RESUMO
No disponible
Assuntos
Humanos , Masculino , Feminino , Bloqueadores Neuromusculares/administração & dosagem , Bloqueadores Neuromusculares/metabolismo , Bloqueadores Neuromusculares/uso terapêutico , Revisão dos Cuidados de Saúde por Pares/métodos , Anestesiologia/métodos , Anestesiologia/estatística & dados numéricos , Dor/terapia , Medicina Baseada em Evidências/métodos , Sociedades/organização & administração , Sociedades/normas , Sociedades Médicas/organização & administração , Anestesiologia/educação , Fármacos Neuromusculares/efeitos adversos , Bloqueadores Neuromusculares/efeitos adversos , Doenças Neuromusculares/induzido quimicamenteRESUMO
Glycine has multiple neurotransmitter functions in the central nervous system (CNS). In the spinal cord and brainstem of vertebrates, it serves as a major inhibitory neurotransmitter. In addition, it participates in excitatory neurotransmission by modulating the activity of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors. The extracellular concentrations of glycine are regulated by Na+/Cl(-)-dependent glycine transporters (GlyTs), which are expressed in neurons and adjacent glial cells. Considerable progress has been made recently towards elucidating the in vivo roles of GlyTs in the CNS. The generation and analysis of animals carrying targeted disruptions of GlyT genes (GlyT knockout mice) have allowed investigators to examine the different contributions of individual GlyT subtypes to synaptic transmission. In addition, they have provided animal models for two hereditary human diseases, glycine encephalopathy and hyperekplexia. Selective GlyT inhibitors have been shown to modulate neurotransmission and might constitute promising therapeutic tools for the treatment of psychiatric and neurological disorders such as schizophrenia and pain. Therefore, pharmacological and genetic studies indicate that GlyTs are key regulators of both glycinergic inhibitory and glutamatergic excitatory neurotransmission. This chapter describes our present understanding of the functions of GlyTs and their involvement in the fine-tuning of neuronal communication.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Glicina/metabolismo , Glicina/metabolismo , Transmissão Sináptica , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Proteínas da Membrana Plasmática de Transporte de Glicina/deficiência , Proteínas da Membrana Plasmática de Transporte de Glicina/genética , Humanos , Hiperglicinemia não Cetótica/genética , Hiperglicinemia não Cetótica/metabolismo , Camundongos , Camundongos Knockout , Receptores de N-Metil-D-Aspartato/metabolismo , Reflexo Anormal/genética , Transcrição GênicaRESUMO
Glycine is a major inhibitory neurotransmitter in the mammalian CNS (central nervous system). Glycinergic neurotransmission is terminated by the uptake of glycine into glycinergic nerve terminals and neighbouring glial cells. This uptake process is mediated by specific Na(+)/Cl(-)-dependent GlyTs (glycine transporters), GlyT1 and GlyT2. GlyT1, in addition, is thought to regulate the concentration of glycine at excitatory synapses containing NMDARs (N-methyl-D-aspartate receptors), which require glycine as a co-agonist. We have analysed the physiological roles and regulation of GlyT1 and GlyT2 by generating transporter-deficient mice and searching for interacting proteins. Our genetic results indicate that at glycinergic synapses, the glial transporter GlyT1 catalyses the removal of glycine from the synaptic cleft, whereas GlyT2 is required for the re-uptake of glycine into nerve terminals, thereby allowing for neurotransmitter reloading of synaptic vesicles. Both GlyT1 and GlyT2 are essential for CNS function, as revealed by the lethal phenotypes of the respective knockout mice. Mice expressing only a single GlyT1 allele are phenotypically normal but may have enhanced NMDAR function. GlyT2 is highly enriched at glycinergic nerve terminals, and Ca(2+)-triggered exocytosis and internalization are thought to regulate GlyT2 numbers in the pre-synaptic plasma membrane. We have identified different interacting proteins that may play a role in GlyT2 trafficking and/or pre-synaptic localization.
