on the spontaneous firing rate. If the Mg²⁺ concentration in the superfusion medium was lowered from 2 mM to nominally zero the response to NMDA was selectively increased. In conclusion, locus coeruleus neurons share with other neurons their sensitivity to agonists of all three types of excitatory amino acid receptors. However, in contrast to other neurons, they do not respond with a bursting type of discharge. Send offprint requests to H.-R. Olpe at the above address     

Distribution of GABAA and GABAB receptors in mammalian brain: Potential targets for drug development

GABA is the major inhibitory neurotransmitter in mammalian brain. GABA receptors and the metabolism of GABA are significant targets for new centrally acting drugs to treat neurological and behavioral disorders. The simple neutral amino acid is likely to subserve a neurotransmitter role at 25–50% of all synapses in the central nervous system. GABA's actions are mediated by two different receptors, GABA_A and GABA_B receptors. GABA_A receptors are ligand-gated chloride channels that are sensitive to the convulsant alkaloid bicuculline and modulated by benzodiazepines and barbiturates. GABA_B receptors affect calcium and potassium conductance through GTP binding proteins and are insensitive to bicuculline and sensitive to the agonist baclofen. Both receptors are widely distributed in cerebral cortex, hippocampus, basal ganglia, thalamus, cerebellum, and brainstem.     

Central GABAA excitatory and GABAB inhibitory receptors regulate gastric acid secretion in rats

The present study investigates the effects of GABAA- and GABAB-receptor agonists and antagonists on gastric secretion after their i.c.v. administration to conscious pylorus-ligated rats and anaesthetized stomach lumen-perfused rats. Muscimol was without effect in pylorus-ligated rats, whereas baclofen produced a significant decrease in acid secretion, which was fully prevented by phaclofen. Under these conditions, a significant decrease in acid secretion was also obtained with bicuculline. In stomach lumen-perfused rats, muscimol caused a marked, dose-dependent increase in acid secretion, which was antagonized by bicuculline. Under the same conditions, baclofen induced a moderate, but significant, bicuculline-sensitive increase in acid secretion. Overall, our results suggest the presence of two central GABA pathways which mediate opposite effects: (a) a bicuculline-sensitive GABAA-receptor, the stimulation of which increases acid secretion under pharmacological depression of central vagal tone (anaesthetized rats); (b) a phaclofen-sensitive GABAB-receptor, the activation of which decreases vagally stimulated acid secretion (pylorus-ligated rats).     

Catecholamine receptors on locus coeruleus neurons: pharmacological characterization.

There is evidence that the noradrenergic neurons of the locus coeruleus (LC) possess α-adrenoreceptors in the vicinity of their cell bodies. To further characterize this receptor, we studied the responses of LC neurons to a series of catecholamine agonist and antagonist drugs using the techniques of single-unit recording and microiontophoresis. The spontaneous firing of LC neurons was inhibited by microiontophoretic application of norepinephrine, epinephrine, the α-adrenoreceptor agonist clonidine and the β-agonist isoproterenol. These inhibitions were blocked by the α-adrenergic antagonist piperoxane but not by the β-antagonist sotalol. In addition these cells were strongly inhibited by dopamine or α-methylnorepinephrine, but only weakly inhibited by phenylephrine or the dopamine agonist apomorphine. The dopamine antagonist trifluoperazine was ineffective in blocking the inhibitions of LC neurons by both dopamine and norepinephrine. The rank order of potencies of agonist drugs in inhibiting LC neurons was clonidine > > α-methylnorepinephrine ? epinephrine = norepinephrine > > phenylephrine. In this respect the LC α-receptor is similar to ‘presynaptic’ or ‘α₂’ receptors or peripheral sympathetic nerves and differs from the classical postsynaptic α-receptor. The noradrenergic neurons of the LC thus appear to possess catecholamine receptors on or near their cell bodies which have pharmacological characteristics of ‘presynaptic’ α-adrenergic receptors. The LC receptors are distinct from central dopamine receptors and from norepinephrine receptors areas of the brain receiving their noradrenergic iput fom the LC.     

Coexistence of GABAA and GABAB receptors on A delta and C primary afferents.

