Autoradiographic Characterisation of [35S]GTPγS Binding Sites in Rat Brain

The binding of [³⁵S]GTPS was characterised with autoradiography in rat brain. The binding was saturable, but the rate of dissociation was very slow. Analysis of binding isotherms revealed one class of binding sites with a Kd of 0.8 M. The specific binding was 98%. Different guanine nucleotides were all able to compete with [³⁵S]GTPS binding. However, no displacement was seen by the ATP-analogue App[NH]p, indicating that [³⁵S]GTPS does not bind to ATP-sites. Autoradiograms showed a highly homogenous distribution of [³⁵S]GTPS binding, in grey as well as in white matter. However, the pattern changed dramatically in the presence of GTP, which, unlike the non-hydrolysable GTP-analogues Gpp[NH]p and GTPS, did not displace [³⁵S]GTPS binding throughout the brain. In white matter areas the binding was potently displaced, while in many grey matter areas, e.g., the striatum, the binding was seen to increase. This GTP-induced increase in [³⁵S]GTPS binding was strongly Mg²⁺-dependent, with an optimum at 10 mM. This, together with the finding that the regional effects of GTP correspond well to previously reported distribution of low Km GTPase, suggest that the levels of binding of [³⁵S]GTPS in the presence of GTP may reflect functional G-protein activity.     

Physicochemical Modulation of Agonist-Induced [35S]GTPγS Binding: Implications for Coexistence of Multiple Functional Conformations of Dopamine D1-Like Receptors

Dopamine agonist-stimulated [³⁵S]GTPγS binding to membrane G proteins was studied in select brain regions under experimental conditions that permit the activation of receptor coupling to the G proteins G_i, G_s, or G_q. Agents studied were agonists known to be effective at various dopamine receptor/effector systems and included quinelorane (D₂-like/G_i), SKF38393 (D₁-like/G_q, D₁-like/G_s), SKF85174 (D₁-like/G_s), and SKF83959 (D₁-like/G_q). Dopamine and SKF38393 significantly stimulated [³⁵S]GTPγS binding to normal striatal membranes by 161% and 67% above controls. Deoxycholate, which enhances agonist-induced phospholipase C (PLC) stimulation, markedly enhanced the agonistic effects of dopamine and SKF38393 to 530% and 637% above controls, respectively. The enhancing effects of deoxycholate were reversed if it was washed off the membranes before agonist addition. The thiol-reducing agent, dithiothreitol, completely abolished the effects of SKF38393 and SKF83959, whereas SKF85174 effects were augmented. Agonist responses were concentration-related, and highest efficacies were obtained in the hippocampus, thus paralleling both the brain regional distribution and agonist efficacies previously observed in phosphoinositide hydrolysis assays. These findings suggest that D₁-like receptor conformations that mediate agonist stimulation of G_s/adenylylcyclase may be structurally different from those that mediate G_q/PLC activation. Although the exact mechanism of deoxycholate's effect awaits elucidation, the results are consistent with the emerging concept of functional selectivity whereby deoxycholate could create a membrane environment that facilitates the transformation of the receptor from a conformation that activates G_s/adenylylcyclase to one that favors G_q/PLC signaling.     

Lysophosphatidylinositol Stimulates [<Superscript>35</Superscript>S]GTPγS Binding in the Rat Prefrontal Cortex and Hippocampus

Lysophosphatidylinositol (LPI) is a biologically active lipid that produces a number of responses in cultured cells, and has been suggested to have neuroprotective properties in vivo. Some of the actions of LPI are mediated by G-protein coupled receptors, but it is not known whether G-protein coupled receptor-mediated responses can be seen in intact brain tissue. In consequence, in the present study, we investigated autoradiographically whether LPI increased the [³⁵S]GTPγS binding level in brain tissue slices. In standard assay conditions, where as a positive control a robust response to cannabinoid receptor activation by the agonist ligand CP55,940 was seen, there was no increase in the autoradiographic density over basal produced by LPI. However, when the conditions were modified (incubation at 4°C rather than at 25°C, incubation time increased to 3 h, GDP concentration reduced from 2 to 0.1 mM), a significant increase in [³⁵S]GTPγS autoradiographic density in response to 10 μM LPI was seen in the prefrontal cortex, hippocampus, and cortex at the level of the hippocampus, although the degree of increase was small and very variable. No significant increases were seen in the hypothalamus or cerebellum. It is concluded that LPI, in the right conditions, can activate a sufficient number of G-proteins in the rat prefrontal cortex and hippocampus to produce a response in the [³⁵S]GTPγS autoradiographic assay of G-protein coupled receptor function.     

