RESUMEN
The medicinal plant Bryophyllum pinnatum was previously shown to block oxytocin (OT)-induced signals in myometrial cells, consistent with its tocolytic effect observed in patients. OT activates not only OT receptors but also V1A receptors, two receptors with high receptor homology that are both expressed in the myometrium and play a crucial role in myometrial contraction signaling. We aimed to study the molecular pharmacology of B. pinnatum herbal preparations using specific receptor ligands, the human myometrial cell line hTERT-C3, and cell lines expressing recombinant human OT and V1A receptors.We found that press juice from B. pinnatum (BPJ) inhibits both OT- and vasopressin (AVP)-induced intracellular calcium increases in hTERT-C3 myometrial cells. In additional assays performed with cells expressing recombinant receptors, BPJ also inhibited OT and V1A receptor-mediated signals with a similar potency (IC50 about 0.5 mg/mL). We further studied endogenous OT- and AVP-sensitive receptors in hTERT-C3 cells and found that OT and AVP stimulated those receptors with similar potency (EC50 of ~ 1 nM), suggesting expression of both receptor subtypes. This interpretation was corroborated by the antagonist potencies of atosiban and relcovaptan that we found. However, using qPCR, we almost exclusively found expression of OT receptors suggesting a pharmacological difference between recombinant OT receptors and native receptors expressed in hTERT-C3 cells.In conclusion, we show that B. pinnatum inhibits both OT and AVP signaling, which may point beyond its tocolytic effects to other indications involving a disbalance in the vasopressinergic system.
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Kalanchoe , Miometrio , Oxitocina , Receptores de Oxitocina , Transducción de Señal , Vasopresinas , Humanos , Oxitocina/farmacología , Femenino , Kalanchoe/química , Receptores de Oxitocina/metabolismo , Miometrio/efectos de los fármacos , Miometrio/metabolismo , Transducción de Señal/efectos de los fármacos , Vasopresinas/farmacología , Vasopresinas/metabolismo , Extractos Vegetales/farmacología , Receptores de Vasopresinas/metabolismo , Receptores de Vasopresinas/genética , Vasotocina/farmacología , Vasotocina/análogos & derivados , Línea Celular , Pirrolidinas/farmacología , Calcio/metabolismo , IndolesRESUMEN
In this work, we investigated the technical feasibility of 'on-demand' production of selected drugs to cover their demand for a time window of 90 days. We focused on two sub-processes 'automated chemical synthesis' and 'formulation in micropellets' to enable personalized dosing. The production of drugs 'on-demand' is challenging, important, but also attractive. Switzerland could thus gain access to an additional instrument for increasing resilience for supply-critical drugs. The biggest challenge in the case study presented here is the scalability of automated chemical synthesis and the application range of micropellet formulations.
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Preparaciones Farmacéuticas , Suiza , Preparaciones Farmacéuticas/provisión & distribuciónRESUMEN
We describe the discovery and optimization of new, brain-penetrant T-type calcium channel blockers. We present optimized compounds with excellent efficacy in a rodent model of generalized absence-like epilepsy. Along the fine optimization of a chemical series with a pharmacological target located in the CNS (target potency, brain penetration, and solubility), we successfully identified an Ames negative aminopyrazole as putative metabolite of this compound series. Our efforts culminated in the selection of compound 20, which was elected as a preclinical candidate.
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Bloqueadores de los Canales de Calcio/uso terapéutico , Canales de Calcio Tipo T/efectos de los fármacos , Descubrimiento de Drogas , Epilepsia Generalizada/tratamiento farmacológico , Animales , Canales de Calcio Tipo T/fisiología , Modelos Animales de Enfermedad , Humanos , Ratones , RatasRESUMEN
A series of dihydropyrazole derivatives was developed as potent, selective, and brain-penetrating T-type calcium channel blockers. An optimized derivative, compound 6c, was advanced to in vivo studies, where it demonstrated efficacy in the WAG/Rij rat model of generalized nonconvulsive, absence-like epilepsy. Compound 6c was not efficacious in the basolateral amygdala kindling rat model of temporal lobe epilepsy, and it led to prolongation of the PR interval in ECG recordings in rodents.