Assuntos
Proteínas da Membrana Plasmática de Transporte de Glicina/metabolismo , Isoformas de Proteínas/metabolismo , Transmissão Sináptica/fisiologia , Animais , Glicina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Glicina/genética , Humanos , Camundongos , Camundongos Knockout , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/metabolismo , Isoformas de Proteínas/genética , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
A large body of evidence indicates that muscarinic acetylcholine receptors (mAChRs) play critical roles in regulating the activity of many important functions of the central and peripheral nervous systems. However, identification of the physiological and pathophysiological roles of the individual mAChR subtypes (M(1)-M(5)) has proven a difficult task, primarily due to the lack of ligands endowed with a high degree of receptor subtype selectivity and the fact that most tissues and organs express multiple mAChRs. To circumvent these difficulties, we used gene targeting technology to generate mutant mouse lines containing inactivating mutations of the M(1)-M(5) mAChR genes. The different mAChR mutant mice and the corresponding wild-type control animals were subjected to a battery of physiological, pharmacological, behavioral, biochemical, and neurochemical tests. The M(1)-M(5) mAChR mutant mice were viable and reproduced normally. However, each mutant line displayed specific functional deficits, suggesting that each mAChR subtype mediates distinct physiological functions. These results should offer new perspectives for the rational development of novel muscarinic drugs.
Assuntos
Receptores Muscarínicos/genética , Animais , Comportamento Animal/fisiologia , Epilepsia/genética , Coração/fisiologia , Camundongos , Camundongos Knockout , Músculo Liso/fisiologia , Receptores Muscarínicos/fisiologiaRESUMO
Among the five different muscarinic receptors that have been cloned and characterized, M2 and M4 receptors are localized both post- and presynaptically and are believed to have a pronounced autoreceptor role. The functional importance of these receptors in the regulation of acetylcholine release in the hippocampus and in cognitive processes was investigated by using M2 and M4 receptor single knockout (KO) as well as M2/M4 receptor double KO mice. We found profound alterations in acetylcholine homeostasis in the hippocampus of both M2- and M4-KO mice as well as of the combined M2/M4-KOs, as assessed by in vivo microdialysis. Basal acetylcholine efflux in the hippocampus was significantly increased in M4-KO and was elevated further in M2/M4-KOs. The increase in hippocampal acetylcholine induced by local administration of scopolamine was markedly reduced in M2-KO and completely abolished in M2/M4-KOs. In M2-KO and much more in M2/M4-KOs, the increase in hippocampal acetylcholine triggered by exposure to a novel environment was more pronounced both in amplitude and duration, with a similar trend observed for M4-KOs. Dysregulation of cholinergic function in the hippocampus, as it could result from perturbed autoreceptor function, may be associated with cognitive deficits. Importantly, M2- and M2/M4-KO, but not M4-KO, animals showed an impaired performance in the passive avoidance test. Together these results suggest a crucial role for muscarinic M2 and M4 receptors in the tonic and phasic regulation of acetylcholine efflux in the hippocampus as well as in cognitive processes.
Assuntos
Acetilcolina/metabolismo , Hipocampo/metabolismo , Receptor Muscarínico M2/fisiologia , Receptor Muscarínico M4/fisiologia , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Aprendizagem da Esquiva/fisiologia , Cognição/fisiologia , Cruzamentos Genéticos , Meio Ambiente , Habituação Psicofisiológica/fisiologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Homeostase , Masculino , Camundongos , Camundongos Knockout , Microdiálise , Antagonistas Muscarínicos/farmacologia , Receptor Muscarínico M2/deficiência , Receptor Muscarínico M2/efeitos dos fármacos , Receptor Muscarínico M2/genética , Receptor Muscarínico M4/deficiência , Receptor Muscarínico M4/efeitos dos fármacos , Receptor Muscarínico M4/genética , Escopolamina/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
A muscarinic receptor radioligand, 3-(3-(3-fluoropropyl)thio) -1,2,5,thiadiazol-4-yl)-1,2,5,6-tetrahydro-1-methylpyridine (fP-TZTP) radiolabeled with the positron emitting radionuclide (18)F ([(18)F]FP-TZTP) displayed regional brain distribution consistent with M2 receptor densities in rat brain. The purpose of the present study is to further elucidate the subtype selectivity of [(18)F]FP-TZTP using genetically engineered mice which lacked functional M1, M2, M3, or M4 muscarinic receptors. Using ex vivo autoradiography, the regional brain localization of [(18)F]FP-TZTP in M2 knockout (M2 KO) was significantly decreased (51.3 to 61.4%; P<0.01) when compared to the wild-type (WT) mice in amygdala, brain stem, caudate putamen, cerebellum, cortex, hippocampus, hypothalamus, superior colliculus, and thalamus. In similar studies with M1KO, M3KO and M4KO compared to their WT mice, [(18)F]FP-TZTP uptakes in the same brain regions were not significantly decreased at P<0.01. However, in amygdala and hippocampus small decreases of 19.5% and 22.7%, respectively, were observed for M1KO vs WT mice at P<0.05. Given the fact that large decreases in [(18)F]FP-TZTP brain uptakes were seen only in M2 KO vs. WT mice, we conclude that [(18)F]FP-TZTP preferentially labels M2 receptors in vivo.