α2A-Adrenoceptor-specific Stimulation of [35S]GTPγS Binding to Membrane Preparations of Rat Frontal Cortex

Functional activation of α_2A adrenergic receptors in the crude membranes from rat frontal cortex was studied by a [³⁵S]-guanosine 5′-O-(γ-thiotriphosphate) ([³⁵S]GTPγS) binding assay. α_2A agonists UK14304 and guanfacine decreased the ability of GDP to compete with [³⁵S]GTPγS binding to the membranes and 0.1 mM GDP was found to be optimal for the following functional experiments. However, even after careful optimization of experimental conditions the specificity of ligands for rat α₂ adrenoceptors were not sufficient, as agonists as well as antagonists became activators of other signal transduction systems before achieving their maximal effect in the α_2A-adrenergic system. Only using compromising concentration of agonist (up to 1 μM UK14304) and antagonist (up to 1 μM RS79948) to inhibit agonist’s effect, allowed us to filtrate out α_2A specific effect for characterization of signal transduction in rat frontal cortex membranes for the comparison efficacies of this system for different animals from behavioral experiments.     

Optimized Binding of [35S]GTPγS to Gq-Like Proteins Stimulated with Dopamine D1-Like Receptor Agonists

Subtypes of dopamine D₁-like receptors are coupled through the G proteins Gs or Gq to stimulate either adenylate cyclase or phospholipase C signaling cascades. In the present study, we have uncovered the marked enhancement by sodium deoxycholate of D1-like agonist-stimulated [³⁵S]GTPS binding to Gq-like G proteins in brain membranes, and determined the optimal experimental conditions for assessing agonist effects on [³⁵S]GTPS binding in the presence of the detergent. Factors and their optimal levels that were found to significantly enhance the sensitivity and robustness of the agonist-stimulated [³⁵S]GTPS binding reaction include protein concentration at 40 g/ml, cationic concentrations of 120 mM Na⁺, 1.8 mM K⁺, and 20 mM Mg²⁺, a molar guanine nucleotide ratio of 100,000 GDP to [³⁵S]GTPS, the presence of 1 mM deoxycholate, and an overall incubation duration of 30–120 min. Under the optimized conditions, the D₁-like agonist SKF38393 induced potent and highly efficacious (up to 1000%) stimulation of [³⁵S]GTPS binding in membrane preparations from the striatum and other rat brain regions. In striatal membranes incubated with drug for 2 h, immunoprecipitation of the [³⁵S]GTPS-bound proteins with specific G antibodies showed that at least 70% of SKF38393-stimulated [³⁵S]GTPS binding was to Gq. The present reaction parameters are consistent with conditions previously found to support dopaminergic stimulation of phospholipase C-mediated signaling in brain slice preparations. These results imply that different but equally physiologically relevant conditions can be obtained under which subtypes of dopaminergic receptors may couple preferentially to Gs and the adenylate cyclase pathway or to Gq and the phospholipase C pathway.     

