Potentiation of sensory responses in ventrobasal thalamus in vivo via selective modulation of mGlu1 receptors with a positive allosteric modulator.

Metabotropic glutamate subtype 1 (mGlu1) receptor is thought to play a role in synaptic responses in thalamic relay nuclei. The aim of this study was to evaluate the positive allosteric modulator (PAM) Ro67-4853 as a tool to modulate thalamic mGlu1 receptors on single thalamic neurones in vivo. Ro67-4853, applied by iontophoresis onto ventrobasal thalamus neurones of urethane-anaesthetised rats, selectively enhanced responses to the agonist (S)-3,5-dihydroxy-phenylglycine (DHPG), an effect consistent with mGlu1 potentiation. The PAM was also able to enhance maintained responses to 10 Hz trains of sensory stimulation of the vibrissae, but had little effect on responses to single sensory stimuli. Thus Ro67-4853 appears to be a highly selective tool that can be useful in investigating how mGlu1 receptor potentiation can alter neural processing in vivo. Our results show the importance of mGlu1 in sensory processing and attention mechanisms at the thalamic level and suggest that positive modulation of mGlu1 receptors might be a useful mechanism for enhancing cognitive and attentional processes.

Characterization of RO4583298 as a novel potent, dual antagonist with in vivo activity at tachykinin NK1 and NK3 receptors.

BACKGROUND AND PURPOSE: Clinical results of osanetant and talnetant (selective-NK3 antagonists) indicate that blocking the NK3 receptor could be beneficial for the treatment of schizophrenia. The objective of this study was to characterize the in vitro and in vivo properties of a novel dual NK1/NK3 antagonist, RO4583298 (2-phenyl-N-(pyridin-3-yl)-N-methylisobutyramide derivative). EXPERIMENTAL APPROACH: RO4583298 in vitro pharmacology was investigated using radioligand binding ([³H]-SP, [³H]-osanetant, [³H]-senktide), [³H]-inositol-phosphate accumulation Schild analysis (SP- or [MePhe7]-NKB-induced) and electrophysiological studies in guinea-pig substantia nigra pars compacta (SNpc). The in vivo activity of RO4583298 was assessed using reversal of GR73632-induced foot tapping in gerbils (GFT; NK1) and senktide-induced tail whips in mice (MTW; NK3). KEY RESULTS: RO4583298 has a high-affinity for NK1 (human and gerbil) and NK3 (human, cynomolgus monkey, gerbil and guinea-pig) receptors and behaves as a pseudo-irreversible antagonist. Unusually it binds with high-affinity to mouse and rat NK3, yet with a partial non-competitive mode of antagonism. In guinea-pig SNpc, RO4583298 inhibited the senktide-induced potentiation of spontaneous activity of dopaminergic neurones with an apparent non-competitive mechanism of action. RO4583298 (p.o.) robustly blocked the GFT response, and inhibited the MTW. CONCLUSIONS AND IMPLICATIONS: RO4583298 is a high-affinity, non-competitive, long-acting in vivo NK1/NK3 antagonist; hence providing a useful in vitro and in vivo pharmacological tool to investigate the roles of NK1 and NK3 receptors in psychiatric disorders.

Characterization of (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one as a positive allosteric modulator of GABAB receptors.