Assuntos
Piridinas/metabolismo , Receptores Muscarínicos/deficiência , Tiazóis/metabolismo , Animais , Encéfalo/metabolismo , Feminino , Radioisótopos de Flúor/metabolismo , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor Muscarínico M1 , Receptor Muscarínico M2 , Receptor Muscarínico M3 , Receptor Muscarínico M4 , Receptores Muscarínicos/genéticaRESUMO
We have previously suggested that presynaptic M(2)-muscarinic receptors (M(2)R) are involved in the control of the time course of evoked acetylcholine release in the frog neuromuscular junction. The availability of knockout mice lacking functional M(2)R (M(2)-KO) enabled us to address this issue in a more direct way. Using the phrenic diaphragm preparation, we show that in wild-type (WT) mice experimental manipulations known to affect Ca(2+) entry and removal, greatly affected the amount of acetylcholine released (quantal content). However, the time course of release remained unaltered under all these experimental treatments. On the other hand, in the M(2)-KO mice, similar experimental treatments affected both the quantal content and the time course of release. In general, a larger quantal content was accompanied by a longer duration of release. Similarly, the rise time of the postsynaptic current produced by axon stimulation was sensitive to changes in [Ca(2+)](o) or [Mg(2+)](o) in M(2)-KO mice but not in WT mice. Measurements of Ca(2+) currents revealed that the shorter rise time of the postsynaptic current seen in high [Mg(2+)](o) in M(2)-KO mice was not produced by a shorter wave of the presynaptic Ca(2+) current. These results support our earlier findings and provide direct evidence for the major role that presynaptic M(2)-muscarinic receptors play in the control of the time course of evoked acetylcholine release under physiological conditions.
Assuntos
Acetilcolina/metabolismo , Cálcio/farmacocinética , Receptores Muscarínicos/genética , Receptores Muscarínicos/metabolismo , Transmissão Sináptica/fisiologia , Potenciais de Ação/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Espaço Extracelular/metabolismo , Camundongos , Camundongos Knockout , Terminações Pré-Sinápticas/metabolismo , Receptor Muscarínico M2 , Sódio/metabolismoRESUMO
Muscarinic acetylcholine receptors (M1-M5) play important roles in the modulation of many key functions of the central and peripheral nervous system. To explore the physiological roles of the two Gi-coupled muscarinic receptors, we disrupted the M2 and M4 receptor genes in mice by using a gene targeting strategy. Pharmacological and behavioral analysis of the resulting mutant mice showed that the M2 receptor subtype is critically involved in mediating three of the most striking central muscarinic effects, tremor, hypothermia, and analgesia. These studies also indicated that M4 receptors are not critically involved in these central muscarinic responses. However, M4 receptor-deficient mice showed an increase in basal locomotor activity and greatly enhanced locomotor responses following drug-induced activation of D1 dopamine receptors. This observation is consistent with the concept that M4 receptors exert inhibitory control over D1 receptor-mediated locomotor stimulation, probably at the level of striatal projection neurons where the two receptors are known to be coexpressed. These findings emphasize the usefulness of gene targeting approaches to shed light on the physiological and pathophysiological roles of the individual muscarinic receptor subtypes.