Comparison of [35S]GTPγS binding to plasma membranes and endomembranes prepared from soybean and rat

Summary Plasma membranes were prepared from soybean hypocotyls and roots by aqueous two-phase partitioning and subsequent free-flow electrophoresis. The highly purified plasma membranes bound [³⁵S]GTPS with a relatively high affinity (K_d10nM). The binding was saturable and specific as it was indicated by the displacement of bound [³⁵S]GTPS by unlabeled GTPS and GTP, but not by ATPS, ATP, UTP or CTP. ITP was intermediate in its ability to displace [³⁵S]GTPS. When soybean plasma membrane proteins were separated by SDS-PAGE and displayed by autoradiography, two major [³⁵S]GTPS binding proteins were revealed with apparent molecular weights of 24 and 28 kDa. Results with plasma membranes from soybean hypocotyls and roots were similar but differed from those with plasma membranes prepared from rat liver and adipocytes where only a single major [³⁵S]GTPS binding activity with a molecular weight of 28 kDa was observed.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - G protein hetero-trimeric GTP binding protein with , , subunits - G_n protein GTP binding protein detected on nitrocellulose blots - GTPS guanosine 5-[-thio]triphosphate - IAA 3-indoleacetic acid - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Europium-labeled GTP as a general nonradioactive substitute for [S]GTPγS in high-throughput G protein studies

[³⁵S]GTPγS, the nonhydrolyzable radioactive GTP analog, has been a powerful tool in G protein studies and has set the standards in this field of research. However, its radioactive nature imposes clear limitations to its use in regular laboratory practice and in high-throughput experimentation. The europium-labeled GTP analog (Eu-GTP) has been used as an alternative in the analysis of G protein activation by G protein-coupled receptors in cellular membrane preparations. Here we expand the usage of Eu-GTP and show that it can be applied in other types of assays where [³⁵S]GTPγS has been previously utilized. We demonstrate the applicability of the modified Eu-GTP binding technology to analysis of heterotrimeric and monomeric G proteins of natural and recombinant sources, from different organisms, in assays with soluble proteins and membrane-containing assays of a high-throughput format. The deci-nanomolar K_D of Eu-GTP for the tested G proteins is similar to that of other fluorescent-modified GTP analogs, while the sensitivity achieved in time-resolved fluorescence analysis of Eu-GTP exceeds that of the radioactive measurements. Overall, the results of our modified Eu-GTP binding assay present Eu-GTP as a general nonradioactive alternative for G protein studies, especially attractive in high-throughput experiments.     

Upregulation of NMDA Receptors in Hippocampus and Cortex in the Pentylenetetrazol-Induced “Kindling” Model of Epilepsy

Kindling is a phenomenon of epileptogenesis, which has been widely used as an experimental model of temporal lobe epilepsy. At the present work we investigated the contribution of NMDA receptors in the Pentylenetetrazol-induced kindling model in the mouse brain, by using quantitative autoradiography and the radioactive ligands [³H]MK801 and [³H]L-glutamate (NMDA-sensitive component). One week after establishment of kindling, a small but significant increase in [³H]MK801 as well as NMDA-sensitive [³H]glutamate binding was seen, being restricted to the molecular layer (ML) of the dentate gyrus (DG) and the CA3 region of the hippocampus. These binding augmentations persisted one month after establishment of kindling. A significant increase of NMDA receptor binding was also observed in the cortex-somatosensory and temporal one week after acquisition of the kindled state. The upregulation of NMDA receptors seen in DG and CA3 region of the hippocampus could be associated with the kindling process of this model especially with its maintenance phase, since it persists at long term, is area-specific and consistent with electrophysiological data. The increase of NMDA receptors seen in the cortex of the kindled animals could underlie the hyperexcitability detected by electrophysiological studies in this area.     

[35S]GTPγS binding: A tool to evaluate functional activity of a cloned opioid receptor transiently expressed in COS cells

In this paper we propose a powerful procedure to measure functional activation of the mouse δ-opioid receptor transiently expressed in mammalian cells. Receptor stimulation was assessed using a population of electroporated COS cells, transfected at a 50% efficiency. Under those conditions, agonist-promoted activation of the receptor was measured by [³⁵S]GTPγS binding. Both BW373U86, an alkaloid compound, and DADLE, a peptide agonist, elicited increase of specific [³⁵S]GTPγS binding representing 300% of basal level. Maximal activation was compared to that obtained for the cloned receptor stably expressed in CHO cells. Agonist efficacy was similar in both expressions systems, demonstrating the high sensitivity of the proposed method applied to transient expression. Finally dose-response curves were found highly reproducible across transfection experiments, opening the possibility for a direct comparison of distinct recombinant receptor preparations. This method represents a powerfull tool for the study of opioid signal transduction at the receptor level. It may also be extended to investigate signalling properties of other Gi/Go coupled receptors. Special issue dedicated to Dr. Eric J. Simon.     

