Subchronic administration and combination metabotropic glutamate and GABAB receptor drug therapy in fragile X syndrome

The most common cause of inherited mental retardation, fragile X syndrome, results from a triplet repeat expansion in the FMR1 gene and loss of the mRNA binding protein, fragile X mental retardation protein (FMRP). In the absence of FMRP, signaling through group I metabotropic glutamate receptors (mGluRs) is enhanced. We previously proposed a mechanism whereby the audiogenic seizures exhibited by FMR1 null mice result from an imbalance in excitatory mGluR and inhibitory GABA(B) receptor (GABA(B)R) signaling (Mol Pharmacol 76:18-24, 2009). Here, we tested the mGluR5-positive allosteric modulator 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB), the mGluR5 inverse agonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), and GABA(B) receptor agonists, alone and in combination on receptor protein expression and audiogenic seizures in FMR1 mice. Single doses of MPEP (30 mg/kg), the GABA(B)R orthosteric agonist R-baclofen (1 mg/kg), or the GABA(B)R-positive allosteric modulator N,N'-dicyclopentyl-2-(methylthio)-5-nitro-4,6-pyrimidine diamine (GS-39783) (30 mg/kg), reduced the incidence of seizures. However, when administered subchronically (daily injections for 6 days), MPEP retained its anticonvulsant activity, whereas R-baclofen and GS-39783 did not. When administered at lower doses that had no effect when given alone, a single injection of MPEP plus R-baclofen also reduced seizures, but the effect was lost after subchronic administration. We were surprised to find that subchronic treatment with R-baclofen also induced tolerance to a single high dose of MPEP. These data demonstrate that tolerance develops rapidly to the antiseizure properties of R-baclofen alone and R-baclofen coadministered with MPEP, but not with MPEP alone. Our findings suggest that cross-talk between the G-protein signaling pathways of these receptors affects drug efficacy after repeated treatment.     

Coexpression of full-length gamma-aminobutyric acid(B) (GABA(B)) receptors with truncated receptors and metabotropic glutamate receptor 4 supports the GABA(B) heterodimer as the functional receptor

Direct evidence is lacking to show whether the gamma-aminobutyric acid (GABA)(B) gb1-gb2 heterodimer is the signaling form of the receptor. In this study, we tested whether gb1a or gb2 subunits when coexpressed with truncated receptors or metabotropic glutamate receptor mGluR4 could form functional GABA receptors. Coexpression of the ligand binding N-terminal domain of gb1a or the C-terminal portion of gb1a composing the seven-transmembrane segments and intracellular loops with gb2 could not reconstitute functional receptors. We next examined whether mGluR4, which forms homodimers and is structurally related to GABA(B), could act as a surrogate coreceptor for gb1 or gb2. The coexpression of mGluR4 and gb1a led to the expression of gb1a monomers on cell surface membranes as determined by immunoblot analysis and flow cytometry. However, mGluR4-gb1a heterodimers were not formed, and membrane-expressed gb1a monomers were not functionally coupled to adenylyl cyclase in human embryonic kidney 293 cells or activated inwardly rectifying potassium (Kir) channels in Xenopus oocytes. Similarly, the coexpression of mGluR4 and gb2 led to nonfunctional GABA receptors. GABA-activated distal signaling events resulted only after the coexpression and heterodimerization of gb1 and gb2. Taken together with the truncated receptor studies, the data suggest that a high degree of structural specificity is required to form the functional GABA(B) receptor that is a gb1-gb2 heterodimer.     

GABAB receptor antagonist-mediated antidepressant-like behavior is serotonin-dependent