Intracellular recordings from adult rat dorsal root ganglion neurones were performed in vitro and the coexistence of two gamma-aminobutyric acid (GABA) receptors on the membrane of identified A delta and C primary afferents was demonstrated. Transient applications of GABA (10(-6)-10(-2) M) evoked dose-dependent depolarizations and increased membrane conductance. The responses were mimicked by muscimol, isoguvacine, THIP and 3 amino propane sulphonic acid (3 APS); they were blocked by bicuculline and picrotoxin. Pentobarbitone induced an increase of GABA-induced depolarizations. Perfusion of tetraethylammonium (TEA, 7.5 mM) and intracellular injection of Cs+ ions unmasked the Ca2+ component of action potentials, which appeared as long-lasting plateau depolarizations. Such action potentials were shortened in the presence of methoxyverapamil (D600, 5 X 10(-6)-10(-5) M) and in a medium without Ca+ ions. Prolonged (5-10 min) perfusion of GABA (10(-9)-10(-5) M) shortened the Ca2+ component of action potentials. This effect was mimicked by baclofen (10(-7)-5 X 10(-6) M) and muscimol (5 X 10(-7)-10(-5) M) and was not affected by bicuculline perfusion (5 X 10(-6)-10(-5) M). Isoguvacine (2.5 X 10(-5) M) did not affect action potential duration. It is concluded that two GABA receptors coexist on the membrane of slow conducting primary afferents: the bicuculline-sensitive GABAA receptor mediates depolarizations and the bicuculline-insensitive GABAB receptor shortens the calcium component of action potentials.     

Role of GABAA and GABAB receptors and peripheral cholinergic mechanisms in the antinociceptive action of taurine

1. Gabaergic and cholinergic mediation in the antinociceptive effect of taurine has been investigated in mice (acetic acid test) and rats (tail-flick test).2. Scopolamine sulfate and methylnitrate exhibit intrinsic antinociceptive activity and increase the effect of taurine in mice.3. Baclofen also increases the antinociceptive effect of taurine in mice.4. Anticholinergic agents and bicuculline but not CGP 35348 antagonize the effect of taurine in rats.5. These results suggest that the antinociceptive effect of taurine may be partly mediated by spinal GABA_A receptors and peripheral cholinergic mechanisms.     

Stimulatory effects of clonidine,cirazoline and rilmenidine on locus coeruleus noradrenergic neurones: possible involvement of imidazoline-preferring receptors

Summary Clonidine and related drugs not only interact with ₂-adrenoceptors but also recognise non-adrenoceptor sites in the brain. The involvement of these imidazoline-preferring receptors in the regulation of the activity of locus coeruleus noradrenergic neurones (NA-LC) was investigated after inactivation of ₂-adrenoceptors with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). In EEDQ-pretreated rats (6 mg/kg, i.p., 6 h), the characteristic inhibitory effect of low doses of clonidine on these neurones was abolished and a paradoxical, dose-dependent increase in firing rate was observed at higher doses (640–5120 g/kg, i.v.) (ED₅₀ = 702 g/kg, E_max = 83 %, n = 14). Guanfacine (0.3–20 mg/kg) did not modify neuronal activity but antagonised the stimulatory effect of clonidine. Cirazoline (80–640 g/kg) and rilmenidine (0.3–10 mg/kg) also stimulatedneuronal activity(ED₅₀ = 192 g/kg, E_max = 102%, n = 5; ED₅₀ = 1563 g/kg, E_max = 70%, n = 1–5, respectively) by an ₂-adrenoceptor-independent mechanism. The results suggest that these drugs can modulate the activity of locus coeruleus noradrenergic neurones through the activation of I₁-imidazoline-preferring receptors.     

Cyclobenzaprine effects on locus coeruleus cells in tissue slice

Tissue slices 400 μ thick were taken from the brain stem, at the level of the locus coeruleus, of 150–250 g Sprague-Dawley rats. Microe1ectrodes were placed in the locus coeruleus under visual control, and a cell whose discharge rate would decrease with microiontophoretic application of norepinephrine or clonidine was sought. Cyclobenzaprine (CBZ) was then introduced into the perfusion medium at a concentration equivalent to 1 mg/kg body weight of the whole animal. Discharge rates before and during CBZ administration were compared. The six cells with initial discharge rates between 2 and 10 Hz decreased firing with CBZ, whereas the four cells with initial rates between 0.5 and 1.5 Hz increased their rates with CBZ.     

