GABAB Receptor Binding Research Articles

Neonatal Clonazepam Administration Induced Long-Lasting Changes in GABAA and GABAB Receptors.

Benzodiazepines (BZDs) are widely used in patients of all ages. Unlike adults, neonatal animals treated with BZDs exhibit a variety of behavioral deficits later in life; however, the mechanisms underlying these deficits are poorly understood. This study aims to examine whether administration of clonazepam (CZP; 1 mg/kg/day) in 7–11-day-old rats affects Gama aminobutyric acid (GABA)ergic receptors in both the short and long terms. Using RT-PCR and quantitative autoradiography, we examined the expression of the selected GABAA receptor subunits (α1, α2, α4, γ2, and δ) and the GABAB B2 subunit, and GABAA, benzodiazepine, and GABAB receptor binding 48 h, 1 week, and 2 months after treatment discontinuation. Within one week after CZP cessation, the expression of the α2 subunit was upregulated, whereas that of the δ subunit was downregulated in both the hippocampus and cortex. In the hippocampus, the α4 subunit was downregulated after the 2-month interval. Changes in receptor binding were highly dependent on the receptor type, the interval after treatment cessation, and the brain structure. GABAA receptor binding was increased in almost all of the brain structures after the 48-h interval. BZD-binding was decreased in many brain structures involved in the neuronal networks associated with emotional behavior, anxiety, and cognitive functions after the 2-month interval. Binding of the GABAB receptors changed depending on the interval and brain structure. Overall, the described changes may affect both synaptic development and functioning and may potentially cause behavioral impairment.

Parafascicular thalamic nucleus deep brain stimulation decreases NMDA receptor GluN1 subunit gene expression in the prefrontal cortex

The rodent parafascicular nucleus (PFn) or the centromedian-parafascicular complex of primates is a posterior intralaminar nucleus of the thalamus related to cortical activation and maintenance of states of consciousness underlying attention, learning and memory. Deep brain stimulation (DBS) of the PFn has been proved to restore arousal and consciousness in humans and to enhance performance in learning and memory tasks in rats. The primary expected effect of PFn DBS is to induce plastic changes in target neurons of brain areas associated with cognitive function. In this study, Wistar rats were stimulated for 20mins in the PFn following a DBS protocol that had previously facilitated memory in rats. NMDA and GABAB receptor binding, and gene expression of the GluN1subunit of the NMDA receptor (NMDAR) were assessed in regions related to cognitive functions, such as the prefrontal cortex and hippocampus. The results showed that PFn DBS induced a decrease in NMDAR GluN1 subunit gene expression in the cingulate and prelimbic cortices, but no significant statistical differences were found in the density of NMDA or GABAB receptors in any of the analyzed regions. Taken together, our findings suggest a possible role for the NMDAR GluN1 subunit in the prefrontal cortex in the procognitive actions of the PFn DBS.

Receptor autoradiographic correlates of deafferentation‐induced reorganization in adult primate somatosensory cortex

The primate somatosensory system provides an excellent model system with which to investigate adult neural plasticity. We have previously shown that transection of the median and ulnar nerves is followed by an expansion in the representation of radial nerve skin, and that this plasticity proceeds in stages. Immediately following nerve injury, new receptive fields are "unmasked" in a fraction of the affected cortex. The remaining deprived cortex regains responsiveness to tactile stimulation over the following days to weeks. Given these progressive changes, it has been suggested that different mechanisms might account for the earlier and later phases of reorganization. In the present experiments, we quantified receptor autoradiographic binding data for GABAA and GABAB, AMPA, and NMDA receptors in the primary somatosensory cortices of adult squirrel monkeys at four postnerve injury survival durations: immediately (1-3 hours), 3 days, 1 month, and 2 months. We found dramatic reductions in GABAA binding in layer IV within hours following nerve injury, and this reduction was maintained across all survival durations. This finding is consistent with the idea that the earliest reorganizational changes are due to a relaxation in tonic inhibitory mechanisms permitting the expression of formerly subthreshold receptive fields. GABAB receptor binding is decreased in layer IV by 1 month after nerve injury, while binding for AMPA receptors is increased in layer IV by this time. These findings are consistent with our previous suggestion that the second stage of reorganization proceeds via mechanisms comparable to those revealed to account for NMDA-dependent long-term potentiation in the hippocampus.