Asunto(s)
Anticonvulsivantes/química , Anticonvulsivantes/uso terapéutico , Bloqueadores de los Canales de Calcio/química , Bloqueadores de los Canales de Calcio/uso terapéutico , Epilepsia/tratamiento farmacológico , Pirazoles/química , Pirazoles/uso terapéutico , Animales , Anticonvulsivantes/farmacocinética , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/fisiopatología , Bloqueadores de los Canales de Calcio/farmacocinética , Canales de Calcio Tipo T/metabolismo , Modelos Animales de Enfermedad , Perros , Electroencefalografía , Epilepsia/metabolismo , Epilepsia/fisiopatología , Humanos , Excitación Neurológica/efectos de los fármacos , Masculino , Pirazoles/farmacocinética , Ratas WistarRESUMEN
Glutamate transmission and synaptic plasticity in the amygdala are essential for the learning and expression of conditioned fear. Glutamate activates both ionotropic glutamate receptors and eight subtypes of metabotropic glutamate receptors (mGlu1-8). In the present study, we investigated the roles of mGlu7 and mGlu8 in amygdala-dependent behavior and synaptic plasticity. We show that ablation of mGlu7 but not mGlu8 attenuates long-term potentiation (LTP) at thalamo-lateral amygdala (LA) synapses where a strong association between LTP and learning has been demonstrated. mGlu7-deficient mice express a general deficit in conditioned fear whereas mGlu8-deficient mice show a dramatic reduction in contextual fear. The mGlu7 agonist AMN082 reduced thalamo-LA LTP and intra-amygdala administration blocked conditioned fear learning. In contrast, the mGlu8 agonist DCPG decreased synaptic transmission but not LTP at thalamo-LA synapses. Intra-amygdala DCPG selectively reduced the expression of contextual fear but did not affect the acquisition and expression of cued fear. Taken together, these data revealed very different roles for mGlu7 and mGlu8 in amygdala synaptic transmission, fear learning and its expression. These receptors seem promising targets for treating anxiety disorders with different underlying pathologies with exaggerated fear learning (mGlu7) or contextual fear (mGlu8).
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Amígdala del Cerebelo/fisiología , Condicionamiento Psicológico/fisiología , Receptores de Glutamato Metabotrópico/metabolismo , Amígdala del Cerebelo/efectos de los fármacos , Animales , Biofisica , Condicionamiento Psicológico/efectos de los fármacos , Estimulación Eléctrica , Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Miedo/efectos de los fármacos , Miedo/fisiología , Técnicas In Vitro , Potenciación a Largo Plazo/efectos de los fármacos , Potenciación a Largo Plazo/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Movimiento/efectos de los fármacos , Movimiento/fisiología , Receptores de Glutamato Metabotrópico/deficiencia , Factores de TiempoRESUMEN
Neurons derived from human-induced pluripotent stem cells were characterized using manual and automated patch-clamp recordings. These cells expressed voltage-gated Na(+) (Na(v)), Ca(2+) (Ca(v)), and K(+) (K(v)) channels as expected from excitable cells. The Na(v) current was TTX sensitive, IC(50) = 12 ± 6 nM (n = 5). About 50% of the Ca(v) current was blocked by 10 µM of the L-type channel blocker nifedipine. Two populations of the K(v) channel were present in different proportions: an inactivating (A-type) and a noninactivating type. The A-type current was sensitive to 4-AP and TEA (IC(50) = 163 ± 93 µM; n = 3). Application of γ-aminobutyric acid (GABA) activated a current sensitive to the GABA(A) receptor antagonist bicuculline, IC(50) = 632 ± 149 nM (n = 5). In both devices, comparable action potentials were generated in the current clamp. With unbiased, automated patch clamp, about 40% of the cells expressed Na(v) currents, whereas visual guidance in manual patch clamp provided almost a 100% success rate of patching "excitable cells." These results show high potential for pluripotent stem cell-derived neurons as a useful model for drug discovery, in combination with automated patch-clamp recordings for high-throughput and high-quality drug assessments at human neuronal ion channels in their correct cellular background.