Assuntos
Analgesia , Química Encefálica , Marcação de Genes , Atividade Motora , Agonistas Muscarínicos/farmacologia , Oxotremorina/farmacologia , Receptores Muscarínicos/fisiologia , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/farmacologia , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Acetilcolina/metabolismo , Analgésicos/farmacologia , Animais , Apomorfina/farmacologia , Dopamina/metabolismo , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Atividade Motora/efeitos dos fármacos , Medição da Dor , Quimpirol/farmacologia , Ensaio Radioligante , Receptor Muscarínico M2 , Receptor Muscarínico M4 , Receptores Dopaminérgicos/metabolismo , Receptores Muscarínicos/genética , Vasodilatadores/metabolismoRESUMO
Determination of muscarinic agonist-induced parasympathomimetic effects in wild type and M2 and M4 muscarinic receptor knockout mice revealed that M2 receptors mediated tremor and hypothermia, but not salivation. The M4 receptors seem to play a modest role in salivation, but did not alter hypothermia and tremor. In the M2 knockout mice, agonist-induced bradycardia in isolated spontaneously beating atria was completely absent compared to their wild type litter mates, whereas agonist-induced bradycardia was similar in the M4 knockout and wild type mice. The potency of carbachol to stimulate contraction of isolated stomach fundus, urinary bladder and trachea was reduced by a factor of about 2 in the M2 knockout mice, but was unaltered in the M4 knockout mice. The binding of the muscarinic agonist, [3H]-oxotremorine-M, was reduced in cortical tissue from the M2 knockout mice and to a lesser extent from the M4 knockout mice, and was reduced over 90% in the brain stem of M2 knockout mice. The data demonstrate the usefulness of knockout mice in determining the physiological function of peripheral and central muscarinic receptors.
Assuntos
Agonistas Muscarínicos/farmacologia , Oxotremorina/farmacologia , Receptores Muscarínicos/fisiologia , Animais , Carbacol/farmacologia , Córtex Cerebral/metabolismo , Coração/efeitos dos fármacos , Coração/fisiologia , Frequência Cardíaca/efeitos dos fármacos , Humanos , Hipotermia/induzido quimicamente , Masculino , Camundongos , Camundongos Knockout , Agonistas Muscarínicos/metabolismo , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Oxotremorina/metabolismo , Ensaio Radioligante , Receptor Muscarínico M2 , Receptor Muscarínico M4 , Receptores Muscarínicos/genética , Salivação/efeitos dos fármacos , Salivação/fisiologia , Tremor/induzido quimicamenteRESUMO
Many different G protein-coupled receptors modulate the activity of Ca2+ and K+ channels in a variety of neuronal types. There are five known subtypes (M1-M5) of muscarinic acetylcholine receptors. Knockout mice lacking the M1, M2, or M4 subtypes are studied to determine which receptors mediate modulation of voltage-gated Ca2+ channels in mouse sympathetic neurons. In these cells, muscarinic agonists modulate N- and L-type Ca2+ channels and the M-type K+ channel through two distinct, G-protein mediated pathways. The fast and voltage-dependent pathway is lacking in the M2 receptor knockout mice. The slow and voltage-independent pathway is absent in the M1 receptor knockout mice. Neither pathway is affected in the M4 receptor knockout mice. Muscarinic modulation of the M current is absent in the M1 receptor knockout mice, and can be reconstituted in a heterologous expression system using cloned channels and M1 receptors. Our results using knockout mice are compared with pharmacological data in the rat.
Assuntos
Canais de Cálcio/metabolismo , Neurônios/metabolismo , Canais de Potássio/metabolismo , Isoformas de Proteínas/metabolismo , Receptores Muscarínicos/metabolismo , Gânglio Cervical Superior/citologia , Animais , Eletrofisiologia , Inibidores Enzimáticos/farmacologia , Etilmaleimida/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Camundongos , Camundongos Knockout , Agonistas Muscarínicos/farmacologia , Neurônios/efeitos dos fármacos , Oxotremorina/farmacologia , Isoformas de Proteínas/genética , Ratos , Receptores Muscarínicos/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Gânglio Cervical Superior/efeitos dos fármacos , Gânglio Cervical Superior/fisiologia , Fatores de TempoRESUMO
Muscarinic receptors have been implicated in the regulation of cognition and psychosis based on pharmacological evidence from pre-clinical and clinical studies. Muscarinic agonists have shown promise in the clinic in improving cognition and reducing psychotic episodes in Alzheimer's patients. However, lack of selective muscarinic ligands has limited their use due to troublesome side effects observed at higher doses. Without selective ligands, it has been difficult to assign a specific muscarinic receptor subtype to these high order mental processes. Recent development of muscarinic receptor knockout mice has provided additional tools to investigate cognition and psychosis in behavioral assays and to determine the receptor subtypes associated with parasympathomimetic physiology. Biochemical studies indicate that the M1 receptor plays a significant role in regulating G alpha q-mediated signal transduction in the hippocampus and cortex. Behavioral studies suggest that the M4 receptor is involved in movement regulation and prepulse inhibition of the startle reflex, a measure of attention. These findings support a role for the development of M1 and M4 receptor agonists for diseases in which symptoms include cognitive impairment and psychotic behaviors.