There is an emerging body of data purporting a role of gamma-aminobutyric acid (GABA) in the pathophysiology of mood disorders. However, the role of metabotropic GABA(B) receptors in depression is not well defined. The modified forced swim test has recently emerged as an excellent tool to assess behaviorally the role of monoamines in antidepressant action. To assess the role of GABA(B) receptors in antidepressant-related behavior, we examined a number of selective GABA(B) receptor ligands (novel positive modulators and antagonists) on behavior in the modified forced swim test. We demonstrate that the selective GABA(B) receptor antagonists CGP56433A [[3-{1-(S)-[{3-cyclohexylmethyl)hydroxy phosphinyl}-2-(S) hydroxy propyl]amino}ethyl]benzoic acid; 1-10 mg/kg] and [3-[[1-(S)-3-dichlorophenyl)ethyl]amino]-2-(S)-hydroxy-propyl]phenylmethyl-phosphinic acid hydrochloride; 3-10 mg/kg] had a similar profile to the selective serotonin reuptake inhibitor fluoxetine; they decreased immobility and increased swimming behavior. The tricyclic antidepressant desipramine decreased immobility but increased climbing behavior. In contrast, the novel GABA(B) receptor-positive modulator GS39783 (10-40 mg/kg) did not display antidepressant-like activity in the modified forced swim test. To further assess the possible interaction between GABA(B) receptor antagonism and serotonin, rats were pretreated with the tryptophan hydroxylase inhibitor para-chlorophenylalanine. 5-Hydroxytryptamine depletion (>90%) abolished the antidepressant-like behavior of CGP56433A (10 mg/kg) by attenuating the increase in swimming. Together, these data demonstrate that GABA(B) receptor antagonists via an interaction with the serotonergic system display antidepressant-like properties and therefore represent a novel approach for the treatment of depression.     

Involvement of inhibitory and excitatory neurotransmitters in levofloxacin- and ciprofloxacin-induced convulsions in mice.

We studied the effects of gamma-aminobutyric acid (GABA)-benzodiazepine receptor agonists and glutamate receptor antagonists on levofloxacin (LVFX)- and ciprofloxacin (CPFX)-induced convulsions using intrathecal (i.t.) injections in mice. We also studied the effects of these agonists and antagonists on exacerbated convulsions induced by coadministration of the quinolone with 4-biphenylacetic acid (BPAA). The agonists or antagonists were injected i.t. 5 min and BPAA was administered orally 30 min before a single i.t. injection of the quinolone (10 microliters per animal). The animals were observed for clonic convulsion and death, and latency times to the appearance of convulsion were determined. Among the agonists, baclofen showed marked inhibition of both LVFX- and CPFX-induced convulsions, while other compounds such as GABA, muscimol, diazepam, and 3-aminopropylphosphonic acid had slight effects. Among the antagonists, kynurenic acid showed the strongest inhibition of convulsions caused by all doses of LVFX and CPFX and prolonged latency times; gamma-glutamyl-aminomethylsulfonic acid (GAMS) also markedly inhibited convulsions. The antagonists D-AP-5, AP-7, and 6,7-dinitroquinoxaline-2,3-dione (DNQX) had slight effects. Additionally, GAMS, DNQX, and MK-801 significantly lowered the incidence of death in the groups treated with CPFX. The enhanced convulsive activities of LVFX or CPFX by pretreatment with BPAA were clearly blocked by baclofen, kynurenic acid, GAMS, and DNQX. D-AP-5 and AP-7 also showed clear effects on the activity of LVFX. These results suggest that LVFX has fewer effects on the brains than CPFX and that convulsions induced by these quinolones alone and by these quinolones administered with BPAA may be mediated largely through glutamate and GABA(B) rather than GABA(A) receptors in mice.     

Effect of antioxidant treatment on spinal GABA neurons in a neuropathic pain model in the mouse

One feature of neuropathic pain is a reduced spinal gamma-aminobutyric acid (GABA)-ergic inhibitory function. However, the mechanisms behind this attenuation remain to be elucidated. This study investigated the involvement of reactive oxygen species in the spinal GABA neuron loss and reduced GABA neuron excitability in spinal nerve ligation (SNL) model of neuropathic pain in mice. The importance of spinal GABAergic inhibition in neuropathic pain was tested by examining the effects of intrathecally administered GABA receptor agonists and antagonists in SNL and naïve mice, respectively. The effects of SNL and antioxidant treatment on GABA neuron loss and functional changes were examined in transgenic GAD67-enhanced green fluorescent protein positive (EGFP+) mice. GABA receptor agonists transiently reversed mechanical hypersensitivity of the hind paw in SNL mice. On the other hand, GABA receptor antagonists made naïve mice mechanically hypersensitive. Stereological analysis showed that the numbers of enhanced green fluorescent protein positive (EGFP+) GABA neurons were significantly decreased in the lateral superficial laminae (I–II) on the ipsilateral L5 spinal cord after SNL. Repeated antioxidant treatments significantly reduced the pain behaviors and prevented the reduction in EGFP+ GABA neurons. The response rate of the tonic firing GABA neurons recorded from SNL mice increased with antioxidant treatment, whereas no change was seen in those recorded from naïve mice, which suggested that oxidative stress impaired some spinal GABA neuron activity in the neuropathic pain condition. Together the data suggest that neuropathic pain, at least partially, is attributed to oxidative stress, which induces both a GABA neuron loss and dysfunction of surviving GABA neurons.     