The amphiphilic peptide adenoregulin enhances agonist binding to A1-adenosine receptors and [35S]GTPγS to brain membranes

Summary 1. Adenoregulin is an amphilic peptide isolated from skin mucus of the tree frog,Phyllomedusa bicolor. Synthetic adenoregulin enhanced the binding of agonists to several G-protein-coupled receptors in rat brain membranes.2. The maximal enhancement of agonist binding, and in parentheses, the concentration of adenoregulin affording maximal enhancement were as follows: 60% (20 µM) for A₁-adenosine receptors, 30% (100 µM) for A_2a-adenosine receptors, 20% (2 µM) for ₂-adrenergic receptors, and 30% (100 µM) for 5HT_1A receptors. High affinity agonist binding for A_1-, _2-, and 5HT_1A-receptors was virtually abolished by GTPS in the presence of adenoregulin, but was only partially abolished in its absence. Magnesium ions increased the binding of agonists to receptors and reduced the enhancement elicited by adenoregulin.3. The effect of adenoregulin on binding of N⁶-cyclohexyladenosine ([³H]CHA) to A₁-receptors was relatively slow and was irreversible. Adenoregulin increased the B_max value for [³H]CHA binding sites, and the proportion of high affinity states, and slowed the rate of [³H]CHA dissociation. Binding of the A₁-selective antagonist, [³H]DPCPX, was maximally enhanced by only 13% at 2 µM adenoregulin. Basal and A₁-adenosine receptor-stimulated binding of [³⁵S]GTPS were maximally enhanced 45% and 23%, respectively, by 50 µM adenoregulin. In CHAPS-solubilized membranes from rat cortex, the binding of both [³H]CHA and [³H]DPCPX were enhanced by adenoregulin. Binding of [³H]CHA to membranes from DDT₁ MF-2 cells was maximally enhanced 17% at 20 µM adenoregulin. In intact DDT₁ MF-2 cells, 20 µM adenoregulin did not potentiate the inhibition of cyclic AMP accumulation mediatedvia the adenosine A₁ receptor.4. It is proposed that adenoregulin enhances agonist binding through a mechanism involving enhancement of guanyl nucleotide exchange at G-proteins, resulting in a conversion of receptors into a high affinity state complexed with guanyl nucleotide-free G-protein.     

[3H]Epibatidine Photolabels Non-equivalent Amino Acids in the Agonist Binding Site of Torpedo and ��4��2 Nicotinic Acetylcholine Receptors

Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo α₂βγδ and expressed human α4β2 nAChRs with [³H]epibatidine and identified by Edman degradation the photolabeled amino acids. Irradiation at 254 nm resulted in photolabeling of αTyr¹⁹⁸ in agonist binding site Segment C of the principal (+) face in both α subunits and of γLeu¹⁰⁹ and γTyr¹¹⁷ in Segment E of the complementary (−) face, with no labeling detected in the δ subunit. For affinity-purified α4β2 nAChRs, [³H]epibatidine photolabeled α4Tyr¹⁹⁵ (equivalent to Torpedo αTyr¹⁹⁰) in Segment C as well as β2Val¹¹¹ and β2Ser¹¹³ in Segment E (equivalent to Torpedo γLeu¹⁰⁹ and γTyr¹¹¹, respectively). Consideration of the location of the photolabeled amino acids in homology models of the nAChRs based upon the acetylcholine-binding protein structure and the results of ligand docking simulations suggests that epibatidine binds in a single preferred orientation within the α-γ transmitter binding site, whereas it binds in two distinct orientations in the α4β2 nAChR.Nicotinic acetylcholine receptors (nAChRs)³ are prototypical members of the Cys loop superfamily of neurotransmitter-gated ion channels that mediate the actions of the neurotransmitter acetylcholine (1). nAChRs from vertebrate skeletal muscle and the electric organs of Torpedo rays are heteropentamers of homologous subunits with a stoichiometry of 2α:β:γ(ϵ):δ that are arranged pseudosymmetrically around central cation-selective ion channels (1, 2). There are 12 mammalian neuronal nAChR subunit genes: nine neuronal α subunits (α2–α10) and three neuronal β subunits (β2–β4). The α4β2 nAChR is the most abundant and widely distributed nAChR subtype expressed in the brain and is a major target for potential therapeutic agents for neurological diseases and conditions, including nicotine dependence and Alzheimer and Parkinson diseases (3, 4). Although the ratio of α4 to β2 subunit in vivo is uncertain, expressed receptors containing either three α4 or three β2 subunits have distinct pharmacological properties (5, 6).The agonist binding sites (ABS) of nAChRs are located within the amino-terminal extracellular domain at the interface of adjacent subunits (α-γ and α-δ in the Torpedo nAChR), and different nAChR subunit combinations form ABS with distinct physical and pharmacological properties (3, 7). Affinity labeling studies with Torpedo nAChR and site-directed mutational analyses of muscle and neuronal nAChRs identified key amino acids delineating the ABS from three noncontiguous stretches of the α subunit (Segments A-C, the principal component (+ face)) and three noncontiguous regions of the non-α subunit (Segments D–F, the complementary component (− face)) (8, 9). The three-dimensional structure of the ABS in the absence and presence of nAChR agonists or competitive antagonists has been determined for snail acetylcholine-binding proteins (AChBPs) that are soluble homopentamers homologous to the extracellular (amino-terminal) domain of a nAChR (10–12). In the AChBP, four aromatic amino acids from Segments A–C that are conserved within α subunits, along with a conserved Trp in Segment D, form a core aromatic “pocket” with a dimension optimal for accommodation of a trimethylammonium group. The other amino acids in the non-α subunits closest to the aromatic pocket, which are generally not conserved among γ, δ, or neuronal β subunits, are on three antiparallel β strands. The AChBP structure was used to refine the structure of the Torpedo nAChR in the absence of agonist to 4 Å resolution (13). In this structure, there is a reorientation of Segments A–C, resulting in the absence of a well defined core aromatic binding pocket.Analysis of agonist interactions with mutant nAChRs containing fluorine-substituted core aromatic residues provides evidence that cation-π interactions, particularly with αTrp¹⁴⁹ in Segment B, are important determinants of agonist binding affinity (14) and for the higher affinity binding of nicotine to α4β2 nAChRs compared with α₂βγδ nAChRs (15). Mutational analyses and molecular docking calculations have also provided evidence that two molecules of very similar structure may actually bind to a single receptor in very different orientations, as seen for two high affinity antagonists, d-tubocurarine and its quaternary ammonium analog metocurine, binding to the AChBP and to the muscle nAChR (16, 17).Photoaffinity labeling provides an alternative means to identify amino acids contributing to a drug binding site (18, 19) and has been used to determine the orientation of drugs bound in the ABS of Torpedo nAChR (20). Epibatidine binds with very high affinity (∼10 pm) to heteromeric neuronal nAChRs (e.g. α4β2) and with nanomolar affinity to α7 and muscle-type/Torpedo nAChRs (3). Utilizing a photoreactive analogue of epibatidine (azidoepibatidine; Fig. 1) and mass spectrometry, Tomizawa et al. (21) identified photolabeled amino acids in the Aplysia AChBP (Tyr¹⁹⁵ in Segment C and Met¹¹⁶ in Segment E), establishing an orientation for bound azidoepibatidine consistent with the orientation of epibatidine in an AChBP crystal structure (12).Open in a separate windowFIGURE 1.Structure of [³H]epibatidine (top) and azidoepibatidine (bottom).In this report, we use [³H]epibatidine as a photoaffinity reagent to identify the amino acids photolabeled in an expressed α4β2 nAChR and in the Torpedo α₂βγδ nAChR. Comparisons of the labeled amino acids seen in the Torpedo nAChR α-γ binding site and in the α4β2 nAChR, in conjunction with the results of docking calculations for epibatidine binding to homology models of the α₂βγδ and α4β2 nAChRs, suggests that epibatidine binds in a single orientation in the α-γ site but in two orientations in the α4β2 ABS.     