Assuntos
Doença de Alzheimer/fisiopatologia , Neurônios/metabolismo , Receptores Muscarínicos/metabolismo , Esquizofrenia/fisiopatologia , Animais , Fracionamento Celular , Linhagem Celular , Membrana Celular/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Antagonistas de Aminoácidos Excitatórios/farmacologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Masculino , Memória/fisiologia , Camundongos , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Oxotremorina/farmacologia , Fenciclidina/farmacologia , Ensaio Radioligante/métodos , Receptores Muscarínicos/genética , Transdução de Sinais/fisiologiaRESUMO
Na(+)/Cl(-)-dependent neurotransmitter transporters form a superfamily of transmembrane proteins that share 12 membrane-spanning regions. To gain information about the quaternary structure of these transporter proteins, we heterologously expressed the glial glycine transporter GlyT1 and its neuronal homolog GlyT2 in Xenopus oocytes. By using metabolic labeling with [(35)S]methionine or surface labeling with a plasma membrane impermeable reagent followed by affinity purification, we separately analyzed the total cellular pools of newly synthesized GlyTs and its functional plasma membrane-bound fractions. Upon blue native gel electrophoresis, the surface-localized transporter proteins were found to exist exclusively in complex-glycosylated monomeric form, whereas a significant fraction of the intracellular GlyT1 and GlyT2 was core-glycosylated and oligomeric. In contrast, even after treatment with the crosslinker glutaraldehyde, surface GlyTs failed to migrate as oligomeric proteins. These results indicate that plasma membrane-bound GlyT1 and GlyT2 are monomeric proteins. Thus, Na(+)/Cl(-)-dependent neurotransmitter transporters do not require oligomerization for substrate translocation.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Proteínas de Transporte/metabolismo , Glicina/metabolismo , Proteínas de Membrana/metabolismo , Animais , Proteínas de Transporte/genética , Membrana Celular/metabolismo , Proteínas da Membrana Plasmática de Transporte de Glicina , Proteínas de Membrana/genética , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Oócitos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Xenopus laevisRESUMO
Activation of extracellular signal-regulated kinases (ERK) is crucial for many neural functions, including learning, memory, and synaptic plasticity. As muscarinic acetylcholine receptors (mAChR) modulate many of the same higher brain functions as ERK, we examined mAChR-mediated ERK activation in mouse hippocampal slices. The cholinergic agonist carbachol caused an atropine-sensitive ERK activation in the dendrites and somata CA1 pyramidal neurons. To determine the responsible mAChR subtype, we combined pharmacologic and genetic approaches. Pretreatment with M1 antagonists inhibited ERK activation. Furthermore, mAChR-induced ERK activation was absent in slices from M1 knockout mice. ERK activation was normal in slices derived from other mAChR subtype knockouts (M2, M3, and M4), although these other subtypes are expressed in many of the same neurons. Thus, we demonstrate divergent functions for the different mAChR subtypes. We conclude that M1 is responsible for mAChR-mediated ERK activation, providing a mechanism by which M1 may modulate learning and memory.