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Células Madre Pluripotentes Inducidas/citología , Activación del Canal Iónico/efectos de los fármacos , Canales Iónicos/metabolismo , Neuronas/metabolismo , Técnicas de Placa-Clamp/métodos , Bicuculina/farmacología , Canales de Calcio/metabolismo , Diferenciación Celular , Descubrimiento de Drogas/métodos , Humanos , Concentración 50 Inhibidora , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas/efectos de los fármacos , Nifedipino/farmacología , Canales de Potasio con Entrada de Voltaje/metabolismo , Tetrodotoxina/farmacología , Canales de Sodio Activados por Voltaje/metabolismo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Links between synaptic plasticity in the lateral amygdala (LA) and Pavlovian fear learning are well established. Neuropeptides including gastrin-releasing peptide (GRP) can modulate LA function. GRP increases inhibition in the LA and mice lacking the GRP receptor (GRPR KO) show more pronounced and persistent fear after single-trial associative learning. Here, we confirmed these initial findings and examined whether they extrapolate to more aspects of amygdala physiology and to other forms of aversive associative learning. GRP application in brain slices from wildtype but not GRPR KO mice increased spontaneous inhibitory activity in LA pyramidal neurons. In amygdala slices from GRPR KO mice, GRP did not increase inhibitory activity. In comparison to wildtype, short- but not long-term plasticity was increased in the cortico-lateral amygdala (LA) pathway of GRPR KO amygdala slices, whereas no changes were detected in the thalamo-LA pathway. In addition, GRPR KO mice showed enhanced fear evoked by single-trial conditioning and reduced spontaneous firing of neurons in the central nucleus of the amygdala (CeA). Altogether, these results are consistent with a potentially important modulatory role of GRP/GRPR signaling in the amygdala. However, administration of GRP or the GRPR antagonist (D-Phe(6), Leu-NHEt(13), des-Met(14))-Bombesin (6-14) did not affect amygdala LTP in brain slices, nor did they affect the expression of conditioned fear following intra-amygdala administration. GRPR KO mice also failed to show differences in fear expression and extinction after multiple-trial fear conditioning, and there were no differences in conditioned taste aversion or gustatory neophobia. Collectively, our data indicate that GRP/GRPR signaling modulates amygdala physiology in a paradigm-specific fashion that likely is insufficient to generate therapeutic effects across amygdala-dependent disorders.
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Amígdala del Cerebelo/fisiología , Miedo/fisiología , Péptido Liberador de Gastrina/metabolismo , Memoria/fisiología , Receptores de Bombesina/metabolismo , Amígdala del Cerebelo/metabolismo , Animales , Bombesina/análogos & derivados , Bombesina/química , Bombesina/farmacología , Condicionamiento Clásico/fisiología , Péptido Liberador de Gastrina/antagonistas & inhibidores , Péptido Liberador de Gastrina/genética , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Neuronas/fisiología , Fragmentos de Péptidos/química , Células Piramidales/metabolismo , Células Piramidales/fisiología , Receptores de Bombesina/antagonistas & inhibidores , Receptores de Bombesina/genética , Transducción de SeñalRESUMEN
A novel series of agonists at the benzodiazepine binding site of the GABA(A) receptor was prepared by functionalizing a known template. Adding substituents to the pyrazolone-oxygen of CGS-9896 led to a number of compounds with selectivities for either α2- or α1-containing GABA(A) receptor subtypes offering an entry into indications such as anxiety and insomnia. In this communication, structure-activity relationship and efforts to increase in vitro stabilities are discussed.
Asunto(s)
Benzodiazepinas/química , Agonistas de Receptores de GABA-A/síntesis química , Pirazoles/química , Receptores de GABA-A , Benzodiazepinas/metabolismo , Sitios de Unión , Estabilidad de Medicamentos , Agonistas de Receptores de GABA-A/química , Agonistas de Receptores de GABA-A/farmacología , Concentración 50 Inhibidora , Ligandos , Estructura Molecular , Pirazoles/metabolismo , Receptores de GABA-A/metabolismo , Relación Estructura-ActividadRESUMEN
GABA(B) receptors are heterodimers of two subunits, GABA(B1) (GB1) and GABA(B2) (GB2). Agonists such as GABA and baclofen bind to the GB1 subunit only, whereas GB2 is essential for G protein activation. Positive allosteric modulators enhance the potency and efficacy of agonists at GABA(B) receptors and are of particular interest because they lack the sedative and muscle relaxant properties of agonists. In this study, we aimed to characterize the interaction of the positive modulator N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783) with the GABA(B) receptor heterodimer. Using functional guanosine 5'-O-(3-[(35)S]thio)triphosphate binding assays, we observed positive modulation by GS39783 in different vertebrate species but not in Drosophila melanogaster. However, coexpression of D. melanogaster GB1 with rat GB2 yielded functional receptors positively modulated by GS39783. Together with data from rat/D. melanogaster GB2 subunit chimeras, this pointed to a critical role of the GB2 transmembrane region for positive modulation. We further characterized GS39783 function using point mutations. GS39783 positively modulated GABA responses but also showed considerable agonistic activity at heterodimers containing a mutant rat GB2 subunit with three amino acid substitutions in transmembrane domain VI. It was surprising that in contrast to wild-type rat GB2, this mutant subunit was also activated by GS39783 when expressed without GB1. The mutations of both G706T and A708P are necessary and sufficient for activation and identify a key region for the effect of GS39783 in the GB2 transmembrane region. Our data show that mutations of specific amino acids in GB2 can induce agonism in addition to positive modulation and facilitate GB2 activation in the absence of GB1.