BACKGROUND AND PURPOSE: As baclofen is active in patients with anxiety disorders, GABAB receptors have been implicated in the modulation of anxiety. To avoid the side effects of baclofen, allosteric enhancers of GABAB receptors have been studied to provide an alternative therapeutic avenue for modulation of GABAB receptors. The aim of this study was to characterize derivatives of (R,S)-5,7-di-tert-butyl-3-hydroxy-3-trifluoromethyl-3H-benzofuran-2-one (rac-BHFF) as enhancers of GABAB receptors. EXPERIMENTAL APPROACH: Enhancing properties of rac-BHFF were assessed in the Chinese hamster ovary (CHO)-Galpha16-hGABA(B1a,2a) cells by Fluorometric Imaging Plate Reader and GTPgamma[35S]-binding assays, and in rat hippocampal slices by population spike (PS) recordings. In vivo activities of rac-BHFF were assessed using the loss of righting reflex (LRR) and stress-induced hyperthermia (SIH) models. KEY RESULTS: In GTPgamma[35S]-binding assays, 0.3 microM rac-BHFF or its pure enantiomer (+)-BHFF shifted the GABA concentration-response curve to the left, an effect that resulted in a large increase in both GABA potency (by 15.3- and 87.3-fold) and efficacy (149% and 181%), respectively. In hippocampal slices, rac-BHFF enhanced baclofen-induced inhibition of PS of CA1 pyramidal cells. In an in vivo mechanism-based model in mice, rac-BHFF increased dose-dependently the LRR induced by baclofen with a minimum effective dose of 3 mg kg(-1) p.o. rac-BHFF (100 mg kg(-1) p.o.) tested alone had no effect on LRR nor on spontaneous locomotor activity, but exhibited anxiolytic-like activity in the SIH model in mice. CONCLUSIONS AND IMPLICATIONS: rac-BHFF derivatives may serve as valuable pharmacological tools to elucidate the pathophysiological roles played by GABAB receptors in the central and peripheral nervous systems.

Positive allosteric modulators of metabotropic glutamate 1 receptor: characterization, mechanism of action, and binding site.

We have identified two chemical series of compounds acting as selective positive allosteric modulators (enhancers) of native and recombinant metabotropic glutamate 1 (mGlu1) receptors. These compounds did not directly activate mGlu1 receptors but markedly potentiated agonist-stimulated responses, increasing potency and maximum efficacy. Binding of these compounds increased the affinity of a radiolabeled glutamate-site agonist at its extracellular N-terminal binding site. Chimeric and mutated receptors were used to localize amino acids in the receptor transmembrane region critical for these enhancing properties. Finally, the compounds potentiated synaptically evoked mGlu1 receptor responses in rat brain slices. The discovery of selective positive allosteric modulators of mGlu1 receptors opens up the possibility to develop a similar class of compounds for other family 3 G protein-coupled receptors.

Identification of essential residues involved in the glutamate binding pocket of the group II metabotropic glutamate receptor.

Metabotropic glutamate (mGlu) receptors are a family of G-protein-coupled receptors that play central roles as modulators of both glutamatergic and other major neurotransmitter systems in CNS. Using molecular modeling, site-directed mutagenesis, [(3)H]LY354740 binding, [(35)S]GTPgammaS binding, and activation of GIRK current, we have been able to identify residues crucial for the binding of LY354740 and glutamate to rat mGlu2 receptors. Several of the crucial residues located in the binding site (Arg-57, Tyr-144, Tyr-216, Asp-295) have not been identified previously. We propose that the gamma-carboxyl group of LY354740 forms H-bonds to Arg-57, whereas the alpha-carboxyl group forms an H-bond with the hydroxyl group of Ser-145. The alpha-amino group of LY354740 forms H-bonds to Asp-295 and to the side-chain hydroxyl group of Thr-168. In addition, Tyr-144 may establish a hydrophobic (C-H/pi)-interaction with the bicyclo-hexane ring of LY354740. Furthermore, the mutation of residues Ser-148 and Arg-183, which are too remote for a direct interaction, affected the ligand affinity dramatically. These results suggest that Ser-148 and Arg-183 may be important for the 3D structure and/or are involved in closure of the domain. Finally, Asp-146, which is also remote from the binding site, was shown to be involved in the differential binding affinity of [(3)H]LY354740 for mGlu2 versus mGlu3 receptors. All the mGlu receptors except mGlu2 are activated by Ca(2+) and have serine instead of aspartic acid at this position, which suggests a critical role of this aspartic acid residue in the binding properties of this unique receptor.

Pharmacological properties of native metabotropic glutamate receptors in freshly dissociated Golgi cells of the rat cerebellum.