Inverse agonism at heptahelical receptors: concept, experimental approach and therapeutic potential

Inverse agonists (negative antagonists) are ligands that stabilize the inactive conformation (R) of receptors according to the two-state receptor model. The active conformation (R*) of heptahelical receptors, i.e. G protein-coupled receptors, has high affinity for G proteins. According to ternary complex models of receptor activation, the R*G complex is in equilibrium with R + G, with spontaneous activity in the absence of agonist. Inverse agonists, having a higher affinity for R, shift R*G towards R + G, decreasing the spontaneous activity of receptors. Agonists have the opposite effect, with a higher affinity for R*. Neutral antagonists have the same affinity for R and R* and compete for both agonists and inverse agonists. Inverse agonists have been recently proposed for a variety of heptahelical receptors. Methods to detect inverse agonists among antagonists are based on the determination of ligand affinity at R and R* with binding experiments, and on the modulation of G protein activity (GTP binding and hydrolysis) or of effector activity. Receptor inverse agonists, but also G protein antagonists and GTPase inhibitors, decrease spontaneous G protein activity corresponding to R*G. Receptor agonists, G protein agonists and GTPase inhibitors increase effector basal activity, but receptor inverse agonists decrease it. The therapeutic potential of inverse agonists is proposed in human diseases ascribed to constitutively active mutant receptors and may be extended to diseases related to wild-type receptor over-expression leading to the increase of R*. Some of the therapeutic effects of presently used receptor antagonists may be related to their inverse agonist properties. Inverse agonists lead to receptor upregulation, offering new approaches to tolerance and dependence to drugs.     

Positive modulation of GABA(B) receptors decreased nicotine self-administration and counteracted nicotine-induced enhancement of brain reward function in rats

Acute administration of gamma-aminobutyric acid (GABA)-B receptor agonists decreases nicotine, cocaine, ethanol, and heroin self-administration and also decreases food-maintained responding and suppresses locomotor activity at high doses. GABA(B) receptor-positive modulators may represent potentially improved therapeutic compounds because of their fewer side effects than receptor agonists. The present study investigated the effects of administration of the GABA(B) receptor-positive modulators 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930) and N-[(1R,2R,4S)-bicyclo[2.2.1]hept-2-yl]-2-methyl-5-[4-(trifluoromethyl)phenyl]-4-pyrimidinamine (BHF177) and coadministration of the GABA(B) receptor-positive modulator N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783) with the GABA(B) receptor agonist (3-amino-2[S]-hydroxypropyl)-methylphosphinic acid (CGP44532) on nicotine- and food-maintained responding under fixed ratio (FR) 5 and progressive ratio schedules of reinforcement. Furthermore, the effects of BHF177 and CGP44532 on nicotine-induced enhancement of brain reward function were evaluated. The results indicated that administration of CGP7930 decreased nicotine self-administration under an FR5 schedule. Administration of either GS39783 or CGP44532 selectively decreased nicotine self-administration, whereas coadministration of these compounds had additive effects. BHF177 administration selectively decreased nicotine- but not food-maintained responding under FR5 and progressive ratio schedules. The nicotine-induced enhancement of brain reward function was blocked by BHF177 or CGP44532, although the highest doses of both compounds, particularly CGP44532, decreased brain reward function when administered alone, suggesting an additive, rather than interactive, effect. Overall, the present results indicate that GABA(B) receptor-positive modulators, similarly to GABA(B) receptor agonists, attenuated the reinforcing and reward-enhancing effects of nicotine, perhaps with higher selectivity than GABA(B) receptor agonists. Thus, GABA(B) receptor-positive modulators may be useful antismoking medications.     