Age-related changes of the GABA-B receptor in the lumbar spinal cord of male rats and penile erection

Dorsal horn neurons of lumbosacral spinal cord innervate penile vasculature and regulate penile erection. GABAergic system is involved in the regulation of male sexual behavior. Because aging is frequently accompanied by a progressive decline in erectile function, the aim of this work was to examine age-related changes of the GABA-B receptor in the lumbar spinal cord. Sprague–Dawley rats of 10 and 21 days old, 3, 9 and 20 months old were used. GABA-B receptors were evaluated by quantitative autoradiography using [ 3H]-Baclofen as ligand with or without GABA (10 μM) to determine the non-specific binding. Ten days after birth a homogeneous neuroanatomical distribution pattern was found in the gray matter, however at 20-day-old adult distribution emerged becoming heterogeneous with the highest binding values at layers II–III and X. In dorsal layers a significant decrease was observed in 9-month-old rats while layer X showed an earlier decrease (21-day-old). GABA-B receptor affinity showed significant age-dependent and regional increase. The GABA-B receptor decrease in aged rats seems not to be related to this receptor inhibitory function in penile erection. Moreover the changes found in GABA-B receptor binding anatomical distribution may indicate its role in the morphological development of the lumbar spinal cord rather than in the decline of the erectile function.

The Absence of Information about Hormones and Absence

Hormonal Regulation of Absence SeizuresPersad V, Ting Wong CG, Cortez MA, Wang YT, Snead OC 3rdAnn Neurol 2004;44:353–361A time course study that examined the effects of the female estrous cycle on the chronic slow spike-and-wave discharges (SSWDs), GABAB-receptor (GABABR) binding, and GABABR protein expression was conducted in Long-Evans hooded rats treated during development with a cholesterol synthesis inhibitor AY9944 (AY). In addition, a pharmacologic study using the hormones progesterone, 17 β-estradiol, mifepristone (intracellular progesterone-receptor antagonist), tamoxifen (intracellular estrogen-receptor antagonist), and allopregnanolone (progesterone metabolite) was performed to determine their effects on AY-induced seizures. The data indicate that a significant increase occurs in both the duration of SSWDs and GABABR binding in the AY model during the proestrus stage of the estrous cycle, the stage during which the levels of progesterone are at their highest. No changes in GABABR1a or R2 protein levels were observed. In addition, the administration of both progesterone and allopregnanolone exacerbated seizures in the AY model, whereas 17 β-estradiol attenuated the SSWD duration. Neither mifepristone nor tamoxifen blocked the effects of progesterone and 17 β-estradiol, respectively, on SSWD duration in the AY model, suggesting that these two sex hormones are working in a manner independent of their intracellular receptors. These data suggest an important role for steroid hormones in the regulation and maintenance of AY-induced atypical absence seizures.

Plasticity of GABABReceptor-Mediated Heterosynaptic Interactions at Mossy Fibers After Status Epilepticus

Several neurotransmitters, including GABA acting at presynaptic GABA(B) receptors, modulate glutamate release at synapses between hippocampal mossy fibers and CA3 pyramidal neurons. This phenomenon gates excitation of the hippocampus and may therefore prevent limbic seizure propagation. Here we report that status epilepticus, triggered by either perforant path stimulation or pilocarpine administration, was followed 24 hr later by a loss of GABA(B) receptor-mediated heterosynaptic depression among populations of mossy fibers. This was accompanied by a decrease in the sensitivity of mossy fiber transmission to the exogenous GABA(B) receptor agonist baclofen. Autoradiography revealed a reduction in GABA(B) receptor binding in the stratum lucidum after status epilepticus. Failure of GABA(B) receptor-mediated modulation of mossy fiber transmission at mossy fibers may contribute to the development of spontaneous seizures after status epilepticus.

Changes in GABAB RECEPTOR mRNA expression in the rodent basal ganglia and thalamus following lesion of the nigrostriatal pathway