We have examined the pharmacological properties of native metabotropic glutamate (mGlu) receptors in freshly isolated rat cerebellar Golgi cells using the whole-cell configuration of the patch-clamp technique. Group II mGlu receptor agonists inhibited voltage-gated Ca(2+) channels (VGCC) currents in a reversible and concentration-dependent manner with a rank order of potency being LY354740> DCG-IV > L-CCG-I > glutamate >>1S,3R-ACPD > NAAG. The maximum degree of inhibition obtained was similar for all drugs tested, saturating at about 33-41%, except for NAAG that had a non saturating effect of 50% at 1mM. Two novel group II mGlu receptor antagonists, LY341495 and Ro 65-3479, reversed VGCC current inhibition by LY354740 with pK(B) values of 7.0 and 6.3, respectively. In a subpopulation of Golgi cells, the antagonistic effect of LY341495 was only partial, suggesting a remaining effect of group I mGlu receptors. This was confirmed by experiments with S-DHPG, a selective group I mGlu receptor agonist. These experiments suggest that Golgi cells of the cerebellum express group II mGlu receptors that couple to the inhibition of VGCCs. Therefore, inhibition of VGCCs in cerebellar Golgi cells is a useful model system to evaluate novel group II mGlu receptor ligands.

Effect of metabotropic glutamate receptor activation on receptor-mediated cyclic AMP responses in primary cultures of rat striatal neurones.

Co-activation of group I metabotropic glutamate (mGlu) receptors and adenosine receptors resulted in an augmented cyclic AMP response in primary cultures of rat striatal neurones. L-glutamate and the selective group I agonist, (S)-dihydroxyphenylglycine (S-DHPG) evoked concentration-dependent potentiations of cyclic AMP accumulation stimulated by the adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA), with EC50 values of 3.41+/-0. 39 and 5.69+/-1.64 microM, respectively, and maximal augmentations of approximately 350% at concentrations of 100 microM. The S-DHPG potentiation was inhibited by group I mGlu receptor antagonists and a protein kinase C inhibitor, Ro 31-8220, implicating products of PI hydrolysis in this effect. Furthermore, L-glutamate and S-DHPG stimulated PI hydrolysis in striatal neuronal cultures with similar EC50 values to those observed for the augmentation of NECA cyclic AMP responses (5.19+/-1.18 and 3.78+/-1.42 microM, respectively). In situ hybridization and immunofluorescence techniques indicate that group I mGlu receptor-evoked potentiations are likely to be mediated via mGlu5 receptors, which are expressed at high levels in these cultures. In contrast to cross-chopped slices of neonatal rat striatum, of equivalent age, the group II mGlu receptor agonist, (2S, 2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) was without effect on NECA- or forskolin-stimulated cyclic AMP responses in primary striatal neuronal cultures. This lack of effect might be due to a low level of expression of group II mGlu receptors in cultured striatal neurones.

Metabotropic glutamate group II receptors activate a G protein-coupled inwardly rectifying K+ current in neurones of the rat cerebellum.

1. The effects of the group II metabotropic glutamate receptor (mGluR) agonists DCG-IV and LY354740 were examined in neurones freshly dissociated from the rat cerebellum and olfactory bulb, using the whole-cell configuration of the patch-clamp technique. 2. Under experimental conditions in which K+ currents would be inward, rapid application of DCG-IV and LY354740 to interneurones expressing the group II mGluRs induced an inward current in a subpopulation of interneurones of the cerebellum, the unipolar brush cells. 3. The currents induced by DCG-IV and LY354740 had the major characteristics of a G protein-coupled inwardly rectifying K+ channel (GIRK) current; namely, rapid activation and deactivation upon agonist application and removal, G protein dependence, strong inward rectification, Cs+ and Ba2+ sensitivity, and K+ selectivity. 4. In Golgi cells of the cerebellum and interneurones of the accessory olfactory bulb, which also express group II mGluRs, LY354740 did not induce GIRK activation but inhibited voltage-gated Ca2+ channel currents. 5. These results demonstrate that, in unipolar brush cells, native group II mGluRs can functionally couple to activation of GIRKs. Thus, the absence of coupling in the majority of CNS neurones examined to date may be due to restricted cellular co-localization or co-expression of the appropriate proteins.

Multiple pathways for regulation of the KCl-induced [3H]-GABA release by metabotropic glutamate receptors, in primary rat cortical cultures.