No evidence for functional imidazoline receptors on locus coeruleus neurons

₂-Adrenoceptor agonists inhibit the firing of locus coeruleus (LC) neurons. It was recently observed that the ₂-adrenoceptor agonists clonidine, rilmenidine and cirazoline, when injected intravenously in anaestbetized rats pretreated with the irreversible ₂-adrenoceptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), excite the LC. The effect was attributed to activation of 11 imidazoline receptors. The aim of the present experiments was to characterize the direct effect of ₂-adrenoceptor and I₁ imidazoline receptor agonists on LC neurons.Electrical activity of LC neurons was extracellularly recorded in midpontine slices prepared from the rat brain. Concentration-response curves were obtained for the ₂-agonist noradrenaline and the mixed I₁/₂-receptor agonists clonidine, rilmenidine and moxonidine in slices without treatment and in slices treated with 6-chloro-N-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SK&F86466) or EEDQ, ₂-adrenoceptor antagonists with low affinity for I₁ and I₂ imidazoline receptors, respectively. All four agonists concentration-dependently reduced the firing rate of the neurons, with full inhibition at higher concentrations. SK&F86466 shifted the concentration-response curves of the agonists to the right; the calculated antagonist dissociation constants are compatible with an effect of the agonists on ₂-adrenoceptors. EEDQ completely prevented the inhibition by the agonists. Neither in SK&F86466- nor in EEDQ-treated slices was an excitation by clonidine, rilmenidine and moxonidine observed.We conclude that the LC neurons do not possess functional I₁ (and also no I₂) imidazoline receptors. The effects of noradrenaline, clonidine, rilmenidine and moxonidine on the neurons can be fully explained with an interaction with inhibitory ₂-adrenoceptors (probably of the _2D subtype). The excitation of the LC by imidazoline receptor agonists under in vivo conditions, hence, is not a direct effect on the neurons of the LC.     

GABAA and GABAB receptors in the nucleus accumbens shell differentially modulate dopamine and acetylcholine receptor-mediated turning behaviour

The ability of GABA_A and GABA_B receptors in the shell of the nucleus accumbens to modulate distinct types of turning behaviour was investigated in freely moving rats, using the unilateral injection technique. The GABA_A receptor agonist muscimol and the GABA_A receptor antagonist bicuculline did not produce turning behaviour; the same holds for the GABA_B agonist baclofen and the GABA_B antagonist 2-hydroxysaclofen. A mixture of the dopamine D₁ receptor agonist SKF 38393 and the dopamine D_2/3 receptor agonist quinpirole has been found to elicit contraversive pivoting, when injected into the shell. This pivoting was dose-dependently inhibited by muscimol, and the inhibitory effect of muscimol was antagonised by bicuculline. Pivoting was also dose-dependently inhibited by baclofen; however, 2-hydroxysaclofen did not antagonise the inhibitory effect. The acetylcholine receptor agonist carbachol has been found to elicit contraversive circling, when injected into the shell. This carbachol-induced circling was inhibited by baclofen, and 2-hydroxysaclofen antagonised the inhibitory effect. Carbachol-induced circling was also partially inhibited by muscimol; however, the inhibitory effect of muscimol was not antagonised by bicuculline. It is concluded that mesolimbic GABA_A receptors exert an inhibitory control on dopamine-dependent pivoting that can be elicited from the shell of the nucleus accumbens, and that GABA_B receptors exert an inhibitory control on acetylcholine-dependent circling that can be elicited from the shell of the nucleus accumbens. This data extends the earlier reported findings that the neurochemical substrate in the shell of the nucleus accumbens that mediates dopamine-dependent pivoting is fundamentally different from the shell substrate that mediates acetylcholine-dependent circling.     

GABAA and GABAB receptor sites involvement in rat thermoregulation

1. Intraperitoneal (i.p.) injection of muscimol (MUS, 2-8 mg kg-1) decreased the core body temperature (BT) of the rats dose-dependently. 2. Intracerebroventricular (i.c.v.) injection of MUS (1 microgram/microliter/rat) also caused a fall in BT. 3. The hypothermia induced by MUS was inhibited by pretreatment of the animals with either bicuculline (BIC) or picrotoxin (PIC). 4. i.p. Injection of baclofen (BAC, 2.5-10 mg kg-1) induced hypothermia. Higher dose of the drug (20 mg kg-1) caused an initial fall followed by a marked increase in BT. 5. i.c.v. Injection of BAC produced a rise in BT. 6. The hypothermic effect of BAC was antagonized in animals pretreated with either BIC or PIC, while hyperthermic effect of the drug was potentiated with PIC pretreatment. 7. i.c.v. Injection of isoguvacine (ISO) induced hypothermia, which was attenuated in rats pretreated with either BIC or PIC. 8. It can be concluded that: activation of GABAA or GABAB receptor sites respectively may induce hypothermia or hyperthermia in rats.     

