GABA(B) receptor activation inhibits dopamine D1 receptor-mediated facilitation of [(3)H]GABA release in substantia nigra pars reticulata

GABA(B) receptors inhibit and dopamine D1 receptors stimulate the release of GABA from striatal terminals in the pars reticulata of the substantia nigra. Here we have studied the interaction between both classes of receptors by exploring the effect of GABA(B) receptors upon the stimulation of depolarization-induced [(3)H]GABA release induced by the activation of D1 receptors in slices of the pars reticulata of the rat substantia nigra. The activation of GABA(B) receptors with baclofen (100 microM) inhibited by 48+/-8% the evoked [(3)H]GABA release in normal slices but did not modify the release in slices from reserpine-treated rats, indicating that the inhibition was dependent on endogenous dopamine. The inhibitory effect of baclofen was also abolished by the D1 receptor antagonist SCH 23390 (1 microM), indicating a D1 receptor-dependence of the baclofen inhibition. Baclofen dose-dependently inhibited (IC(50)=3.6 microM) the stimulation of release induced by the D1 agonist SKF 38393 (1 microM). Baclofen also blocked the stimulation of release induced by forskolin but not that induced by 8-Br-cAMP, indicating that the inhibitory effect was exerted before cAMP synthesis. N-ethylmaleimide (NEM), a selective inactivator of PTX-sensitive G-proteins, abolished the baclofen inhibition of the SKF 38393-induced stimulation of the release without affecting the stimulation induced by the D1 agonist, suggesting that the baclofen effect was mediated by Galpha(i/o) proteins. These results might have relevance in the control motor disorders associated with D1 receptor supersensitivity.     

Characterization of [(3)H]-CGP54626A binding to heterodimeric GABA(B) receptors stably expressed in mammalian cells

Functional human GABA(B(1a,2)) and GABA(B(1b,2)) receptors have been stably expressed in mammalian CHO K1 cells. Detailed characterization of GABA(B) ligand binding at each of the receptors has been compared using [(3)H]-CGP54626A. In cell membranes fractions, [(3)H]-CGP54626A bound to a single site with a K(D) of 1. 51+/-1.12 nM, B(max) of 2.02+/-0.17 pmoles mg protein(-1) and 0. 86+/-0.20 nM, B(max) of 5.19+/-0.57 pmoles mg protein(-1) for GABA(B(1a,2)) and GABA(B(1b,2)) respectively. In competition binding assays the rank order was identical for both GABA(B) receptors. For known GABA(B) agonists the rank order was CGP27492>SKF97541=CGP46381>GABA>Baclofen and for GABA(B) antagonists the rank order was CGP54262A>CGP55845>CGP52432>SCH 50911>CGP51176>CGP36742=CGP35348 > or =2-OH Saclofen > or =ABPA. The allosteric effect of calcium cations was also investigated. The effect of removal of CaCl(2) from the binding assay conditions was ligand dependent to either cause a decrease in ligand affinity or to have no significant effect. However, these effects were similar for both GABA(B) receptors. A whole cell, scintillation proximity binding assay was used to determine agonist affinity at exclusively heterodimeric GABA(B) receptors. In competition assays, the rank order was the same for both GABA(B(1a,2)) and GABA(B(1b,2)) and consistent with that seen with cell membrane fractions. These data suggest that, in terms of ligand binding, the currently identified isoforms of the GABA(B) receptor are pharmacologically indistinguishable.     

Properties of a high affinity binding site for [3H]avermectin B1a

The specific high affinity binding of [3H]avermectin B1a was investigated in membranes from several rat brain regions. Binding occurred rapidly, was reversible and partially dependent on the presence of chloride ions in the incubation medium. Specific high affinity binding of [3H]avermectin B1a was partially inhibited by GABA receptor agonists and this effect was blocked by GABA receptor antagonists. Pentobarbital and etazolate inhibited, and picrotoxin, picrotoxinin and IPTBO stimulated high affinity binding of [3H]avermectin B1a. All these effects were influenced by the presence of chloride ions in the incubation medium. The results indicate that the high affinity binding site of [3H]avermectin B1a is associated with the GABA-benzodiazepine receptor-chloride ion channel complex.     