In rat cortical primary cultures, group II- and III-metabotropic glutamate receptor-selective agonists concentration-dependently reduced KCl-induced [3H]GABA release, with IC50 values of 11 nM for LY354740, 80 nM for L(+)-2-amino-4-phosphonobutyric acid (L-AP4), 180 nM for DCG-IV, and 330 nM for L-SOP. The group II antagonists, LY341495 and EGLU, reversed the effect of LY354740, and the group III antagonist MTPG reversed the effect of L-AP4. In the presence of omega-conotoxin GVIA, LY354740 inhibited the remaining [3H]GABA release, whereas L-AP4 was inactive. In contrast, in the presence of nifedipine, L-AP4 inhibited the remaining [3H]GABA release, but LY354740 was no longer active. The PKA inhibitor, H89, blocked the effects of both L-AP4 and LY354740, whereas the PKC inhibitor Ro 31-8220 blocked only the effect of LY354740. Both Ro 31-8220 and H89 reduced the [3H]GABA release to 60% of control. In whole-cell, voltage-clamp experiments, LY354740 and L-AP4 inhibited voltage-gated calcium channel currents with IC50 values of 28 nM and 22 microM, respectively. The results suggest that, in these cells, KCl-induced [3H]GABA release is modulated by two different mechanisms, one involving group II receptors and a direct control of the Ca2+ channel activity, and the other mediated by group III receptors and possibly involving a regulation located downstream of the Ca2+ channel activation.

Modulation of voltage-gated calcium channels by orphanin FQ in freshly dissociated hippocampal neurons.

Orphanin FQ (OFQ) has recently been reported to be an endogenous ligand for the opioid-like LC132 receptor. The effect of OFQ on high voltage-gated calcium channels (VGCCs) was examined in freshly dissociated rat pyramidal neurons using the whole-cell configuration of the patch-clamp technique. High-threshold Ba2+ currents were reversibly inhibited by OFQ. The depression of the currents was associated with a slowed rate of activation and a change in the activation I-V relationship at step potentials higher than +30 mV. In concentration-response experiments, a mean (+/-SEM) pEC50 value of 7.0 +/- 0.07 and a Hill coefficient of 1.5 +/- 0.08 (n = 5) were obtained. The near-maximum inhibition of the Ba2+ currents by OFQ (1 microM) amounted to 31 +/- 2.2% of control (n = 15). Opioid receptors could not account for the effects of OFQ on VGCCs, because naloxone, a broad spectrum mu-, delta-, and kappa-receptor antagonist, did not reduce the effectiveness of OFQ. When GTP-gamma-S was included in the pipette, the depression of the currents by OFQ was irreversible, whereas currents from neurons preincubated with pertussis toxin were not inhibited by OFQ, consistent with the involvement of a PTX-sensitive G-protein. When selective blockers of VGCCs were used, it was demonstrated that all subtypes of VGCCs were affected by OFQ. In conclusion, the effect of OFQ on VGCCs expressed in hippocampal CA3 and CA1 neurons may play an important role in the regulation of hippocampal cell excitability and neurotransmitter release.

Pharmacological modulation of the diazepam-insensitive recombinant gamma-aminobutyric acidA receptors alpha 4 beta 2 gamma 2 and alpha 6 beta 2 gamma 2.

We characterized modulation of the gamma-aminobutyric acid (GABA)-evoked responses of the diazepam-insensitive alpha 4 beta 2 gamma2 and alpha 6 beta 2 gamma 2 recombinant GABAA receptors. The partial agonist bretazenil potentiated the responses of both receptors with similar dose dependence but with a higher maximal enhancement at the alpha 4 beta 2 gamma 2 receptor. The bretazenil-induced potentiation was reduced by the benzodiazepine antagonist flumazenil. At a high concentration (10 microM), flumazenil was a weak potentiator of the GABA response. The partial agonist imidazenil was inactive. The imidazobenzodiazepine inverse agonist Ro 15-4513, which is known to bind with high affinity to the alpha 6 beta 2 gamma 2 receptor, potentiated the GABA responses of the alpha 4 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 receptor subtypes with similar dose dependence over the concentration range of 0.1-10 microM. Methyl-6, 7-dimethoxy-4-ethyl-beta-carboline, a beta-carboline inverse agonist, had a similar potentiating effect when tested at a concentration of 10 microM. The alpha 4 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 receptor-mediated currents had equal sensitivities to furosemide and Zn2+ ions, both of which reduced the GABA-evoked responses. The alpha 6 beta 2 gamma 2 receptor but not the alpha 4 beta 2 gamma 2 receptor exhibited a low level of spontaneous activity in the absence of GABA; this resting current could be directly potentiated by Ro 15-4513, methyl-6,7-dimethoxy-4-ethyl-beta-carboline, bretazenil and flumazenil and was blocked by picrotoxin. Thus, although the alpha 4 beta 2 gamma 2 receptors are insensitive to benzodiazepine binding site full agonists, such as diazepam, they can be modulated by certain ligands acting as partial and inverse agonists at diazepam-sensitive receptors and thereby contribute to the respective pharmacological profiles.