Loss of striatal dopaminergic innervation in Parkinson's disease (PD) is accompanied by widespread alterations in GABAergic activity within the basal ganglia and thalamus. Accompanying changes in GABAB receptor binding have been noted in some basal ganglia regions in parkinsonian primates, suggesting that plasticity of this receptor may also occur in PD. However, the molecular mechanisms underlying the changes in receptor binding and the manner and extent to which different GABAB receptor mRNA subunits and splice-variants are affected remain unknown. This study used in situ hybridisation to examine the full profile of changes in expression of the known rat GABAB receptor genes and gene variants in the basal ganglia and thalamus of rats, brought about by degeneration of the nigrostriatal tract. All of the GABAB mRNA species examined showed unique expression patterns throughout the basal ganglia and thalamus. In addition, all exhibited a marked loss of expression (between 46 and 80%) in the substantia nigra pars compacta of animals bearing a complete 6-hydroxydopamine-induced lesion of the nigrostriatal tract, confirming the presence of these variants in dopaminergic neurones in this region. Further analysis of autoradioagrams revealed additional changes only in GABAB(1a) mRNA in discrete anatomical regions. Expression of the GABAB(1a) variant was significantly increased in the substantia nigra pars reticulata (33±2%), entopeduncular nucleus (26±1%) and the subthalamic nucleus (16±1%). Since these regions all receive reduced GABAergic innervation following nigrostriatal tract lesioning, it is possible that the increased expression occurs as a compensatory measure. In conclusion, these data demonstrate that GABAB receptor genes exhibit regional- and subunit/variant-specific plasticity at the molecular level under parkinsonian conditions.

Effects of repeated administration of baclofen to rats on GABAB receptor binding sites and subunit expression in the brain.

Repeated stimulation of the GABAB receptor with baclofen frequently produces tolerance, the underlying mechanisms of which are poorly understood. The purpose of the present work was to determine whether repeated administration of baclofen to rats is accompanied by changes in cerebral GABAB receptor binding sites, mRNA for the subunits GABAB(1) and GABAB(2), and protein levels for these subunits. Rats were injected with placebo or baclofen (20 micromol/kg subcutaneously) once daily for 14 days. Decreases in body temperature were measured as an index of pharmacological effects of baclofen. Binding of radiolabeled GABA to GABAB receptors was quantitated in brain membranes, mRNA levels were determined using quantitative real-time PCR, and GABAB receptor protein levels were assessed with Western blot analysis. Baclofen caused a decline in temperature amounting to approximately 2.5 degrees C after the first dose. This effect was partly lost after the fifth and abolished after the seventh injection. Despite the complete development of tolerance, there were no significant alterations in GABAB receptor binding sites (number or affinity) or mRNA levels for the subtypes GABAB(1a), GABAB(1b), or GABAB(2). Receptor protein levels were also unchanged. It is concluded that baclofen induces tolerance through mechanisms other than down-regulation of GABAB receptor transcription or translation.

Maturational Aspects of Epilepsy Mechanisms and Consequences for the Immature Brain

Maturational Aspects of Epilepsy Mechanisms and Consequences for the Immature Brain

An in situ hybridization study of the distribution of the GABAB2 protein mRNA in the rat CNS

γ-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system. GABA exerts its actions through two classes of receptors: GABAA, multimeric ligand-gated Cl− ion channels (a class which has been proposed to include the homomeric variant previously called GABAC, to be designated GABAA0r); and GABAB, G-protein coupled receptors which regulate Ca2+ and K+ channels. Currently, within the GABAB receptor family two proteins have been identified through molecular cloning techniques and designated GABAB1 and GABAB2. Two N-terminal variants of GABAB1 were isolated and designated GABAB1a and GABAB1b. The distribution of neurons in the rat CNS expressing the mRNA for the GABAB1 isoforms have been previously described by in situ hybridization histochemistry. The recent isolation and identification of the GABAB2 protein by homology cloning has enabled the use of radiolabeled oligonucleotides to detect the distribution of the expression of GABAB2 mRNA in the rat CNS. The expression of GABAB2 mRNA was observed to be primarily related to neuronal profiles. The highest levels of GABAB2 mRNA expression were detected in the piriform cortex, hippocampus, and medial habenula. GABAB2 mRNA was abundant in all layers of the cerebral cortex, the thalamus and in cerebellar Purkinje cells. Moderate expression was observed in several hypothalamic and brainstem nuclei. In contrast to the distribution of GABAB1 mRNA, only a weak hybridization signal for GABAB2 was detected over cells of the basal ganglia, including the caudate–putamen, nucleus accumbens, olfactory tubercle and throughout most of the hypothalamus. Moderate-to-heavy GABAB2 mRNA expression was also seen over dorsal root and trigeminal ganglion cells. In general, the pattern of GABAB2 mRNA expression in the rat brain overlaps considerably with the distributions described for both GABAB1 mRNAs, and is concordant with the distribution described for GABAB receptor binding sites. However, differences between GABAB2 expression levels and GABAB binding sites were observed in the basal ganglia.

GABAB-receptor splice variants GB1a and GB1b in rat brain: developmental regulation, cellular distribution and extrasynaptic localization.