GABAB1 receptor subunit isoforms exert a differential influence on baseline but not GABAB receptor agonist-induced changes in mice

GABA(B) receptor agonists produce hypothermia and motor incoordination. Two GABA(B(1)) receptor subunit isoforms exist, but because of lack of specific molecular or pharmacological tools, the relevance of these isoforms in controlling basal body temperature, locomotor activity, or in vivo responses to GABA(B) receptor agonists has been unknown. Here, we used mice deficient in the GABA(B(1a)) and GABA(B(1b)) subunit isoforms to examine the influence of these isoforms on both baseline motor behavior and body temperature and on the motor-incoordinating and hypothermic responses to the GABA(B) receptor agonists l-baclofen and gamma-hydroxybutyrate (GHB). GABA(B(1b))(-/-) mice were hyperactive in a novel environment and showed slower habituation than either GABA(B(1a))(-/-) or wild-type mice. GABA(B(1b))(-/-) mice were hyperactive throughout the circadian dark phase. Hypothermia in response to l-baclofen (6 and 12 mg/kg) or GHB (1 g/kg), baclofen-induced ataxia as determined on the fixed-speed Rotarod, and GHB-induced hypolocomotion were significantly, but for the most part similarly, attenuated in both GABA(B(1a))(-/-) and GABA(B(1b))(-/-) mice. We conclude that l-baclofen and GHB are nonselective for either GABA(B(1)) receptor isoform in terms of in vivo responses. However, GABA(B(1)) receptor isoforms have distinct and different roles in mediating locomotor behavioral responses to a novel environment. Therefore, GABA(B(1a)) and GABA(B(1b)) isoforms are functionally relevant molecular variants of the GABA(B(1)) receptor subunit, which are differentially involved in specific neurophysiological processes and behaviors.     

Incidence of mononeuropathy in rats is influenced by pre-emptive alteration of spinal excitability.

Both clinical and experimental data support the notion that the development of neuropathic pain is related to the state of excitability at the time of nerve injury. The present study was performed to investigate whether altering spinal excitability immediately before creation of a chronic constriction nerve injury in rats can influence the incidence of tactile hypersensitivity ('allodynia') by using pre-emptive: (1) intrathecal injection of a GABAB agonist or antagonists; (2) intrathecal lidocaine; or (3) C-fibre activation by electric stimulation. The incidence of tactile hypersensitivity was significantly reduced by the GABA(B) agonist baclofen while it was markedly enhanced by the administration of the GABA(B) antagonists 5-AVA and CGP 55845, as well as by C-fibre stimulation. Intrathecal administration of lidocaine did not influence the incidence of hypersensitivity. The results suggest that GABAergic mechanisms play an important role in the development of tactile hypersensitivity, and suggest that GABA(B) receptor agonists may be used as pre-emptive treatment to prevent the development of postinjury neuropathic pain.     

Ethanol potentiation of GABAergic transmission in cultured spinal cord neurons involves gamma-aminobutyric acidA-gated chloride channels

The interaction of ethanol with gamma-aminobutyric acid (GABA)-mediated 36-Cl-influx and its modulation by various drugs was investigated in C57 mice spinal cord cultured neurons. Ethanol (5-100 mM) potentiated the effect of GABA on 36Cl-influx; whereas at concentrations greater than or equal to 50 mM ethanol activated Cl- channels directly. The effect of ethanol was specific for GABAA receptor-gated Cl- channels, as ethanol did not potentiate glycine-induced 36Cl-influx in the same neurons. Both the enhancing and direct effects of ethanol on 36Cl-influx were blocked by GABA antagonists like bicuculline, picrotoxinin and inverse agonists of the benzodiazepine site like the imidazodiazepine R015-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5 alpha], [1,4]benzodiazepine-3-carboxylate) and N-methyl-beta-carboline-3-carboxamide (FG-7142). Ethanol potentiating effect of GABA-induced 36Cl-influx was also reversed by methyl-6,7-dimethyl-4-ethyl-beta-carboline-3-carboxylate. The effects of the inverse agonists were blocked by the benzodiazepine receptor antagonist R015-1788. Both R015-4513 and FG-7142 reversed direct and GABA potentiating effects of ethanol effect at concentrations lower than those that exhibit inverse agonistic activity in the 36Cl-influx assay in cultured neurons. These results suggest that ethanol facilitation of GABAAergic transmission involves GABA receptor-gated Cl- channels and that this interaction may be responsible for some of the pharmacological effects of ethanol.     