Specific [(3)H]-guanosine binding sites in rat brain membranes

1. Extracellular guanosine has diverse effects on many cellular components of the central nervous system, some of which may be related to its uptake into cells and others to its ability to release adenine-based purines from cells. Yet other effects of extracellular guanosine are compatible with an action on G-protein linked cell membrane receptors. 2. Specific binding sites for [(3)H]-guanosine were detected on membrane preparations from rat brain. The kinetics of [(3)H]-guanosine binding to membranes was described by rate constants of association and dissociation of 2.6122 x 10(7) M(-1) min(-1) and 1.69 min(-1), respectively. A single high affinity binding site for [(3)H]-guanosine with a K(D) of 95.4 +/- 11.9 nM and B(max) of 0.57 +/- 0.03 pmol mg(-1) protein was shown. This site was specific for guanosine, and the order of potency in displacing 50 nM [(3)H]-guanosine was: guanosine=6-thio-guanosine > inosine > 6-thio-guanine > guanine. Other naturally occurring purines, such as adenosine, hypoxanthine, xanthine caffeine, theophylline, GDP, GMP and ATP were unable to significantly displace the radiolabelled guanosine. Thus, this binding site is distinct from the well-characterized receptors for adenosine and purines. 5. The addition of GTP produced a small concentration-dependent decrease in guanosine binding, suggesting this guanosine binding site was linked to a G-protein. 6. Our results therefore are consistent with the existence of a novel cell membrane receptor site, specific for guanosine.     

Flunitrazepam photoaffinity labeling of the GABA(A) receptor reduces inhibition of [3H]Ro15-4513 binding by GABA

The benzodiazepine drugs modulate gamma-aminobutyric acid (GABA)-mediated synaptic transmission via a high-affinity binding site that is part of the GABA(A) receptor complex, but which is distinct from the GABA binding site. Ro15-4513 is a benzodiazepine negative modulator of GABA action that displays unique anti-ethanol properties both in vivo and in vitro. Ro15-4513 has been reported to photoaffinity label nearly 100% of the benzodiazepine binding sites in rat brain homogenates. In contrast, the benzodiazepine positive modulator flunitrazepam photoaffinity labels only 25% of the sites. Here, we have examined the reversible binding of [3H]Ro15-4513, [3H]flumazenil (Ro15-1788), and [3H]flunitrazepam to embryonic chick brain membranes, and to membranes that have been photoaffinity labeled with nonradioactive flunitrazepam. Photoaffinity labeling with flunitrazepam decreased the subsequent reversible binding of [3H]flunitrazepam and [3H]flumazenil, but increased the binding of [3H]Ro15-4513. The increase in [3H]Ro15-4513 binding after flunitrazepam photoaffinity labeling was due to a decrease in the apparent Kd, with no change in Bmax. Following photoaffinity labeling, negative modulation of [3H]Ro15-4513 binding by GABA was lost, whereas positive modulation of residual [3H]flunitrazepam binding was retained. We conclude that the site photoaffinity labeled by flunitrazepam is distinct from the site responsible for reversible binding of [3H]Ro15-4513.     

Distribution of [(3)H]BU224, a selective imidazoline I(2) binding site ligand,in rat brain

BU224 (2-(4,5-dihydroimidaz-2-yl)-quinoline) is a selective imidazoline I(2) binding site ligand characterised in both competition binding assays and functional studies. However, in some studies, BU224 has been reported to have a different functional effect from that seen with another selective imidazoline I(2) binding site ligand 2-BFI (2-(2-benzofuranyl)-2-imidazoline). This effect may reflect differing efficacies of the ligands or a difference in their brain distribution. The present study has investigated the distribution of the tritiated form of BU224 in rat brain and correlated this distribution with other imidazoline I(2) binding site ligands, [(3)H]idazoxan and [(3)H]2-BFI. Saturation studies revealed binding of [(3)H]BU224 was of high affinity and saturable. The central distribution of [(3)H]BU224 was similar to that previous reported for imidazoline I(2) binding site in rat brain. Autoradiography revealed that the highest levels of binding were in the arcuate nucleus, interpeduncular nucleus, area postrema, pineal gland and ependymal cell layer lining the ventricles. Correlation analysis of the binding distribution with our previous published studies revealed a highly significant correlation between [(3)H]BU224 and both [(3)H]idazoxan (r=0.94) and [(3)H]2-BFI (r=0.96). These data indicate [(3)H]BU224 labels the same population of imidazoline I(2) binding site in rat brain as seen with [(3)H]idazoxan and [(3)H]2-BFI. Therefore, the differences in functional effects observed with these compounds may reflect agonist and antagonist properties.     