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Ciclopentanos/farmacología , Agonistas de Receptores GABA-B , Mutación Puntual , Pirimidinas/farmacología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , Dimerización , Drosophila melanogaster , Datos de Secuencia Molecular , Ratas , Receptores de GABA-B/química , Receptores de GABA-B/genética , Receptores de GABA-B/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Functional gamma-aminobutyric acid type B (GABA(B)) receptors are normally only observed upon coexpression of GABA(B1) with GABA(B2) subunits. A C-terminal arginine-based endoplasmic reticulum (ER) retention/retrieval signal, RSRR, prevents escape of unassembled GABA(B1) subunits from the ER and restricts surface expression to correctly assembled heteromeric receptors. The RSRR signal in GABA(B1) is proposed to be shielded by C-terminal coiled-coil interaction of the GABA(B1) with the GABA(B2) subunit. Here, we investigated whether the RSRR motif in GABA(B1) remains functional when grafted to ectopic sites. We found that the RSRR signal in GABA(B1) is inactive in any of the three intracellular loops but remains functional when moved within the distal zone of the C-terminal tail. C-terminal deletions that position the RSRR signal closer to the plasma membrane drastically reduce its effectiveness, supporting that proximity to the membrane restricts access to the RSRR motif. Functional ectopic RSRR signals in GABA(B1) are efficiently inactivated by the GABA(B2) subunit in the absence of coiled-coil dimerization, supporting that coiled-coil interaction is not critical for release of the receptor complex from the ER. The data are consistent with a model in which removal of RSRR from its active zone rather than its direct shielding by coiled-coil dimerization triggers forward trafficking. Because arginine-based intracellular retention signals of the type RXR, where X represents any amino acid, are used to regulate assembly and surface transport of several multimeric complexes, such a mechanism may apply to other proteins as well.
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Membrana Celular/fisiología , Retículo Endoplásmico/fisiología , Receptores de GABA-B/fisiología , Receptores X Retinoide/fisiología , Transducción de Señal/fisiología , Línea Celular , Membrana Celular/genética , Retículo Endoplásmico/genética , Humanos , Receptores de GABA-B/química , Receptores de GABA-B/genética , Receptores X Retinoide/química , Receptores X Retinoide/genéticaRESUMEN
The neuronal nicotinic acetylcholine receptor alpha7 (nAChR alpha7) may be involved in cognitive deficits in Schizophrenia and Alzheimer's disease. A fast pharmacological characterization of homomeric alpha7 receptors is mostly hampered by their low functional expression levels in heterologous expression systems. In the present study expression of homomeric nAChR alpha7 was achieved in GH3 rat pituitary cells. Alpha7 subunits were heterologously expressed as components of [125I]-labeled alpha-bungarotoxin binding nAChRs (Bmax: 1.2 pmol/mg protein). Function of the expressed alpha7 ion channels was assessed by patch-clamp recording and calcium imaging. While acetylcholine-induced currents desensitized within much less than 1 s, calcium-sensitive fluorescence transients peaked after 5-10 s and returned to background levels within 30 s only. The fluorescence signal was blocked by isradipine and removal of extracellular sodium indicated that in these cells opening of rapidly desensitizing alpha7 nAChR triggers calcium influx via voltage-gated, DHP-sensitive calcium channels. In this cellular system, agonists revealed the following rank order of potency: epibatidine>anatoxin A>AAR17779>ABT-594>DMPP>nicotine>GTS-21>cytisine>ABT-418>acetylcholine>choline>ABT-089. All of the signals were inhibited by the alpha7 antagonists alpha-bungarotoxin (pIC50: 7.4) and methyllycaconitine (pIC50: 7.8). Further, marketed antidepressants showed antagonistic activity with the following rank order of potency: fluoxetine>imipramine>paroxetine>sertraline. These data illustrate that coupling to voltage-gated calcium channels allows a rapid and reliable functional examination of nAChR alpha7.