NAD-299 ANTAGONISES 5-HT-STIMULATED AND SPIPERONE-INHIBITED [35S]GTPγS BINDING IN CLONED 5-HT1A RECEPTORS

ABSTRACT

In Chinese Hamster Ovary (CHO) cells expressing cloned human 5-hydroxy-tryptamine_1A (5-HT_1A) receptors, (R)-3-N,N-dicyclobutylamino-8-fluoro-[6-³H]-3,4-dihydro-2H-1-benzopyran-5-carboxamide ([³H]NAD-299) exhibited high affinity (K_d = 0.16?nM) and labeled 34% more receptors than 8-hydroxy-2-([2,3-³H]di-n-propylamino)tetralin ([³H]8-OH-DPAT). NAD-299 behaved as a silent antagonist in [³⁵S]GTPγS binding similar to N-tert-butyl-3-(4-(2-methoxyphenyl)-piperazin-1-yl)-2-phenylpropanamide (WAY-100635) and (S)-5-fluoro-8-hydroxy-2-(di-n-propylamino)tetralin ((S)UH-301). 5-HT and 5-carboxamidotryptamine (5-CT) stimulated [³⁵S]GTPγS binding 2.5-fold while spiperone and methiothepin inhibited [³⁵S]GTPγS binding 1.4-fold. Furthermore, NAD-299 antagonised both the 5-HT stimulated and the spiperone inhibited [³⁵S]GTPγS binding to basal levels. The K_iL/K_iH ratios for spiperone (0.66), methiothepin (0.39), WAY-100635 (0.32), (S)UH-301 (0.94), NAD-299 (1.29), NAN-190 (1.23), (S)pindolol (5.85), ipsapirone (13.1), buspirone (24.6), (±)8-OH-DPAT (47.3), flesinoxan (55.8), 5-HT (200) and 5-CT (389) correlated highly significantly with the intrinsic activity obtained with [³⁵S] GTPγS (r = 0.97).     

Perturbation of the vesicle cycle in reticulospinal synapses of lamprey after presynaptic microinjections of GTPγS

The synaptic vesicle cycle sustains neurotransmission and keeps exo- and endocytosis in synapses in dynamic equilibrium. GTP-binding proteins function as key regulators of this cycle. The large GTPase dynamin is implicated in the fission of clathrin-coated vesicles from presynaptic membrane during endocytosis. The present study addresses the effect of the nonhydrolysable GTP analog GTPγS on assembly of the dynamin fission complex in situ. Intraaxonal microinjections of GTPγS induced the following distinct ultrastructural changes in the synapses: the number of synaptic vesicles in a cluster decreased while the number of the docked vesicles at the active zone increased; at the same time, the clathrin-coated intermediates also increased in number, indicating the inhibition of synaptic vesicle recycling. Unusual clathrin-coated intermediates were found. At low concentrations of GTPγS, they were presented by long tubules wreathed with a dynamin helix (spiral) and topped with a clathrin-coated vesicle. At high concentrations of GTPγS the tubular structures were much shorter and branched, with each branch topped with a clathrin-coated vesicle. The spiral pitch and the tubule diameter were significantly reduced as the concentration of GTPγS built up (23.1 ± 0.4 and 26.6 ± 0.4 nm, respectively, at low and 19.0 ± 0.5 and 23.3 ± 0.4 nm at high concentration of GTPγS, p < 0.001). We suggest that these ultrastructural changes reflect different steps in dynamin-mediated fission of clathrin-coated vesicles and propose a model for this process. The model implies that at first, GTP hydrolysis leads to a fast elongation of the helix due to a straightening of its dynamin dimmers. This entails an increase both in a pitch and a diameter of the dynamin helix. The shift in diameter disrupts local hydrophobic interactions between the inner and the outer lipid layers of the membrane at the sites of dynamin binding. Concurrent stretching of the helix and the clathrin-coated vesicle’s neck disintegrates the neck membrane and results finally in a release of the clathrincoated vesicle.     