Ethanol enhances gamma-aminobutyric acid responses in a subpopulation of nucleus accumbens neurons: role of metabotropic glutamate receptors

The nucleus accumbens (NAcc) may be a key area in the rewarding effects of abused drugs. We previously showed that low ethanol concentrations decreased both N-methyl-D-aspartate (NMDA)-induced and kainate-induced currents in NAcc core neurons. To explore the effects of ethanol on gamma-aminobutyric acid (GABA) responses in NAcc, we used intracellular voltage-clamp recordings and locally applied GABA in a slice preparation containing the NAcc. Ethanol (11-200 mM) had no effect on resting membrane properties, but 11, 22, 44, 100, and 200 mM ethanol increased GABA currents in 17, 33, 45, 50, and 22% of cells, respectively. Superfusion of low glutamate concentrations that had no direct effect on membrane properties enhanced ethanol potentiation of GABA currents in more than half the NAcc cells. Neither alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate receptor nor NMDA receptor antagonists affected the percentage of cells showing ethanol enhancement of GABA responses or the degree of ethanol enhancement of GABA currents in NAcc neurons. However, in ethanol-sensitive cells, the metabotropic receptor antagonist alpha-methyl-4-carboxyphenylglycine (MCPG) blocked the ethanol enhancement of GABA currents. In addition, the metabotropic receptor agonist trans-1-aminocyclopentane-1,3-dicarboxylic acid enhanced GABA responses in 50% of cells tested, an effect blocked by MCPG. These data suggest that NAcc core neurons possess both ethanol-sensitive and -insensitive GABA receptors and that glutamate can mimic and enhance the ethanol potentiation of GABA currents in many of these neurons. Furthermore, the ethanol potentiation of GABA currents may involve metabotropic glutamate receptors, perhaps via a phosphorylation mechanism that regulates ethanol sensitivity of GABA receptors in some NAcc neurons.     

gamma-Aminobutyric acid receptor-regulated 36Cl- flux in mouse cortical slices

An improved neurochemical assay for gamma-aminobutyric acid (GABA) function has been developed using tracer-radioactive chloride efflux in mouse cortical slices. Careful maintenance of the brain slice viability resulted in a 3-fold stimulation of 36Cl- efflux rate by the GABA agonist muscimol (EC50 = 3 microM), comparable to electrophysiologic and other chloride flux preparations. The shape of the muscimol dose-response curve was shallow, suggestive of negative cooperativity or heterogeneous receptors, but tissue uptake of agonist, possible diffusion barriers and apparent functional desensitization complicated these results. The response to muscimol was inhibited by GABAA receptor antagonists such as RU5135 and was enhanced by barbiturates and benzodiazepines. As observed previously, barbiturates stimulated 36Cl- efflux rate on their own and potentiated the response (potency and maximal effect) to muscimol in a stereospecific and picrotoxin-sensitive manner. Benzodiazepine receptor ligands alone did not alter 36Cl- flux, but agonists such as flunitrazepam enhanced the response to muscimol, an effect sensitive to the antagonist Ro15-1788. The inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate did not inhibit muscimol-activated 36Cl- flux. The anthelminthic-insecticide avermectin B1a stimulated 36Cl- flux by itself, and this response was apparently additive with that of muscimol. This brain slice chloride flux assay is therefore suitable for the assessment of activity including dose-response curves for GABAA agonists, antagonists and modulators including benzodiazepines.     