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Canales de Calcio/metabolismo , Hipófisis/metabolismo , Receptores Nicotínicos/metabolismo , Animales , Bloqueadores de los Canales de Calcio/metabolismo , Línea Celular , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Humanos , Isradipino/metabolismo , Hipófisis/citología , Unión Proteica/fisiología , Ratas , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
In the mammalian central nervous system, slow inhibitory neurotransmission is largely mediated by metabotropic GABA(B) receptors (where GABA stands for gamma-aminobutyric acid), which belong to the G-protein-coupled receptor gene family. Functional GABA(B) receptors are assembled from two subunits GABA(B1) (GABA(B) receptor subtype 1) and GABA(B2). For the GABA(B1) subunit, which binds the neurotransmitter GABA, two variants GABA(B1a) (GABA(B) receptor subtype 1 variant a) and GABA(B1b) have been identified. They differ at the very N-terminus of their large glycosylated ECD (extracellular domain). To simplify the structural characterization, we designed truncated GABA(B1) receptors to identify the minimal functional domain which still binds a competitive radioligand and leads to a functional, GABA-responding receptor when co-expressed with GABA(B2). We show that it is necessary to include all the portion of the ECD encoded by exon 6 to exon 14. Furthermore, we studied mutant GABA(B1b) receptors, in which single or all potential N-glycosylation sites are removed. The absence of oligosaccharides does not impair receptor function, suggesting that the unglycosylated ECD of GABA(B1) can be used for further functional or structural investigations.
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Receptores de GABA-B/química , Receptores de GABA-B/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Línea Celular , Dimerización , Exones/genética , Agonistas de Receptores GABA-B , Glicosilación , Humanos , Ligandos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Ratas , Receptores de GABA-B/genética , Alineación de Secuencia , Eliminación de Secuencia/genética , Transducción de SeñalRESUMEN
GABA(B) receptors are broadly expressed in the nervous system and have been implicated in a wide variety of neurological and psychiatric disorders. The cloning of the first GABA(B) receptor cDNAs in 1997 revived interest in these receptors and their potential as therapeutic targets. With the availability of molecular tools, rapid progress was made in our understanding of the GABA(B) system. This led to the surprising discovery that GABA(B) receptors need to assemble from distinct subunits to function and provided exciting new insights into the structure of G protein-coupled receptors (GPCRs) in general. As a consequence of this discovery, it is now widely accepted that GPCRs can exist as heterodimers. The cloning of GABA(B) receptors allowed some important questions in the field to be answered. It is now clear that molecular studies do not support the existence of pharmacologically distinct GABA(B) receptors, as predicted by work on native receptors. Advances were also made in clarifying the relationship between GABA(B) receptors and the receptors for gamma-hydroxybutyrate, an emerging drug of abuse. There are now the first indications linking GABA(B) receptor polymorphisms to epilepsy. Significantly, the cloning of GABA(B) receptors enabled identification of the first allosteric GABA(B) receptor compounds, which is expected to broaden the spectrum of therapeutic applications. Here we review current concepts on the molecular composition and function of GABA(B) receptors and discuss ongoing drug-discovery efforts.
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Receptores de GABA-B/química , Receptores de GABA-B/metabolismo , Animales , Clonación Molecular , Epilepsia/metabolismo , Enfermedades Gastrointestinales/metabolismo , Ligamiento Genético , Humanos , Estructura Molecular , Nociceptores/metabolismo , Receptores de GABA-B/genética , Relación Estructura-Actividad , Trastornos Relacionados con Sustancias/metabolismoRESUMEN
GABAB receptors mediate slow synaptic inhibition in the nervous system. In transfected cells, functional GABAB receptors are usually only observed after coexpression of GABAB(1) and GABAB(2) subunits, which established the concept of heteromerization for G-protein-coupled receptors. In the heteromeric receptor, GABAB(1) is responsible for binding of GABA, whereas GABAB(2) is necessary for surface trafficking and G-protein coupling. Consistent with these in vitro observations, the GABAB(1) subunit is also essential for all GABAB signaling in vivo. Mice lacking the GABAB(1) subunit do not exhibit detectable electrophysiological, biochemical, or behavioral responses to GABAB agonists. However, GABAB(1) exhibits a broader cellular expression pattern than GABAB(2), suggesting that GABAB(1) could be functional in the absence of GABAB(2). We now generated GABAB(2)-deficient mice to analyze whether GABAB(1) has the potential to signal without GABAB(2) in neurons. We show that GABAB(2)-/- mice suffer from spontaneous seizures, hyperalgesia, hyperlocomotor activity, and severe memory impairment, analogous to GABAB(1)-/- mice. This clearly demonstrates that the lack of heteromeric GABAB(1,2) receptors underlies these phenotypes. To our surprise and in contrast to GABAB(1)-/- mice, we still detect atypical electrophysiological GABAB responses in hippocampal slices of GABAB(2)-/- mice. Furthermore, in the absence of GABAB(2), the GABAB(1) protein relocates from distal neuronal sites to the soma and proximal dendrites. Our data suggest that association of GABAB(2) with GABAB(1) is essential for receptor localization in distal processes but is not absolutely necessary for signaling. It is therefore possible that functional GABAB receptors exist in neurons that naturally lack GABAB(2) subunits.