Distinct binding properties of the AT(1) receptor antagonist [(3)H]candesartan to intact cells and membrane preparations

[(3)H]-2-Ethoxy-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]-1H-benzimidazoline-7-carboxylic acid ([(3)H]candesartan), a non-peptide angiotensin II type 1 receptor (AT(1) receptor) antagonist bound with high affinity and specificity to intact adherent human AT(1) receptor transfected Chinese hamster ovary cells. The binding characteristics were preserved when cells were suspended, but the dissociation was 3-4-fold faster and the affinity 2-fold lower, while examining [(3)H]candesartan binding to cell membranes. These data suggested the role of the intracellular organisation of living CHO-hAT(1) cells in antagonist-AT(1) receptor interactions. Yet, a specific role of microtubule or actin filaments of the cytoskeleton, receptor phosphorylation by Protein Kinase C, membrane polarity, cytoplasmic components like ATP and the need of an intact cell membrane could be excluded. The potential effect of protease degradation or receptor oxidation during the membrane preparation was also unlikely. The dissociation rate and the equilibrium dissociation constant of [(3)H]candesartan increased with the temperature for both intact cells and membranes. Thermodynamic studies suggested that the bonds between candesartan and the hAT(1) receptor may be of different nature in intact CHO-hAT(1) cells and membranes thereof. Whereas the binding was almost completely enthalpy-driven on intact cells, there was a mixed contribution of both enthalpy and entropy on membranes.     

Changes in [(3)H]glibenclamide binding to mouse forebrain membranes during morphine tolerance

The characteristics of specific binding of the ATP-sensitive K(+) (K(ATP)) channel blocker [3H]glibenclamide to forebrain membranes (P(2) fraction, 4 degrees C) obtained from morphine-naive and -tolerant mice were evaluated. Morphine tolerance was induced by osmotic minipumps that released 45 mg/kg/day of morphine subcutaneously for 6 days. This treatment enhanced the antinociceptive ED(50) of morphine without changing its E(max). In morphine-naive animals, (1) both the association and the dissociation of [3H]glibenclamide were biphasic; (2) [3H]glibenclamide was displaced by other sulfonylureas (order of potency: glibenclamide>glipizide&z.Gt;tolbutamide) with pseudo-Hill coefficients lower than unity and biphasic Hofstee plots; and (3) Scatchard plots of saturation experiments were curvilinear, showed a Hill coefficient of 0.81+/-0.04 and suggested the presence of two binding sites with a K(D) of 0.13 and 3.17 nM and a B(max) of 12.30 and 84.47 fmol/mg protein, respectively. By contrast, in membranes obtained from morphine-tolerant animals, (1) the Scatchard plots showed only one population of binding sites with a K(D) of 0.87 nM and a B(max) of 77.99 fmol/mg protein, and the Hill coefficient was very close to unity (0.96+/-0.1); (2) competition experiments (using glibenclamide as displacer) showed a pseudo-Hill coefficient of 0.99+/-0.04; and (3) dissociation experiments showed only one phase of dissociation. These results suggest that [3H]glibenclamide binds to two different sites in membranes obtained from morphine-naive animals, but to only one site in morphine-tolerant animals. Consequently, it seems that morphine tolerance in mice involves adaptive changes in K(ATP) channels.     