Interactions of carbon monoxide and metabotropic glutamate receptor groups in the nucleus tractus solitarii of rats

Carbon monoxide has been shown to act as a neurotransmitter and neuronal messenger in the brain. Heme oxygenase catalyzes the conversion of heme to carbon monoxide and biliverdin. We have recently reported that carbon monoxide was involved in central cardiovascular regulation. Carbon monoxide modulated the baroreflex and may affect glutamatergic neurotransmission. In addition, metabotropic glutamate receptors may be coupled to the activation of heme oxygenase in the nucleus tractus solitarii of rats. The present study was designed to investigate the possible interactions of carbon monoxide and metabotropic glutamate receptor groups in the nucleus tractus solitarii. Unilateral microinjection of several agonists for metabotropic glutamate receptor groups such as (R,S)-3,5-dihydroxyphenylglycine (DHPG) (group I) (0.03 nmol), 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (APDC) (group II) (0.3 nmol), and l-(+)-2-amino-4-phosphonobutyric acid (l-AP4) (group III) (0.3 nmol) produced a significant decrease in blood pressure and heart rate. Among the metabotropic glutamate receptor agonists, prior administration of zinc protoporphyrin IX, an inhibitor of heme oxygenase activity, significantly attenuated the cardiovascular effects of APDC and l-AP4, and failed to attenuate the cardiovascular responses of DHPG. These results indicated interactions between carbon monoxide and group II and III metabotropic glutamate receptors in central cardiovascular regulation.     

A functional assay to measure postsynaptic gamma-aminobutyric acidB responses in cultured spinal cord neurons: heterologous regulation of the same K+ channel.

The stimulation of postsynaptic gamma-aminobutyric acid (GABA)B receptors leads to slow inhibitory postsynaptic potentials due to the influx of K(+)-ions. This was studied biochemically, in vitro in mammalian cultured spinal cord neurons by using 86Rb as a substitute for K+. (-)-Baclofen, a GABAB receptor agonist, produced a concentration-dependent increase in the 86Rb-influx. This effect was stereospecific and blocked by GABAB receptor antagonists like CGP 35 348 (3-aminopropyl-diethoxymethyl-phosphonic acid) and phaclofen. Apart from the GABAB receptors, both adenosine via adenosine1 receptors and 5-hydroxytryptamine (5-HT) via 5-HT1 alpha agonists also increased the 86Rb-influx. These agonists failed to show any additivity between them when they were combined in their maximal concentration. In addition, their effect was antagonized specifically by their respective antagonists without influencing the others. These findings suggest the presence of GABAB, adenosine1 and 5-HT1 alpha receptors in the cultured spinal cord neurons, which exhibit a heterologous regulation of the same K(+)-channel. The effect of these agonists were antagonized by phorbol 12,13-didecanoate, an activator of protein kinase C, and pretreatment with pertussis toxin. This suggests that these agonists by acting on their own receptors converge on the same K(+)-channel through the Gi/Go proteins. In summary, we have developed a biochemical functional assay for studying and characterizing GABAB synaptic pharmacology in vitro, using spinal cord neurons.     

Metabotropic glutamate receptor ligands as possible anxiolytic and antidepressant drugs

Depression and anxiety represent a major problem. However, the current treatment of both groups of diseases is not satisfactory. As the glutamatergic system may play an important role in pathophysiology of both depression and anxiety, we decided to discuss the recent data on possible anxiolytic and/or antidepressant effects of metabotropic glutamate (mGlu) receptor ligands. Preclinical data indicated that antagonists of group I mGlu receptors, particularly antagonists of mGlu5 receptors, produced both anxiolytic-like and antidepressant-like effects. Clinical data also demonstrated that mGlu5 receptor antagonist, fenobam, was an active anxiolytic drug. The anxiolytic effects exerted by mGlu5 receptor antagonists are profound, comparable with or stronger than those of benzodiazepines. However, the problem with the psychotomimetic activity of mGlu5 receptor antagonists and their possible influence on memory has to be further investigated. Among all mGlu receptor ligands, group II mGlu receptor agonists seem to be the drugs with the most promising therapeutic potential and a good safety profile. Animal studies showed anxiolytic-like effects of group II mGlu receptor agonists. Currently, group II mGlu receptor agonists are in phase III clinical trials for potential treatment of anxiety disorders. On the other hand, data has been accumulated, indicating that antagonists of group II mGlu receptors have an antidepressant potential. Group III mGlu receptor ligands represent the least investigated group of mGlu receptors. However, preclinical data also indicates that ligands of these receptors, both agonists and antagonists, may have an anxiolytic-like and antidepressant-like potential.     