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Hipocampo/fisiopatología , Hiperalgesia/genética , Hipercinesia/genética , Trastornos de la Memoria/genética , Receptores de GABA-B/metabolismo , Convulsiones/genética , Animales , Encéfalo/metabolismo , Encéfalo/patología , Química Encefálica , Dimerización , Electroencefalografía , Agonistas del GABA/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hiperalgesia/patología , Hipercinesia/patología , Trastornos de la Memoria/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Dimensión del Dolor , Técnicas de Placa-Clamp , Canales de Potasio/efectos de los fármacos , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Transporte de Proteínas/genética , Transporte de Proteínas/fisiología , Ensayo de Unión Radioligante , Receptores de GABA-B/genética , Convulsiones/patología , Transducción de Señal/genética , Transducción de Señal/fisiologíaRESUMEN
Based on recent reports describing enhancing actions of arylalkylamines (fendiline [N-(3,3-diphenylpropyl)-alpha-methylbenzylamine] and prenylamine [N-(3,3-diphenylpropyl)-alpha-methylphenethylamine]), amino acids (L-phenylalanine, L-leucine and L-isoleucine), and dipeptides (L-Phe-Phe and L-Phe-Leu) on baclofen-induced responses in cortical slices, we have examined whether these compounds might act as positive allosteric modulators at GABA(B) receptors. Unlike the previously described allosteric GABA(B) receptor modulator CGP7930 (2,6-Di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol), these compounds did not enhance GABA(B) receptor-mediated guanosine 5'-O-(3-thiotriphosphate) [GTP(gamma)35S] binding in native or recombinant cell membrane preparations. Similarly, in a competition binding assay using the antagonist radioligand [3H]CGP62349, CGP7930, but not the other compounds, enhanced the affinities of gamma-aminobutyric acid (GABA) for native GABA(B) receptors from rat brain cortex. Finally, in a cellular assay (Ca(2+) signaling in a recombinant cell line), CGP7930 was again the only compound found to enhance the GABA response. It is concluded that the arylalkylamines, amino acids and dipeptides tested do not act as allosteric modulators at native and recombinant GABA(B) receptors.