GABA preincubation of rat brain sections increases [3H]GABA binding to the GABAA receptor and compromises the modulatory interactions

Receptor autoradiography has been employed to investigate the effect of gamma-aminobutyric acid (GABA) preincubation on the interaction of the GABAA receptor with its ligands. [3H]GABA (50 nM) binding to the GABAA receptors is increased by 60% compared to control sections after GABA (100 microM) preincubation. Receptor autoradiography shows that the increase is more pronounced in certain brain areas. The allosteric interactions between the GABA and benzodiazepine recognition sites were also examined. An increase in [3H]GABA (50 nM) binding to rat brain sections by co-incubation with the benzodiazepine, flunitrazepam (FNZ) has been observed autoradiographically. This effect has been quantitated in several brain regions; the overall brain increase in [3H]GABA binding induced by 1 microM FNZ was 20%. The increase in [3H]FNZ (1 nM) binding by co-incubation with GABA has also been observed autoradiographically, and the effect quantitated in four brain regions. The overall brain increase in [3H]FNZ binding induced by 100 microM GABA was 34%. After GABA preincubation these allosteric responses are significantly reduced in size. The increase in the [3H]GABAA binding as a consequence of GABA preincubation appears to reflect an increase in receptor affinity for [3H]GABA with no significant change in the maximum number of binding sites. We suggest that GABA preincubation converts the GABAA receptor to a higher affinity desensitised receptor conformation.     

Allosteric modulation of [(3)H]gabapentin binding by ruthenium red

Gabapentin is an anticonvulsant with an unknown mechanism of action. However, it has been proposed that gabapentin acts by binding to voltage-gated calcium channels. To further characterize the interaction of gabapentin with its endogenous binding site in cerebral cortex, we tested for competitive and allosteric interactions between [(3)H]gabapentin and a variety of calcium channel binding ligands. Most ligands for voltage- or ligand-gated calcium channels (verapamil, the omega-conotoxins MVIIC and GVIA, ryanodine, caffeine, capsaicin, MK-801) had no significant effect on [(3)H]gabapentin binding. However, ruthenium red, a relatively nonselective calcium channel ligand, was found to robustly modulate [(3)H]gabapentin binding. Ruthenium red slowed the association and dissociation kinetics of [(3)H]gabapentin while increasing the number of detectable binding sites. Spermine and MgCl(2), which also bind to calcium channels and modulate [(3)H]gabapentin binding, were found to act in a similar manner. These findings support the contention that the principal endogenous binding site for gabapentin is a calcium channel; they characterize the nature of the allosteric interaction of spermine, MgCl(2) and ruthenium red with this binding site; and they suggest possible mechanisms by which gabapentin may modulate calcium channel function and ultimately produce therapeutic actions.     

Regional localization and developmental profile of acetylcholinesterase-evoked increases in [(3)H]-5-fluororwillardiine binding to AMPA receptors in rat brain

In addition to its role in hydrolyzing the neurotransmitter acetylcholine, the synaptically enriched enzyme acetylcholinesterase (AChE) has been reported to play an important role in the development and remodelling of neural processes and synapses. We have shown previously that AChE causes an increase in binding of the specific AMPA receptor ligand (S)-[(3)H]-5-fluorowillardiine ([(3)H]-FW) to rat brain membranes. In this study we have used quantitative autoradiography to investigate the regional distribution and age-dependence of AChE-evoked increases in the binding of [(3)H]-FW in rat brain. Pretreatment of rat brain sections with AChE caused a marked enhancement of [(3)H]-FW binding to many, but not all, brain areas. The increased [(3)H]-FW binding was blocked by the specific AChE inhibitor BW 284c51. The maximal potentiation of [(3)H]-FW binding occurred at different developmental age-points in different regions with a profile consistent with the peak periods for synaptogenesis in any given region. In addition to its effects on brain sections, AChE also strongly potentiated [(3)H]-FW binding to detergent solubilized AMPA receptors suggesting a direct action on the receptors themselves rather than an indirect effect on the plasma membrane. These findings suggest that modulation of AMPA receptors could provide one molecular mechanism for the previously reported effects of AChE on synapse formation, synaptic plasticity and neurodegeneration.     