Benzodiazepine-insensitive mice generated by targeted disruption of the gamma 2 subunit gene of gamma-aminobutyric acid type A receptors.

Vigilance, anxiety, epileptic activity, and muscle tone can be modulated by drugs acting at the benzodiazepine (BZ) site of gamma-aminobutyric acid type A (GABAA) receptors. In vivo, BZ sites are potential targets for endogenous ligands regulating the corresponding central nervous system states. To assess the physiological relevance of BZ sites, mice were generated containing GABAA receptors devoid of BZ sites. Following targeted disruption of the gamma 2 subunit gene, 94% of the BZ sites were absent in brain of neonatal mice, while the number of GABA sites was only slightly reduced. Except for the gamma 2 subunit, the level of expression and the regional and cellular distribution of the major GABAA receptor subunits were unaltered. The single channel main conductance level and the Hill coefficient were reduced to values consistent with recombinant GABAA receptors composed of alpha and beta subunits. The GABA response was potentiated by pentobarbital but not by flunitrazepam. Diazepam was inactive behaviorally. Thus, the gamma 2 subunit is dispensable for the assembly of functional GABAA receptors but is required for normal channel conductance and the formation of BZ sites in vivo. BZ sites are not essential for embryonic development, as suggested by the normal body weight and histology of newborn mice. Postnatally, however, the reduced GABAA receptor function is associated with retarded growth, sensorimotor dysfunction, and drastically reduced life-span. The lack of postnatal GABAA receptor regulation by endogenous ligands of BZ sites might contribute to this phenotype.

Heterogeneity of GABAA-receptors: cell-specific expression, pharmacology, and regulation.

Vigilance, anxiety, memory, epileptogenic activity and muscle tension can be regulated by a modulation of GABAA-receptor function. A multitude of different GABAA-receptors exist in the brain due to the combinational assembly of various subunits encoded by at least 15 genes. The clarification of the physiological and pharmacological significance of GABAA-receptor subtypes, in combination with their cellular localization, will make it possible to identify the neuronal circuits regulating the respective CNS states and to provide strategies for the development of subtype-specific drugs for selective therapies.

Full and partial agonism displayed by benzodiazepine receptor ligands at recombinant gamma-aminobutyric acidA receptor subtypes.

The differences in intrinsic activity and receptor subtype specificity of the newly developed benzodiazepine receptor ligands bretazenil, divaplon and abecarnil were assessed in recombinant gamma-aminobutyric acidA (GABAA) receptors expressed in mammalian cells from the subunit-cDNA combinations alpha 3 beta 2 gamma 2 and alpha 5 beta 2 gamma 2. Chloride currents induced by rapid application of GABA in the presence or absence of drugs were measured using the whole-cell configuration of the patch-clamp technique. Bretazenil displayed an intrinsic activity which amounted only to 58 +/- 7% and 35 +/- 11% of that of flunitrazepam at the alpha 3 beta 2 gamma 2 and alpha 5 beta 2 gamma 2 combination, respectively. The maximum potentiation by divaplon was only 28 +/- 5% and 21 +/- 9% of that of flunitrazepam at the respective subunit combinations. Thus, the partial agonism postulated for bretazenil and divaplon on pharmacological grounds is shown to be operative on the level of single GABAA receptors. Most strikingly, abecarnil potentiated the GABA response to the same degree as flunitrazepam at the alpha 3 beta 2 gamma 2 combination but to only 52 +/- 14% compared to flunitrazepam at the alpha 5 beta 2 gamma 2 combination. This finding demonstrates that the intrinsic activity of benzodiazepine receptor ligands can vary among the receptor subtypes with the degree of receptor modulation being influenced by the type of alpha subunit.