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Regulación Alostérica/fisiología , Aminoácidos/metabolismo , Receptores de GABA-B/metabolismo , Regulación Alostérica/efectos de los fármacos , Aminoácidos/farmacología , Animales , Células CHO , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Cricetinae , Dipéptidos/metabolismo , Dipéptidos/farmacología , Relación Dosis-Respuesta a Droga , Agonistas de Receptores GABA-B , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Ratas , Ácido gamma-Aminobutírico/metabolismo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
gamma-Hydroxybutyrate (GHB), a metabolite of gamma-aminobutyric acid (GABA), is proposed to function as a neurotransmitter or neuromodulator. gamma-Hydroxybutyrate and its prodrug, gamma-butyrolactone (GBL), recently received increased public attention as they emerged as popular drugs of abuse. The actions of GHB/GBL are believed to be mediated by GABAB and/or specific GHB receptors, the latter corresponding to high-affinity [3H]GHB-binding sites coupled to G-proteins. To investigate the contribution of GABAB receptors to GHB actions we studied the effects of GHB in GABAB(1)-/- mice, which lack functional GABAB receptors. Autoradiography reveals a similar spatial distribution of [3H]GHB-binding sites in brains of GABAB(1)-/- and wild-type mice. The maximal number of binding sites and the KD values for the putative GHB antagonist [3H]6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene acetic acid (NCS-382) appear unchanged in GABAB(1)-/- compared with wild-type mice, demonstrating that GHB- are distinct from GABAB-binding sites. In the presence of the GABAB receptor positive modulator 2,6-di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol GHB induced functional GTPgamma[35S] responses in brain membrane preparations from wild-type but not GABAB(1)-/- mice. The GTPgamma[35S] responses in wild-type mice were blocked by the GABAB antagonist [3-[[1-(S)-(3,4dichlorophenyl)ethyl]amino]-2-(S)-hydroxy-propyl]-cyclohexylmethyl phosphinic acid hydrochloride (CGP54626) but not by NCS-382. Altogether, these findings suggest that the GHB-induced GTPgamma[35S] responses are mediated by GABAB receptors. Following GHB or GBL application, GABAB(1)-/- mice showed neither the hypolocomotion, hypothermia, increase in striatal dopamine synthesis nor electroencephalogram delta-wave induction seen in wild-type mice. It, therefore, appears that all studied GHB effects are GABAB receptor dependent. The molecular nature and the signalling properties of the specific [3H]GHB-binding sites remain elusive.
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Unión Competitiva , Receptores de GABA-B/metabolismo , Oxibato de Sodio/farmacología , 4-Butirolactona/farmacocinética , Adyuvantes Anestésicos/farmacología , Animales , Anticonvulsivantes/farmacocinética , Autorradiografía , Baclofeno/farmacología , Conducta Animal/efectos de los fármacos , Benzocicloheptenos/farmacocinética , Peso Corporal/efectos de los fármacos , Encéfalo/anatomía & histología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cromatografía Líquida de Alta Presión , Dihidroxifenilalanina/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Electroquímica , Electroencefalografía , Agonistas de Receptores GABA-B , Guanosina 5'-O-(3-Tiotrifosfato)/farmacocinética , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Compuestos Organofosforados/farmacocinética , Fenoles/farmacocinética , Ensayo de Unión Radioligante , Receptores de GABA-B/deficiencia , Receptores de GABA-B/genética , Factores de Tiempo , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Extracellular matrix molecules are important cues in the shaping of nervous system structure and function. Here, we describe a novel mechanism by which the HNK-1 carbohydrate carried by recognition molecules regulates perisomatic inhibition in the hippocampus. Neutralization of HNK-1 activity by an HNK-1 antibody results in GABA(B) receptor-mediated activation of K(+) currents in CA1 pyramidal cells, which elevates extracellular K(+) concentration and reduces evoked GABA release in perisomatic inhibitory synapses. This mechanism is supported by pharmacological analysis in hippocampal slices and data showing that the HNK-1 carbohydrate binds to GABA(B) receptors and inhibits GABA(B) receptor-activated K(+) currents in a heterologous expression system. We suggest that the HNK-1 carbohydrate is involved in homeostatic regulation of GABA(A) receptor-mediated perisomatic inhibition by suppression of postsynaptic GABA(B) receptor activity.
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Antígenos CD57/metabolismo , Homeostasis/fisiología , Receptores de GABA-B/metabolismo , Sinapsis/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismo , Animales , Línea Celular , Hipocampo/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratas , Sinapsis/metabolismoRESUMEN
N,N'-Dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783) and structurally related compounds are described as novel allosteric enhancers of GABA(B) receptor function. They potentiate GABA-stimulated guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding to membranes from a GABA(B)(1b/2)-expressing Chinese hamster ovary cell line at low micromolar concentrations, but do not stimulate [35S]GTPgammaS binding by themselves. Similar effects of GS39783 are seen on native GABA(B) receptors in rat brain membranes. Concentration-response curves with GABA in the presence of different fixed concentrations of GS39783 reveal an increase of both the potency and maximal efficacy of GABA at the GABA(B)(1b/2) heterodimer. In radioligand binding experiments, GS39783 reduces the kinetic rate constants of the association and dissociation of [3H]3-aminopropylphosphinic acid, resulting in a net increase in affinity for the agonist radioligand. In equilibrium binding experiments (displacement of the antagonist ligand [3H]CGP62349), GS39783 increases agonist affinities. Agonist displacement curves are biphasic, probably reflecting the G protein-coupled and uncoupled states of the receptor. The proportion of the high-affinity component is increased by GS39783, suggesting that the G protein coupling of the receptor is also promoted by the positive modulator. We also show that GS39783 has modulatory effects in cellular assays such as GABA(B) receptor-mediated activation of inwardly rectifying potassium channels in Xenopus oocytes and Ca2+ signaling in human embryonic kidney 293 cells. In a more physiological context, GS39783 is shown to suppress paired pulse inhibition in rat hippocampal slices. This effect is reversed by the competitive GABA(B) receptor antagonist CGP55845A and is produced most likely by enhancing the effect of synaptically released GABA at presynaptic GABA(B) receptors.