Participation of GABAA,GABAB receptors and neurosteroids in toluene-induced hypothermia: Evidence of concentration-dependent differences in the mechanism of action

Toluene is a misused substance that modifies γ-aminobutyric acid (GABA) release and shares behavioral and molecular effects with GABA_A and GABA_B receptor agonists. GABAergic compounds are involved in thermoregulation processes and volatile substance users have reported that one of the reasons to inhale is to avoid feeling cold. At present, no studies have analyzed the effects of inhalants on body temperature and the mechanism of action involved. Thus, the main purpose of this study was to evaluate the effects of a (60 min) acute toluene inhalation (2000, 4000 and 6000 ppm) in core temperature. In addition, we tried to prevent the changes of temperature induced by toluene with the specific GABA_A receptor blockers picrotoxin (0.01–0.1 mg/kg), bicuculline (0.1–0.3 mg/kg), and flumazenil (3–30 mg/kg); the GABA_B receptor antagonist phaclofen (10–30 mg/kg) and the neurosteroid synthesis inhibitor finasteride (10–30 mg/kg). Results show that toluene reduced core temperature in mice in a concentration-dependent manner. The hypothermia produced by 4000 ppm toluene was prevented by picrotoxin, bicuculline, phaclofen and finasteride but not by flumazenil. In contrast none of these antagonists tested blocked the effects of 6000 ppm toluene. In conclusion, toluene decreases core temperature, GABA receptors and neurosteroids participate in toluene’s action at 4000 ppm; but other mechanisms of action are involved in the hypothermic effects of 6000 ppm toluene.     

Panicolytic-like effect induced by the stimulation of GABAA and GABAB receptors in the dorsal periaqueductal grey of rats

Activation of GABA(A) and benzodiazepine receptors within the dorsal periaqueductal grey inhibits the escape behaviour evoked by the electrical stimulation of this midbrain area, a defensive reaction that has been related to panic. Nevertheless, there is no evidence indicating whether the same antiaversive effect is also observed in escape responses evoked by species-specific threatening stimuli. In the present study, male Wistar rats were injected intra-dorsal periaqueductal grey with the benzodiazepine receptor agonist midazolam (10, 20 and 40 nmol), the GABA(A) receptor agonist muscimol (2, 4 and 8 nmol), the GABA(B) receptor agonist baclofen (2, 4 and 8 nmol), or with the benzodiazepine inverse agonist FG 7142 (20, 40 and 80 pmol) and tested in an ethologically-based animal model of anxiety, the elevated T-maze. Besides escape, this test also allows the measurement of inhibitory avoidance which has been related to generalised anxiety disorder. Midazolam, muscimol and baclofen impaired escape, a panicolytic-like effect, without altering inhibitory avoidance. FG 7142, on the other hand, facilitated both avoidance and escape reactions, suggesting an anxiogenic and panicogenic-like effect, respectively. The data suggest that GABA(A)/benzodiazepine and GABA(B) receptors within the dorsal periaqueductal grey are involved in the control of escape behaviour and that a failure in this regulatory mechanism may be of importance in panic disorder.     

Electrophysiological effects of dermorphin on locus coeruleus neurons of rat

Intracellular recording was used to study the effects of dermorphin on neurons of the locus coeruleus in the rat, in a totally submerged brain slice preparation. Dermorphin caused the inhibition of spontaneous firing of all neurons of the locus coeruleus tested, with an IC50 of 7 nM. Based on the inhibition of spontaneous firing rate, dermorphin was 16.5 times more potent than morphine. Larger concentrations of dermorphin (30-100 nM) further hyperpolarized the neurons of the locus coeruleus and simultaneously caused a reduction in input resistance. These effects were antagonized by naloxone, with a dissociation equilibrium constant of 0.8 nM. The hyperpolarization of neurons of the locus coeruleus, caused by dermorphin, was reversed at a membrane potential of -112 mV in this preparation. Furthermore, this hyperpolarization was blocked by cesium chloride and barium chloride. Thus, these data suggest that dermorphin binds to mu-opioid receptors on the cell membrane of neurons of the locus coeruleus. This leads to opening of the inward-going rectification potassium channels, resulting in the observed hyperpolarization of the membrane.