Pharmacological and Electrophysiological Properties of Recombinant GABAA Receptors Comprising the alpha3, beta1 and gamma2 Subunits.

To assess the role of subunits for channel function and drug modulation in recombinant GABAA receptors, the alpha3beta1gamma2 subunits and the dual combinations alpha3beta1, beta1gamma2 and alpha3gamma2 were expressed by transfection of human embryonic kidney cells and by RNA injection in Xenopus oocytes (alpha3beta1gamma2 combination). GABA-induced chloride currents were recorded using the whole-cell configuration of the patch-clamp technique (transfected cells) or the voltage-clamp technique (oocytes). The currents recorded from the alpha3beta1gamma2 subunit combination in transfected cells were reduced by bicuculline and picrotoxin, enhanced by flunitrazepam in a flumazenil-sensitive manner and reduced by beta-carboline-3-carboxylic acid methyl ester (beta-CCM). The GABA-induced current was reduced by beta-CCM in all combinations containing the gamma2 subunit, but potentiation by flunitrazepam was only obtained when the gamma2 subunit was coexpressed in the presence of the alpha3 subunit (alpha3beta1gamma2 or alpha3gamma2). The GABA sensitivities of the receptors were similar when the alpha3beta1gamma2 combination was expressed in oocytes (half-maximum effective concentration=240 microM) or in the kidney cell line (270 microM). However, the currents were less potentiated by flunitrazepam in oocytes (129% of controls) than in transfected cells (189%). These results suggest that the alpha3beta1gamma2 subunit combination, which is coexpressed in various brain regions as shown by in situ hybridization histochemistry, may represent a building block of functional GABAA receptors in situ.

The gamma 3-subunit of the GABAA-receptor confers sensitivity to benzodiazepine receptor ligands.

The gamma 3-subunit of the GABAA-receptor in rat brain has been identified by molecular cloning. When co-expressed with the alpha 5- and beta 2-subunits in transfected cells a high potency for GABA (Ka = 4.9 +/- 1.2 microM) and a strong cooperativity in gating the channel (H = 1.9 +/- 0.2) was observed. The GABA response was potentiated in the presence of flunitrazepam and reduced by beta CCM. An analogous bi-directional modulation of the GABA response was observed with diazepam and DMCM as tested with the subunit combinations alpha 1 beta 2 gamma 3 and alpha 3 beta 2 gamma 3 expressed in Xenopus oocytes. Since the benzodiazepine receptor ligands were virtually inactive in the absence of the gamma 3-subunit, as tested with the alpha 3 beta 2- and alpha 5 beta 2-subunit combinations, the gamma 3-subunit is a prerequisite for the benzodiazepine receptor sensitivity of the expressed GABAA-receptors. The gamma 3-subunit could functionally replace the gamma 2-subunit with regard to the bi-directional allosteric drug modulation.

Regulation of acetylcholine receptor function by the phorbol ester TPA in rat skeletal muscle.

(1) The effect of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a specific activator of the protein kinase C (PrkC), on the function of junctional nicotinic acetylcholine receptors (nAChR) was examined on muscle fibres isolated from the M. flexor digitorum brevis of the rat. (2) In the presence of TPA the sensitivity of the whole endplates to iontophoretically applied ACh exhibited multiphasic oscillations: an early decrease followed by a delayed increase and, at the end again, a decrease to below pretreatment levels. This effect was more pronounced as the TPA concentration was increased in the range of 0.1-1 microM and was blocked by the PrkC-inhibitor 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7). (3) TPA (0.1-0.5 microM) shortly applied to patch-clamped fibres caused a slight decrease in nAChR-channel slope conductance without affecting the mean lifetime. In a patch the opening frequency increased over time, after an initial decrease. (4) It is concluded that specific activation of the PrkC may be of regulatory significance on nAChR function.