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Ciclopentanos/farmacología , Agonistas del GABA/farmacología , Agonistas de Receptores GABA-B , Pirimidinas/farmacología , Regulación Alostérica/efectos de los fármacos , Animales , Baclofeno/farmacología , Células CHO , Cricetinae , Ciclopentanos/química , Agonistas del GABA/química , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Humanos , Cinética , Oocitos , Canales de Potasio/metabolismo , Pirimidinas/química , Ensayo de Unión Radioligante , Transducción de Señal , Relación Estructura-Actividad , Radioisótopos de Azufre , Xenopus , Ácido gamma-AminobutíricoRESUMEN
Mammals detect temperature with specialized neurons in the peripheral nervous system. Four TRPV-class channels have been implicated in sensing heat, and one TRPM-class channel in sensing cold. The combined range of temperatures that activate these channels covers a majority of the relevant physiological spectrum sensed by most mammals, with a significant gap in the noxious cold range. Here, we describe the characterization of ANKTM1, a cold-activated channel with a lower activation temperature compared to the cold and menthol receptor, TRPM8. ANKTM1 is a distant family member of TRP channels with very little amino acid similarity to TRPM8. It is found in a subset of nociceptive sensory neurons where it is coexpressed with TRPV1/VR1 (the capsaicin/heat receptor) but not TRPM8. Consistent with the expression of ANKTM1, we identify noxious cold-sensitive sensory neurons that also respond to capsaicin but not to menthol.
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Canales de Calcio/metabolismo , Frío , Neuronas Aferentes/metabolismo , Nociceptores/metabolismo , Termorreceptores/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Secuencia de Aminoácidos , Animales , Ancirinas/química , Células CHO , Capsaicina/farmacología , Células Cultivadas , Cricetinae , Femenino , Proteínas de la Membrana/química , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Canal Catiónico TRPA1 , Canales Catiónicos TRPC , Termorreceptores/químicaRESUMEN
The actions of the anticonvulsant gabapentin [1-(aminomethyl)cyclohexaneacetic acid, Neurontin] have been somewhat enigmatic until recently, when it was claimed to be a gamma-aminobutyric acid-B (GABA(B)) receptor agonist acting exclusively at a heterodimeric complex containing the GABA(B(1a)) splice variant (Mol Pharmacol 2001;59:144-152). In this study, we have investigated the effects of gabapentin on recombinant GABA(B(1a)) and GABA(B(1b)) receptors coexpressed with GABA(B(2)) in five different functional recombinant assays, its ability to inhibit [(3)H]GABA binding in a GABA(B) receptor-selective binding assay using rat synaptic membranes, and its ability to inhibit transient lower esophageal sphincter relaxations in Labrador retriever dogs. Up to a concentration of 1 mM, gabapentin displayed no agonistic effects on either the GABA(B(1a,2)) or the GABA(B(1b,2)) heterodimer, when these were expressed in Xenopus laevis oocytes or mammalian cells and assayed by means of electrophysiology, calcium mobilization, inositol phosphate, and fluorometry assays. Gabapentin did not displace [(3)H]GABA from GABA(B) receptor sites in rat synaptic membranes. Finally, in contrast to the classic GABA(B) receptor agonist baclofen, gabapentin was unable to inhibit transient lower esophageal sphincter relaxations in dogs. Because of high levels of GABA(B(1a)) in the canine nodose ganglion, this finding indirectly supports the inactivity of gabapentin on the GABA(B(1a,2)) heterodimer demonstrated in various in vitro assays. In light of these results, we find it highly questionable that gabapentin is a GABA(B) receptor agonist. Hence, the anticonvulsive effects of the compound have to arise from GABA(B) receptor-independent mechanisms. This also implies that the first GABA(B) receptor splice variant-selective ligand remains to be discovered.