Developmental and Regional Alteration of κ-Opioid Receptors in Seizure-Susceptible EL Mouse Brain

The binding of [³H]ethylketocyclazocine ([³H]EKC) under the suppression of and sites in the brain of EL mice (seizure-susceptible) was examined to determine the relationship between seizures and the dynorphinergic system. The density of -opioid receptors in the cerebrum of adult EL mice during interictal periods significantly increased, without changes in apparent affinities, compared with that of adult ddY mice (seizure-nonsusceptible; the mother strain of EL). Subsequently, the binding of 0.8 nM [³H]EKC in 8 brain regions was examined in the 2 strains. The [³H]EKC binding in 25-day-old EL mice that had no seizures significantly increased in the hippocampus and amygdala. At the age of 50 days, EL mice displayed abortive seizures, and the number of sites in EL mice was significantly greater in the hippocampus, amygdala and cerebral cortex. It was further shown that the binding of [³H]EKC in 150-day-old adult EL mice during interictal periods was markedly increased in the hippocampus, amygdala, cerebral cortex and striatum, compared with the corresponding regions in ddY mice. The up-regulation of receptors in the EL mouse brain may result from deficits in endogenous dynorphins, which could be involved in the pathogenesis of seizure diathesis and seizure manifestations in the EL mouse.     

Expression of Transforming Growth Factor-β Receptors in Meningeal Fibroblasts of the Injured Mouse Brain

The fibrotic scar which is formed after traumatic damage of the central nervous system (CNS) is considered as a major impediment for axonal regeneration. In the process of the fibrotic scar formation, meningeal fibroblasts invade and proliferate in the lesion site to secrete extracellular matrix proteins, such as collagen and laminin. Thereafter, end feet of reactive astrocytes elaborate a glia limitans surrounding the fibrotic scar. Transforming growth factor-β1 (TGF-β1), a potent scar-inducing factor, which is upregulated after CNS injury, has been implicated in the formation of the fibrotic scar and glia limitans. In the present study, expression of receptors to TGF-β1 was examined by in situ hybridization histochemistry in transcortical knife lesions of the striatum in the mouse brain in combination with immunofluorescent staining for fibroblasts and astrocytes. Type I and type II TGF-β receptor mRNAs were barely detected in the intact brain and first found in meningeal cells near the lesion 1 day postinjury. Many cells expressing TGF-β receptors were found around the lesion site 3 days postinjury, and some of them were immunoreactive for fibronectin. After 5 days postinjury, many fibroblasts migrated from the meninges to the lesion site formed the fibrotic scar, and most of them expressed TGF-β receptors. In contrast, few of reactive astrocytes expressed the receptors throughout the postinjury period examined. These results indicate that meningeal fibroblasts not reactive astrocytes are a major target of TGF-β1 that is upregulated after CNS injury.     

The Role of Interchain Heterodisulfide Formation in Activation of the Human Common β and Mouse βIL-3 Receptors

The cytokines, interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibit overlapping activities in the regulation of hematopoietic cells. In humans, the common β (βc) receptor is shared by the three cytokines and functions together with cytokine-specific α subunits in signaling. A widely accepted hypothesis is that receptor activation requires heterodisulfide formation between the domain 1 D-E loop disulfide in human βc (hβc) and unidentified cysteine residues in the N-terminal domains of the α receptors. Since the development of this hypothesis, new data have been obtained showing that domain 1 of hβc is part of the cytokine binding epitope of this receptor and that an IL-3Rα isoform lacking the N-terminal Ig-like domain (the “SP2” isoform) is competent for signaling. We therefore investigated whether distortion of the domain 1-domain 4 ligand-binding epitope in hβc and the related mouse receptor, β_IL-3, could account for the loss of receptor signaling when the domain 1 D-E loop disulfide is disrupted. Indeed, mutation of the disulfide in hβc led to both a complete loss of high affinity binding with the human IL-3Rα SP2 isoform and of downstream signaling. Mutation of the orthologous residues in the mouse IL-3-specific receptor, β_IL-3, not only precluded direct binding of mouse IL-3 but also resulted in complete loss of high affinity binding and signaling with the mouse IL-3Rα SP2 isoform. Our data are most consistent with a role for the domain 1 D-E loop disulfide of hβc and β_IL-3 in maintaining the precise positions of ligand-binding residues necessary for normal high affinity binding and signaling.     