GABAB (gamma-aminobutyric acid)-receptors have been implicated in central nervous system (CNS) functions, e.g. cognition and pain perception, and dysfunctions including spasticity and absence epilepsy. To permit an analysis of the two known GABAB-receptor splice variants GABAB-R1a (GB1a) and GABAB-R1b (GB1b), their distribution pattern has been differentiated in the rat brain, using Western blotting and immunohistochemistry with isoform-specific antisera. During postnatal maturation, the expression of the two splice variants was differentially regulated with GB1a being preponderant at birth. In adult brain, GB1b-immunoreactivity (-IR) was predominant, and the two isoforms largely accounted for the pattern of GABAB-receptor binding sites in the brain. Receptor heterogeneity was pronounced in the hippocampus, where both isoforms occurred in CA1, but only GB1b in CA3. Similarly, in the cerebellum, GB1b was exclusively found in Purkinje cells in a zebrin-like pattern. The staining was most pronounced in Purkinje cell dendrites and spines. Using electron microscopy, over 80% of the spine profiles in which a synaptic contact with a parallel fibre was visible contained GB1b-IR at extrasynaptic sites. This subcellular localization is unrelated to GABAergic inputs, indicating that the role of GABAB-receptors in vivo extends beyond synaptic GABAergic neurotransmission and may, in the cerebellum, involve taurine as a ligand.

Characteristics of GABAB receptor binding sites on rat whole brain synaptic membranes

Characteristics of GABA_B receptor binding sites on rat whole brain synaptic membranes

GABAA receptor binding in the aging rat inferior colliculus.

The inhibitory neurotransmitter GABA has been shown to be critically involved in shaping neuronal responses to simple and complex acoustic stimuli in the inferior colliculus. Studies in the rat and human inferior colliculus have suggested significant changes in functions related to GABA neurotransmission occur in the aged. These changes include significant decreases in GABA content, GABA release, GABA neurons, glutamate decarboxylase enzymatic activity, and GABAB receptor binding. Such changes within the inferior colliculus may affect the ability of elderly listeners to process complex acoustic signals, particularly in the presence of background noise. The present study was designed to examine the regional distribution and effects of aging on GABAA receptor binding sites in the Fischer 344 rat inferior colliculus using in vitro quantitative receptor autoradiography. [3H]GABA binding to GABAA receptors was significantly reduced in the inferior colliculus of young adult (3 months) and aged (18-26 months) rats when compared to 2-month animals. However, no significant changes were observed after 3 months of age. Single concentrations of tritiated GABAA receptor ligands (muscimol, t-butylbicycloorthobenzoate, and flunitrazepam) revealed no significant age-related changes in receptor binding in the inferior colliculus between 3 and 26 months of age. To characterize further the pharmacology of the GABAA receptor in the inferior colliculus, GABA modulation of the picrotoxin binding site was examined using [3H]t-butylbicycloorthobenzoate. When increasing concentrations of GABA were added to the incubation buffer, a significant decrease in binding was observed in the inferior colliculus of rats in each age group. In aged rats, the dose-response curve was shifted to the left, indicating an increase in the potency of GABA to inhibit [3H]t-butylbicycloorthobenzoate binding. Although no changes in GABAA receptor binding were detected in the inferior colliculus after 3 months of age, a significant alteration in interaction between the GABA and picrotoxin binding sites was observed in the inferior colliculus of aged rats when compared to 3-month-old young adults. This difference appears to reflect an increased sensitivity of the receptor to GABA modulation in aged rats and, thus, may serve as a compensatory mechanism to enhance GABAA receptor function in response to a presynaptic loss of inhibition.

GABAB Receptor‐Mediated Effects in Synaptosomes of Lethargic (lh/lh) Mice

Previously, we have shown a significant increase in number of GABAB receptor binding sites in neocortex and thalamus of lethargic (lh/lh) mice, a mutant strain exhibiting absence seizures. This study was performed to test our hypothesis that presynaptic GABAB receptors would inhibit [3H]GABA release to a greater degree in lh/lh mice compared with their nonepileptic littermates (designated +/+). Synaptosomes isolated from neocortex and thalamus of age-matched male lh/lh and +/+ mice were similar in uptake of [3H]GABA. In the neocortical preparation, baclofen dose-dependently inhibited [3H]GABA release evoked by 12 mM KCl, an effect mediated by GABAB receptors. The maximal inhibition (Imax) value was significantly greater (80%) in lh/lh than +/+ mice, whereas the IC50 (3 microM) was unchanged. In the thalamic preparation, the effect of baclofen (50 microM) was 58% less robust in lh/lh mice. Other effects mediated by GABAB receptors (inhibitions in Ca2+ uptake and cyclic AMP formation) were also significantly reduced in thalamic synaptosomes from lh/lh mice. These data suggest a greater presynaptic GABAB receptor-mediated effect in neocortex and a reduced effect in thalamic nuclei of lh/lh mice. It is possible that selective effects of presynaptic GABAB receptors or GABA release in neocortex and thalamic nuclei of lh/lh mice may contribute to mechanisms underlying absence seizures.