Assuntos
Hipocampo/enzimologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células Piramidais/enzimologia , Receptores Muscarínicos/deficiência , Animais , Atropina/farmacologia , Carbacol/farmacologia , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Agonistas Colinérgicos/farmacologia , Dendritos/metabolismo , Dendritos/ultraestrutura , Relação Dose-Resposta a Droga , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , MAP Quinase Quinase 1 , Masculino , Mecamilamina/farmacologia , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Antagonistas Muscarínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Células Piramidais/citologia , Células Piramidais/efeitos dos fármacos , Receptor Muscarínico M1 , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/genéticaRESUMO
Peripheral muscarinic receptors play key roles in the control of heart rate and smooth muscle activity. In this study, bradycardic and smooth muscle contractile responses to the muscarinic agonist carbamylcholine were compared in isolated tissues from M(2) and M(4) muscarinic receptor knockout mice and their wild-type littermates. Carbamylcholine (1 x 10(-8)-3 x 10(-5) M) produced similar concentration-dependent bradycardia in spontaneously beating atria from M(4) receptor knockout and wild-type control mice. In contrast, carbamylcholine did not produce bradycardia in atria derived from M(2) receptor knockout mice, whereas such atria were responsive to adenosine-induced bradycardia. Carbamylcholine-induced contractile responses were similar in stomach fundus, urinary bladder, and tracheal preparations from M(4) receptor knockout mice and their wild-type littermates for each tissue (-logEC(50) values ranging from 6.20 +/- 0.10 to 6.76 +/- 0.08), suggesting that M(4) receptors do not participate in smooth muscle contraction in these tissues. In contrast, approximately 2-fold higher carbamylcholine concentration was required for contraction of stomach fundus, urinary bladder, and trachea from M(2) receptor knockout mice (-logEC(50) = 6.39 +/- 0.05, 6.07 +/- 0.06, and 6.27 +/- 0.12, respectively) than from wild-type littermates (-logEC(50) = 6.68 +/- 0.07, 6.27 +/- 0.07, and 6.56 +/- 0.06, respectively). Furthermore, the affinity of the M(2) "selective" receptor antagonist AF-DX116 in inhibiting carbamylcholine-induced smooth muscle contraction was significantly reduced in M(2) receptor knockout mice compared with tissues from wild-type littermates. Collectively, these results provide direct and unambiguous evidence that M(2) receptors mediate muscarinic receptor-induced bradycardia and play a role in smooth muscle contractility, whereas M(4) receptors are not involved in stomach fundus, urinary bladder, or tracheal contractility.
Assuntos
Frequência Cardíaca/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Receptores Muscarínicos/fisiologia , Adenosina/farmacologia , Animais , Carbacol/farmacologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Knockout , Contração Muscular/efeitos dos fármacos , Músculo Liso/fisiologia , Pirenzepina/análogos & derivados , Pirenzepina/farmacologia , Receptor Muscarínico M2 , Receptor Muscarínico M4RESUMO
There are five known subtypes of muscarinic receptors (M(1)-M(5)). We have used knockout mice lacking the M(1), M(2), or M(4) receptors to determine which subtypes mediate modulation of voltage-gated Ca(2+) channels in mouse sympathetic neurons. Muscarinic agonists modulate N- and L-type Ca(2+) channels in these neurons through two distinct G-protein-mediated mechanisms. One pathway is fast and membrane-delimited and inhibits N- and P/Q-type channels by shifting their activation to more depolarized potentials. The other is slow and voltage-independent and uses a diffusible cytoplasmic messenger to inhibit both Ca(2+) channel types. Using patch-clamp methods on acutely dissociated sympathetic neurons, we isolated each pathway by pharmacological and kinetic means and found that each one is nearly absent in a particular knockout mouse. The fast and voltage-dependent pathway is lacking in the M(2) receptor knockout mice; the slow and voltage-independent pathway is absent from the M(1) receptor knockout mice; and neither pathway is affected in the M(4) receptor knockout mice. The knockout effects are clean and are apparently not accompanied by compensatory changes in other muscarinic receptors.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Receptores Muscarínicos/classificação , Animais , Etilmaleimida/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Oxotremorina/metabolismo , Técnicas de Patch-Clamp , Fatores de Tempo , Fatores de Virulência de Bordetella/metabolismoRESUMO
Muscarinic acetylcholine receptors (M(1)-M(5)) regulate many key functions of the central and peripheral nervous system. Primarily because of the lack of receptor subtype-selective ligands, the precise physiological roles of the individual muscarinic receptor subtypes remain to be elucidated. Interestingly, the M(4) receptor subtype is expressed abundantly in the striatum and various other forebrain regions. To study its potential role in the regulation of locomotor activity and other central functions, we used gene-targeting technology to create mice that lack functional M(4) receptors. Pharmacologic analysis of M(4) receptor-deficient mice indicated that M(4) receptors are not required for muscarinic receptor-mediated analgesia, tremor, hypothermia, and salivation. Strikingly, M(4) receptor-deficient mice showed an increase in basal locomotor activity and greatly enhanced locomotor responses (as compared with their wild-type littermates) after activation of D1 dopamine receptors. These results indicate that M(4) receptors exert inhibitory control on D1 receptor-mediated locomotor stimulation, probably at the level of striatal projection neurons where the two receptors are coexpressed at high levels. Our findings offer new perspectives for the treatment of Parkinson's disease and other movement disorders that are characterized by an imbalance between muscarinic cholinergic and dopaminergic neurotransmission.