Increases in [3H]Muscimol and [3H]Flumazenil Binding in the Dorsolateral Prefrontal Cortex in Schizophrenia Are Linked to α4 and γ2S mRNA Levels Respectively

Background

GABA_A receptors (GABA_AR) are composed of several subunits that determine sensitivity to drugs, synaptic localisation and function. Recent studies suggest that agonists targeting selective GABA_AR subunits may have therapeutic value against the cognitive impairments observed in schizophrenia. In this study, we determined whether GABA_AR binding deficits exist in the dorsolateral prefrontal cortex (DLPFC) of people with schizophrenia and tested if changes in GABA_AR binding are related to the changes in subunit mRNAs. The GABA orthosteric and the benzodiazepine allosteric binding sites were assessed autoradiographically using [³H]Muscimol and [³H]Flumazenil, respectively, in a large cohort of individuals with schizophrenia (n = 37) and their matched controls (n = 37). We measured, using qPCR, mRNA of β (β1, β2, β3), γ (γ1, γ2, γ2S for short and γ2L for long isoform, γ3) and δ subunits and used our previous measurements of GABA_AR α subunit mRNAs in order to relate mRNAs and binding through correlation and regression analysis.

Results

Significant increases in both [³H]Muscimol (p = 0.016) and [³H]Flumazenil (p = 0.012) binding were found in the DLPFC of schizophrenia patients. Expression levels of mRNA subunits measured did not show any significant difference in schizophrenia compared to controls. Regression analysis revealed that in schizophrenia, the [³H]Muscimol binding variance was most related to α4 mRNA levels and the [³H]Flumazenil binding variance was most related to γ2S subunit mRNA levels. [³H]Muscimol and [³H]Flumazenil binding were not affected by the lifetime anti-psychotics dose (chlorpromazine equivalent).

Conclusions

We report parallel increases in orthosteric and allosteric GABA_AR binding sites in the DLPFC in schizophrenia that may be related to a “shift” in subunit composition towards α4 and γ2S respectively, which may compromise normal GABAergic modulation and function. Our results may have implications for the development of treatment strategies that target specific GABA_AR receptor subunits.     

Pharmacological Characterisation of [35S]-GTPγS Binding to Chinese Hamster Ovary Cell Membranes Stably Expressing Cloned Human 5-HT1D Receptor Subtypes

Abstract

[³⁵S]-GTPγS binding has been used to study the function of cloned human 5-HT_1D receptor subtypes stably expressed in chinese hamster ovary (CHO) cells. 5-HT stimulated [³⁵S]-GTPγS binding to membranes from cells expressing 5-HT_1Dα or 5-HT_1Dβ receptors. In membranes containing 5-HT_1Dβ receptors, 5-CT and sumatriptan stimulated binding to a similar extent as 5-HT while yohimbine, metergoline and 8-OHDPAT were partial agonists. The order of potency for agonists was 5-CT > 5-HT > metergoline > sumatriptan > yohimbine > 8-OHDPAT. The stimulation of binding by 5-HT in membranes containing 5-HT_1Dβ receptors was potently antagonised by methiothepin (pA₂ 8.9 ± 0.1). The overall pharmacological profile for the human 5-HT_1Dβ receptor, defined using [³⁵S]-GTPγS binding, agreed well with that reported for inhibition of forskolin-stimulated adenylyl cyclase. In addition, methiothepin and ketanserin inhibited basal [³⁵S]-GTPγS binding to membranes containing 5-HT_1Dα or 5-HT_1Dβ receptors, suggesting that these compounds show negative efficacy at 5-HT_1D receptor subtypes. The data show that [³⁵S]-GTPγS binding is a suitable method for studying the interaction between cloned human 5-HT_1D receptors and G-proteins.