Deletion of the fyn-kinase gene alters sensitivity to GABAergic drugs: dependence on beta2/beta3 GABAA receptor subunits

Tyrosine phosphorylation can modulate GABA(A) receptor function, and deletion of the fyn-kinase gene alters GABAergic function in olfactory bulb neurons, as reported by Kitazawa, Yagi, Miyakawa, Niki, and Kawai (J Neurophysiol 1998;79:137-142). Our goal was to determine whether fyn gene deletion altered behavioral and functional actions of compounds that act on GABA(A) receptors. Such evidence might suggest a role for fyn-kinase in modulating GABA(A) receptor function, possibly via direct interactions between the kinase and receptor. Using the loss of righting reflex test, we found that null mutants were less sensitive to the hypnotic effects of THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol), a GABA(A) receptor agonist. Subunit specificity was suggested by the observation that null mutants were also less sensitive to the hypnotic effects of etomidate, a GABAergic compound that is selective for receptors possessing beta2 and/or beta3 receptor subunits. The genotypes did not differ in sensitivity to zolpidem, an alpha1-selective GABAergic drug. GABA(A) receptor functional assays ((36)Cl(-) influx) supported our behavioral results; the actions of the GABA(A) agonists, THIP and muscimol, were reduced in the cerebellar membranes of fyn-null mutant mice. Importantly, similar results were seen with etomidate. Binding of [(3)H]flunitrazepam supported the idea that this is due to a decrease in functional GABA(A) receptor density. These data suggest that fyn-kinase may alter the function of GABA(A) receptors, perhaps via actions on beta2 and/or beta3 receptor subunits.     

Constitutive activity of histamine h(3) receptors stably expressed in SK-N-MC cells: display of agonism and inverse agonism by H(3) antagonists.

Agonist-independent activity of G-protein-coupled receptor, also referred to as constitutive activity, is a well-documented phenomenon and has been reported recently for both the histamine H(1) and H(2) receptors. Using SK-N-MC cell lines stably expressing the human and rat H(3) receptors at physiological receptor densities (500-600 fmol/mg of protein), we show that both the rat and human H(3) receptors show a high degree of constitutive activity. The forskolin-mediated cAMP production in SK-N-MC cells is inhibited strongly upon expression of the G(i)-coupled H(3) receptor. The cAMP production can be further inhibited upon agonist stimulation of the H(3) receptor and can be enhanced by a variety of H(3) antagonists acting as inverse agonists at the H(3) receptor. Thioperamide, clobenpropit, and iodophenpropit raise the cAMP levels in SK-N-MC cells with potencies that match their receptor binding affinities. Surprisingly, impentamine and burimamide act as effective H(3) agonists. Modification of the amine group of impentamine dramatically affected the pharmacological activity of the ligand. Receptor affinity was reduced slightly for most impentamine analogs, but the functional activity of the ligands varied from agonist to neutral antagonist and inverse agonist, indicating that subtle changes in the chemical structures of impentamine analogs have major impact on the (de)activation steps of the H(3) receptor. In conclusion, upon stable expression of the rat and human H(3) receptor in SK-N-MC cells constitutive receptor activity is detected. In this experimental system, H(3) receptors ligands, previously identified as H(3) antagonists, cover the whole spectrum of pharmacological activities, ranging from full inverse agonists to agonists.     

Dopamine-independent locomotion following blockade of N-methyl-D-aspartate receptors in the ventral tegmental area.

Compounds acting in the ventral tegmental area to increase motor activity are thought to do so by activating mesolimbic dopamine transmission. The present report demonstrates that the microinjection of N-methyl-D-aspartate (NMDA) antagonists into the ventral tegmental area produces a dose-dependent increase in motor activity. This effect was not mimicked by antagonizing either alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate or metabotropic glutamate receptors in the ventral tegmental area. Three experiments were conducted that indicated that the capacity of NMDA receptor antagonists to elevate motor activity did not involve increased dopamine transmission. 1) The systemic administration of a D1 dopamine receptor antagonist did not inhibit the motor stimulant response to NMDA antagonist injection into the ventral tegmental area except at doses that also inhibited motor activity after an injection of saline into the ventral tegmental area. 2) Stimulating orphanin receptors in the ventral tegmental area selectively inhibits dopamine cells, and this did not alter NMDA antagonist-induced motor activity. Whereas, stimulating gamma-aminobutyric acid (GABA)(B) receptors hyperpolarizes both dopamine and GABA cells in the ventral tegmental area, and this abolished NMDA antagonist-induced motor activity. 3) The microinjection of an NMDA antagonist into the ventral tegmental area did not increase dopamine metabolism in dopamine terminal fields, including the accumbens, striatum, or prefrontal cortex. Also consistent with a lack of dopamine involvement, repeated administration of NMDA antagonist into the ventral tegmental area did not produce behavioral sensitization. These data identify a mechanism to elicit a motor stimulant response from the ventral tegmental area that does not involve activating dopamine transmission.     