Assuntos
Encéfalo/fisiologia , Atividade Motora/fisiologia , Oxotremorina/farmacologia , Receptores de Dopamina D1/fisiologia , Receptores Muscarínicos/fisiologia , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/farmacologia , Analgesia , Animais , Apomorfina/farmacologia , Corpo Estriado/fisiologia , Agonistas de Dopamina/farmacologia , Hipotermia/fisiopatologia , Camundongos , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Prosencéfalo/fisiologia , Quimpirol/farmacologia , Ensaio Radioligante , Receptor Muscarínico M4 , Receptores Muscarínicos/deficiência , Receptores Muscarínicos/genética , Salivação/efeitos dos fármacos , Tremor/induzido quimicamente , Tremor/fisiopatologiaRESUMO
Members of the muscarinic acetylcholine receptor family (M1-M5) are known to be involved in a great number of important central and peripheral physiological and pathophysiological processes. Because of the overlapping expression patterns of the M1-M5 muscarinic receptor subtypes and the lack of ligands endowed with sufficient subtype selectivity, the precise physiological functions of the individual receptor subtypes remain to be elucidated. To explore the physiological roles of the M2 muscarinic receptor, we have generated mice lacking functional M2 receptors by using targeted mutagenesis in mouse embryonic stem cells. The resulting mutant mice were analyzed in several behavioral and pharmacologic tests. These studies showed that the M2 muscarinic receptor subtype, besides its well documented involvement in the regulation of heart rate, plays a key role in mediating muscarinic receptor-dependent movement and temperature control as well as antinociceptive responses, three of the most prominent central muscarinic effects. These results offer a rational basis for the development of novel muscarinic drugs.
Assuntos
Encéfalo/metabolismo , Oxotremorina/farmacologia , Receptores Muscarínicos/fisiologia , Tremor/fisiopatologia , Analgesia , Animais , Embrião de Mamíferos , Feminino , Expressão Gênica , Biblioteca Genômica , Homozigoto , Hibridização In Situ , Masculino , Camundongos , Camundongos Knockout , Morfina/farmacologia , Mutagênese Sítio-Dirigida , Especificidade de Órgãos , Dor/genética , Dor/fisiopatologia , Receptor Muscarínico M2 , Receptores Muscarínicos/deficiência , Receptores Muscarínicos/genética , Mapeamento por Restrição , Salivação/efeitos dos fármacos , Salivação/genética , Salivação/fisiologia , Células-Tronco , Tremor/genéticaRESUMO
Among phospholipase C-coupled metabotropic glutamate receptors (mGluRs), some have a surprisingly long carboxyl-terminal intracellular domain (mGluR1a, -5a, and -5b), and others have a short one (mGluR1b, -1c, and -1d). All mGluR1 sequences are identical up to 46 residues following the 7th transmembrane domain, followed by 313, 20, 11, and 26 specific residues in mGluR1a, mGluR1b, mGluR1c, and mGluR1d, respectively. Several functional differences have been described between the long isoforms (mGluR1a, -5a, and -5b) and the short ones (mGluR1b, -1c, and -1d). Compared with the long receptors, the short ones induce slower increases in intracellular Ca2+, are activated by higher concentration of agonists, and do not exhibit constitutive, agonist-independent activity. To identify the residues responsible for these functional properties, a series of truncated, chimeric, and mutated receptors were constructed. We found that the deletion of the last 19 carboxyl-terminal residues in mGluR1c changed its properties into those of mGluR1a. Moreover, the exchange of the long carboxyl-terminal domain of mGluR5a with that of mGluR1c generated a chimeric receptor that possessed functional properties similar to those of mGluR1c. Mutagenesis of specific residues within the 19 carboxyl-terminal residues of mGluR1c revealed the importance of a cluster of 4 basic residues in defining the specific properties of this receptor. Since this cluster is part of the sequence common to all mGluR1 variants, we conclude that the long carboxyl-terminal domain of mGluR1a suppresses the inhibitory action of this sequence element.