(+)-[3H]3-(3-hydroxyphenyl)-N-(1-propyl)-piperidine binding to sigma receptors in mouse brain in vivo

Binding of i.v. administered (+)-[3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ([3H]3-PPP) in the brain of intact mice is antagonized dose responsively by sigma receptor ligands. The correlation of potencies for inhibition of binding in vivo and in vitro indicates that sigma receptors in mouse brain are labeled in vivo by i.v. [3H]3-PPP. 3-PPPP, the N-phenylpropyl derivative of norpropyl-3-PPP exhibits very high affinity for sigma receptors in vitro and in vivo.     

Synaptosomal [3H]GABA uptake and [3H]nipecotic acid binding in audiogenic seizure susceptible (DBA/2) and resistant (C57 B1/6) mice

The kinetics of [3H]GABA uptake into synaptosomes prepared from the cerebral cortex, cerebellum and pons-medulla of DBA/2 (audiogenic seizure susceptible) and C57 B1/6 (audiogenic seizure resistant) mice were determined at various ages. Vmax of uptake decreased with age in all regions of both strains, Km was unchanged. There were no regional differences in the Vmax between strains at any age but Km was lower in DBA/2 mice at 21-23 days in cerebellum and at 40-43 days in pons-medulla. There were no strain differences in the binding of [3H]nipecotic acid to crude cortical membrane fractions at any age. The age related audiogenic seizure susceptibility of DBA/2 mice is not related to an abnormality of neuronal GABA uptake.     

Pharmacological re-evaluation of a GABA(B) receptor antagonist CGP 47332A in rat brain

In rat neocortical slices maintained in Mg(2+)-free Krebs medium, the gamma-aminobutyric acid (GABA(B)) receptor agonist baclofen concentration-dependently depressed the frequency of spontaneous discharges (EC(50)=12 microM). This was reversibly antagonised by (R, S)-3-amino-2-hydroxy-propyl-P-n-butyl-phosphinic acid (CGP 47332A) (25, 100, 300 microM) which produced rightwards shifts of the baclofen concentration-response curves (pA(2) value=4.8+/-0.1). In electrically stimulated slices preloaded with [3H]GABA, CGP 47332A increased its release (EC(150)=100 microM) through antagonism of GABA(B) autoreceptors. Although CGP 47332A was some six times weaker on GABA(B) auto- than on heteroreceptors, yet its congener lacking the beta-hydroxy substituent displays equal potency in both binding (IC(50)=38 microM) and GABA(B) autoreceptor functional studies (EC(150)=38 microM) as previously reported [Froestl, W., Mickel, S.J. , Von Sprecher, G., Diel, P.J., Hall, R.G., Maier, L., Strub, D., Melillo, V., Baumann, P.A., Bernasconi, R., Gentsch, C., Hauser, K., Jaekel, J., Karlsson, G., Klebs, K., Maitre, L., Marescaux, C., Pozza, M.F., Schmutz, M., Steinmann, M.W., Van Riezen, H., Vassout, A., Mondadori, C., Olpe, H.R., Waldmeier, P.C., Bittiger, H., Phosphinic acid analogues of GABA: 2. Selective, orally active GABA(B) antagonists. J. Med. Chem. 38 (1995) 3313-3331.].     

Identification of a novel nicotinic binding site in mouse brain using [(125)I]-epibatidine