Partial GABAA receptor agonists. Synthesis and in vitro pharmacology of a series of nonannulated analogs of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol.

5-(4-Piperidyl)isoxazol-3-ol (4-PIOL, 10), a structural analog of 4-aminobutanoic acid (GABA, 1) and the GABAA agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 5), is a low-efficacy partial GABAA agonist. A number of compounds bioisosterically derived from 10, including 5-(4-piperidyl)isothiazol-3-ol (11), 3-(4-piperidyl)isoxazol-5-ol (12), 5-(1,2,3,6-tetrahydropyrid-4-yl)isoxazol-3-ol (13), and 5-(1,2,3,6-tetrahydropyrid-4-yl)isothiazol-3-ol (14), were synthesized and tested as GABAA receptor ligands. Whereas none of these compounds significantly affected GABAB receptor binding or GABA uptake, they showed affinities for GABAA receptor sites in the low-micromolar range. Using cultured cerebral cortical neurons and whole-cell patch-clamp techniques, the efficacies of these compounds relative to that of the full GABAA agonist, isoguvacine (8) (20 microM), were determined. The relative efficacy of 11, which has a higher receptor affinity (IC50 = 1.3 +/- 0.3 microM) than 10 (IC50 = 9.3 +/- 2.6 microM), was comparable with that of 10 (30-35%). The tetrahydropyridine analog of 10, compound 13, showed a markedly lower receptor affinity (IC50 = 32 +/- 10 microM) and apparently a lower relative efficacy than 10. The corresponding unsaturated analog of 11, compound 14, showed a slightly weaker receptor affinity (IC50 = 4.0 +/- 2.0 microM) but a significantly higher relative efficacy (50-55%) than 11. The 5-isoxazolol isomer of 10, compound 12, showed a reduced receptor affinity (IC50 = 26 +/- 7 microM) and a very low relative efficacy. Substitution of propanoic or propenoic acid moieties for the acidic heterocyclic units of these compounds gave the monocyclic amino acids 15-18, which have very little or no affinity for GABAA receptor sites.

Central auditory aging: GABA changes in the inferior colliculus.

Age-related hearing loss (presbycusis) is a complex state that reflects pathologic changes along the entire auditory neuraxis. Loss of speech understanding, decreased ability to localize sounds, and a decreased ability to detect and extract signals in noise are characteristic problems encountered by the elderly. Central (neural) presbycusis frequently results in a dramatic loss in speech understanding without a parallel change in pure-tone thresholds. In spite of evidence that suggests these deficits cannot be fully explained by peripheral changes alone, few studies have examined the neurochemical basis of central auditory dysfunction in aging. Age-related alterations in neural circuits involved in the processing of acoustic information could reflect changes in the synthesis, degradation, uptake, release, and receptor sensitivity of neurotransmitters, perhaps secondary to cell loss and/or progressive deafferentation. A series of studies designed to test this hypothesis has examined aging in the central auditory system of the F344 rate. Age-related changes associated with GABA neurotransmitter function in an important auditory midbrain structure, the inferior colliculus, have been investigated. These studies found: (1) decreased numbers of GABA immunoreactive neurons; (2) decreased basal levels (concentrations) of GABA; (3) decreased GABA release; (4) decreased glutamic acid decarboxylase activity; (5) decreased GABAB receptor binding; (6) decreased numbers of presynaptic terminals; and (7) subtle GABAA receptor binding changes. Collectively, these age-related changes suggest altered GABA neurotransmitter function in the IC. Identification of specific neurotransmitter changes in structures important in speech processing could eventually lead to the development of pharmacotherapy for selective types of age-related hearing loss.

Repeated administration of desipramine and a GABAB receptor antagonist, CGP 36742, discretely up-regulates GABAB receptor binding sites in rat frontal cortex.