Assuntos
Receptores de Glutamato Metabotrópico/metabolismo , Fosfolipases Tipo C/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Humanos , Dados de Sequência Molecular , Ligação Proteica , Splicing de RNA , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Three splice variants of the rat metabotropic glutamate receptor 1 (mGlu1a, 1b and 1c receptors) have been characterized so far. All have the same sequence up to the 46th residue following the 7th transmembrane domain, followed by different carboxyl-terminal tails. Whereas mGlu1b and mGlu1c receptors possess a short intracellular carboxyl-terminal tail, the mGlu1a receptor has a very long one. Compared to cells expressing mGlu1b or mGlu1c receptors, a higher agonist potency and basal phospholipase C activity were detected in cells expressing mGlu1a receptors. Another variant with a short carboxyl-terminal tail, the HmGlu1d receptor, has been recently isolated from human brain. Here we show that the mGlu1d receptor variant also exists in the rat. Like all rat mGlu1 receptor variants, the mGlu1d receptor activates phospholipase C upon stimulation with mGlu1 receptor agonists. Although the rank order of agonist potency is the same on mGlu1a and mGlu1d receptors, agonists are less potent in stimulating phospholipase C in mGlu1d receptor-expressing cells than in cells expressing mGlu1a receptors. Moreover, like the other short variants it has no significant constitutive activity. These results indicate that the mGlu1d receptor shares similar functional properties with the other short mGlu1 receptor splice variants, and further suggests that the long carboxyl-terminal tail of the mGlu1a receptor increases phospholipase C coupling efficacy.
Assuntos
Splicing de RNA , Receptores de Glutamato Metabotrópico/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , DNA Complementar , Humanos , Isomerismo , Dados de Sequência Molecular , Ratos , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/metabolismo , Fosfolipases Tipo C/metabolismoRESUMO
Many different G protein-linked receptors are preferentially coupled to G proteins of the Gq/11 family. To elucidate the molecular basis underlying this selectivity, different Gq/11-coupled receptors (m3 muscarinic, V1a vasopressin, and gastrin-releasing peptide receptor) were coexpressed (in COS-7 cells) with mutant alphas subunits in which residues present at the C terminus of alphas were replaced with the corresponding alphaq/11 residues. Remarkably, whereas none of the receptors was able to interact with wild type alphas to a significant extent, all three receptors gained the ability to productively couple to a mutant alphas subunit containing a single Glu --> Asn point mutation at position -3. Moreover, the m3 muscarinic and the V1a vasopressin receptors but not the GRP receptor also gained the ability to interact with a mutant alphas subunit containing a single Gln --> Glu point mutation at position -5, indicating that the alphaq/11 residues present in these mutant G protein constructs play key roles in determining the selectivity of receptor recognition. To identify the site(s) on Gq/11-coupled receptors that can functionally interact with the C terminus of alphaq/11 subunits, we next analyzed the ability of a series of hybrid m2/m3 muscarinic receptors to interact with a mutant alphas subunit (sq5) in which the last five amino acids of alphas were replaced with the corresponding alphaq/11 sequence. Similar to the wild type m2 and m3 muscarinic receptors, none of the investigated hybrid receptors was able to efficiently interact with wild type alphas. Interestingly, however, three mutant m2 receptors in which different segments of the second and third intracellular loops were replaced with the corresponding m3 receptor sequences were identified, which, in contrast to the Gi/o-coupled wild type m2 receptor, gained the ability to efficiently activate the sq5 subunit. This observation suggests that multiple intracellular receptor domains form a binding pocket for the C terminus of G protein alphaq/11 subunits.