GABA(B) receptor activation in the ventral tegmental area inhibits the acquisition and expression of opiate-induced motor sensitization

Opiate-induced motor sensitization refers to the progressive and enduring motor response that develops after intermittent drug administration, and results from neuroadaptive changes in ventral tegmental area (VTA) and nucleus accumbens (NAc) neurons. Repeated activation of mu-opioid receptors localized on gamma-aminobutyric acid (GABA) neurons in the VTA enhances dopaminergic cell activity and stimulates dopamine release in the nucleus accumbens. We hypothesize that GABA(B) receptor agonist treatment in the VTA blocks morphine-induced motor stimulation, motor sensitization, and accumbal Fos immunoreactivity by inhibiting the activation of dopaminergic neurons. First, C57BL/6 mice were coadministered a single subcutaneous injection of morphine with intra-VTA baclofen, a GABA(B) receptor agonist. Baclofen produced a dose-dependent inhibition of opiate-induced motor stimulation that was attenuated by 2-hydroxysaclofen, a GABA(B) receptor antagonist. Next, morphine was administered on days 1, 3, 5, and 9 and mice demonstrated sensitization to its motor stimulant effects and concomitant induction of Fos immunoreactivity in the NAc shell (NAcS) but not NAc core. Intra-VTA baclofen administered during morphine pretreatment blocked the acquisition of morphine-induced motor sensitization and Fos activation in the NAcS. Intra-VTA baclofen administered only on day 9 blocked the expression of morphine-induced motor sensitization and Fos activation in the NAcS. A linear relationship was found between morphine-induced motor activity and accumbal Fos in single- and repeated-dose treatment groups. In conclusion, GABA(B) receptor stimulation in the VTA blocked opiate-induced motor stimulation and motor sensitization by inhibiting the activation of NAcS neurons. GABA(B) receptor agonists may be useful pharmacological treatments in altering the behavioral effects of opiates.     

Multiple differences in agonist and antagonist pharmacology between human and guinea pig histamine H1-receptor

Species isoforms of histamine H2-, H3-, and H4-receptors differ in their pharmacological properties. The study aim was to dissect differences between the human H1R (hH1R) and guinea pig H1R (ghH1R). We coexpressed hH1R and gpH1R with regulators of G-protein signaling in Sf9 insect cells and analyzed the GTPase activity of Gq-proteins. Small H1R agonists showed similar effects at hH1R and gpH1R, whereas bulkier 2-phenylhistamines and histaprodifens were up to approximately 10-fold more potent at gpH1R than at hH1R. Most 2-phenylhistamines and histaprodifens were more efficacious at gpH1R than at hH1R. Several first-generation H1R antagonists were approximately 2-fold, and arpromidine-type H1R antagonists up to approximately 10-fold more potent at gpH1R than at hH1R. [3H]Mepyramine competition binding studies confirmed the potency differences of the GTPase studies. Phe-153-->Leu-153 or Ile-433-->Val-433 exchange in hH1R (hH1R-->gpH1R) resulted in poor receptor expression, low [3H]mepyramine affinity, and functional inactivity. The Phe-153-->Leu-153/Ile-433-->Val-433 double mutant expressed excellently but only partially changed the pharmacological properties of hH1R. Small H1R agonists and 2-phenylhistamines interacted differentially with human and guinea pig H2R in terms of potency and efficacy, respectively. Our data show the following: 1) there are differences in agonist- and antagonist-pharmacology of hH1R and gpH1R encompassing diverse classes of bulky ligands. These differences may be explained by higher conformational flexibility of gpH1R relative to hH1R; 2) Phe-153 and Ile-433 are critical for proper folding and expression of hH1R; and 3) H2R species isoforms distinguish between H1R agonists.