1. GABAB receptor binding site densities within laminar regions of the rat frontal cortex were examined autoradiographically following repeated administration (21 days) of the antidepressants desipramine, paroxetine and amitriptyline in addition to the GABAB receptor antagonists, CGP 35348 and CGP 36742. beta 1-Adrenoceptor autoradiography was studied in parallel with that for GABAB receptor sites. 2. The effects of these compounds were examined concomitantly on the GABAB receptor-mediated inhibition of forskolin- and enhancement of noradrenaline-stimulated cyclic AMP production. 3. GABAB receptor binding was increased by both desipramine (20 mg kg-1, p.o. and 10 mg kg-1, i.p.) and CGP 36742 (100 mg kg-1, i.p.) in the outer laminar region of the frontal cortex by around 50% above control levels. Conversely, no significant changes were mediated by paroxetine, amitriptyline, CGP 35348 or the GABAB receptor agonist, baclofen. 4. With the exception of paroxetine, all compounds down-regulated the total beta-adrenoceptor population throughout frontal cortical laminae which was attributable to the beta 1-adrenoceptor subtype. In contrast, the reduction in beta-adrenoceptors mediated by CGP 35348 and CGP 36742 did not occur as a consequence of reduced beta 1-adrenoceptor numbers. 5. Protracted treatment with CGP 35348, failed to influence forskolin-stimulated cyclic AMP production; however, a significant increase in the accumulation of cyclic AMP produced in response to forskolin was seen after treatment with CGP 36742. 6. Such discretely localized changes in GABAB receptor densities induced by desipramine and CGP 36742 may provide an explanation for the discrepancies reported in membrane binding studies and possibly implicate a role for GABAB receptor antagonists in antidepressant therapy.

Spinal cord transection produces a long‐term increase in GABAB binding in the rat substantia gelatinosa

Quantitative autoradiography was used to determine changes in GABAB receptor binding in the substantia gelatinosa of the lumbar spinal cord at 4 days and at 6 weeks after a midthoracic spinal transection in rats. In the 4 day lesion animals, there was no significant change in either the density or the affinity of the GABAB binding. At 6 weeks, however, there was a 35% increase in binding density, with no significant change in affinity. The results suggest that alterations in spinal synaptic mechanisms can slowly evolve following loss of descending input to the spinal cord.

Effects of pentobarbital tolerance to and dependence on GABAB receptor-binding.

Effects of pentobarbital pellet implantation on [3H]baclofen binding in the frontal cortex of cerebellum of rat brains were examined. In the frontal cortex, pentobarbital tolerance caused an increase in the number of binding sites (Bmax) without changing their affinity (KD). Twenty-four hours after withdrawal of the pentobarbital pellets, there was a significant increase in the KD and Bmax values. Cerebellar binding, in contrast, was not significantly changed in any of the treatment groups. Addition of 1 mM of pentobarbital directly to binding assays using cortical membrane produced as increase in KD without a change in Bmax. In vitro, pentobarbital affected neither the KD nor the Bmax in the cerebellar [3H]baclofen binding. These results suggest that like the GABAA receptor, [3H]baclofen binding to the GABAB receptor in rat frontal cortex was affected by pentobarbital tolerance and dependence, and that there are regional differences in the properties of the GABAB receptor.

Pharmacological and biochemical characteristics of partially purified GABAB receptor.

Pharmacological and biochemical characteristics of the partially purified gamma-aminobutyric acid (GABA)B receptor using baclofen affinity column chromatography have been examined. The Scatchard analysis of [3H]GABA binding to the purified GABAB receptor showed a linear relationship and the KD and Bmax values were 60 nM and 118 pmol/mg of protein, respectively. Although GTP and Mg2+ did not affect on the GABAB receptor binding, Ca2+ significantly increased [3H]GABA binding to the purified GABAB receptor in a dose-dependent manner and showed its maximum effect at 2 mM. The enhancement of the binding by Ca2+ was found to be due to the increase of Bmax by the Scatchard analysis. The treatments with pronase and trypsin significantly decreased the binding of [3H]GABA, but phospholipase A2 had no significant effect on the binding. In addition, treatment with glycosidases such as glycopeptidase A and beta-galactosidase significantly decreased the binding of [3H]GABA to the purified GABAB receptor. These results suggest that purification of the solubilized GABAB receptor by the affinity column chromatography may result in the functional uncoupling of GABAB receptor with GTP-binding protein. Furthermore, the present results suggest that cerebral GABAB receptor may be a glycoprotein and membrane phospholipids susceptible to phospholipase A2 treatment may not be involved in the exhibition of the binding activity.