[(125)I]-Epibatidine binds to multiple nicotinic acetylcholine receptor (nAChR) subtypes with high affinity. In this study, [(125)I]-epibatidine was used to label and characterize a novel nAChR subtype found in mouse brain inferior colliculus, interpeduncular nucleus, and olfactory bulb homogenates. Binding of [(125)I]-epibatidine was saturable and apparently monophasic in each brain region (K:(D:)=71+/-12 pM mean+/-s.e.mean across regions) but inhibition of [(125)I]-epibatidine binding (200 pM) by A85380, cytisine and (-)-nicotine was biphasic, indicating the presence of multiple binding sites. The sites with lower agonist affinity comprised 30.0+/-2.2, 58.6+/-0.1 and 48.7+/-3.3% of specific [(125)I]-epibatidine (200 pM) binding in inferior colliculus, interpeduncular nucleus, and olfactory bulb homogenates, respectively. The affinity difference between A85380-sensitive and -resistant binding sites was particularly marked (approximately 1000 fold). Thus A85380 was used to differentiate agonist-sensitive and -resistant sites. The pharmacological profiles of the A85380-resistant sites in each region were assessed with inhibition binding experiments, using 14 agonists and five antagonists. The profiles were indistinguishable across regions, implying that A85380-resistant [(125)I]-epibatidine binding sites in inferior colliculus, interpeduncular nucleus, and olfactory bulb represent a single nAChR subtype. The pharmacological profile of the A85380-resistant sites is very different from that previously reported for high affinity (-)-[(3)H]-nicotine-, [(125)I]-alpha-bungarotoxin-, or [(125)I]-alpha-conotoxin MII-binding sites, suggesting that they represent a novel nAChR population in mouse brain.     

The insecticide fipronil and its metabolite fipronil sulphone inhibit the rat alpha1beta2gamma2L GABA(A) receptor

Background and purpose:

Fipronil is the active ingredient in a number of widely used insecticides. Human exposure to fipronil leads to symptoms (headache, nausea and seizures) typically associated with the antagonism of GABA_A receptors in the brain. In this study, we have examined the modulation of the common brain GABA_A receptor subtype by fipronil and its major metabolite, fipronil sulphone.

Experimental approach:

Whole-cell and single-channel recordings were made from HEK 293 cells transiently expressing rat α1β2γ2L GABA_A receptors.

Key results:

The major effect of fipronil was to increase the rate of current decay in macroscopic recordings. In single-channel recordings, the presence of fipronil resulted in shorter cluster durations without affecting the intracluster open and closed time distributions or the single-channel conductance. The α1V256S mutation, previously shown alleviate channel inhibition by inhibitory steroids and several insecticides, had a relatively small effect on channel block by fipronil. The mode of action of fipronil sulphone was similar to that of its parent compound but the metabolite was less potent at inhibiting the α1β2γ2L receptor.

Conclusions and implications:

We conclude that exposure to fipronil induces accumulation of receptors in a novel, long-lived blocked state. This process proceeds in parallel with and independently of, channel desensitization. The lower potency of fipronil sulphone indicates that the conversion serves as a detoxifying process in mammalian brain.     

Inhibition of [3H]GABA binding to rat brain synaptic membranes by bicuculline related alkaloids

The binding of 45 bicuculline related phthalideisoquinoline alkaloids to the GABAA receptor was studied using rat brain synaptic membranes prepared both in Tris-HCl and in Tyrode buffers. The IC50 values determined in Tyrode for phthalideisoquinolines are lower (by about one order of magnitude) than and correlate well (r2 = 0.95) with the IC50 data obtained by [3H]GABA displacement in Tris-HCl. Applying Tyrode, the activities of GABA agonists relative to Tris-HCl are decreased. It can be recognized that activities in receptor binding are dependent on the conformations phthalideisoquinolines prefer in solution. On the basis of systematic alterations in the phthalideisoquinoline molecule the main structural elements involved in the binding of phthalideisoquinoline alkaloids appear to be identical with those of GABA agonists, suggesting that the same binding conformation of the GABAA receptor may be implicated for both agonists and antagonists. The opposite shift in relative potencies of agonists and antagonists may be the consequence of an alteration in the "ionic status" rather than that in the conformation of the GABAA receptor.     

Comparative aspects of cerebellar [3H]flunitrazepam and [3H]GABA binding

[3H]Flu and [3H]GABA binding has been studied in mice and Rabbit cerebellum (Ce). The Bmax of [3H]Flu binding in Ce membranes is similar in all mice strains examined and Rabbit. However, in Ce homogenate there are significant differences in both kd and Bmax. Subcellular distribution shows higher [3H]Flu binding in the nuclear than in the membranous fraction. However, the [3H]GABA binding is lower in the nuclear than the membranous fraction.