RESUMO
Cholinesterase inhibitors, the current frontline symptomatic treatment for Alzheimer's disease (AD), are associated with low efficacy and adverse effects. M1 muscarinic acetylcholine receptors (M1 mAChRs) represent a potential alternate therapeutic target; however, drug discovery programs focused on this G protein-coupled receptor (GPCR) have failed, largely due to cholinergic adverse responses. Employing novel chemogenetic and phosphorylation-deficient, G protein-biased, mouse models, paired with a toolbox of probe molecules, we establish previously unappreciated pharmacologically targetable M1 mAChR neurological processes, including anxiety-like behaviors and hyper-locomotion. By mapping the upstream signaling pathways regulating these responses, we determine the importance of receptor phosphorylation-dependent signaling in driving clinically relevant outcomes and in controlling adverse effects including 'epileptic-like' seizures. We conclude that M1 mAChR ligands that promote receptor phosphorylation-dependent signaling would protect against cholinergic adverse effects in addition to driving beneficial responses such as learning and memory and anxiolytic behavior relevant for the treatment of AD.
Assuntos
Receptor Muscarínico M1/genética , Receptor Muscarínico M1/metabolismo , Acetilcolinesterase/metabolismo , Doença de Alzheimer/tratamento farmacológico , Animais , Colinérgicos/farmacologia , Inibidores da Colinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Modelos Animais de Doenças , Desenho de Fármacos , Feminino , Técnicas de Introdução de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , FosforilaçãoRESUMO
Fear memories are acquired through neuronal plasticity, an orchestrated sequence of events regulated at circuit and cellular levels. The conventional model of fear acquisition assumes unimodal (for example, excitatory or inhibitory) roles of modulatory receptors in controlling neuronal activity and learning. Contrary to this view, we show that protease-activated receptor-1 (PAR1) promotes contrasting neuronal responses depending on the emotional status of an animal by a dynamic shift between distinct G protein-coupling partners. In the basolateral amygdala of fear-naive mice PAR1 couples to Gαq/11 and Gαo proteins, while after fear conditioning coupling to Gαo increases. Concurrently, stimulation of PAR1 before conditioning enhanced, but afterwards it inhibited firing of basal amygdala neurons. An initial impairment of the long-term potentiation (LTP) in PAR1-deficient mice was transformed into an increase in LTP and enhancement of fear after conditioning. These effects correlated with more frequent 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid (AMPA) receptor-mediated miniature post synaptic events and increased neuronal excitability. Our findings point to experience-specific shifts in PAR1-G protein coupling in the amygdala as a novel mechanism regulating neuronal excitability and fear.
Assuntos
Tonsila do Cerebelo/fisiologia , Medo/fisiologia , Potenciação de Longa Duração/fisiologia , Receptor PAR-1/fisiologia , Tonsila do Cerebelo/química , Animais , Condicionamento Clássico/efeitos dos fármacos , Condicionamento Clássico/fisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Comportamento Exploratório/efeitos dos fármacos , Comportamento Exploratório/fisiologia , Medo/efeitos dos fármacos , Reação de Congelamento Cataléptica/efeitos dos fármacos , Reação de Congelamento Cataléptica/fisiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Potenciais Pós-Sinápticos em Miniatura/efeitos dos fármacos , Potenciais Pós-Sinápticos em Miniatura/fisiologia , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/fisiologia , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/fisiologia , Limiar da Dor , Técnicas de Patch-Clamp , Pirróis/farmacologia , Quinazolinas/farmacologia , Receptor PAR-1/antagonistas & inibidores , Receptor PAR-1/deficiência , Receptor PAR-1/genética , Reconhecimento Psicológico/efeitos dos fármacos , Reconhecimento Psicológico/fisiologiaRESUMO
There is now substantial evidence, from single-cell imaging, that complex patterns of release from Ca(2+) stores play an important role in regulating synaptic efficacy and plasticity. Moreover, the major mechanism of store release depends on the generation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] through the action of phospholipase(s) C on phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)], and several neurotransmitters can enhance receptor-mediated activation of this enzyme. The recent development of techniques to image real-time changes in PtdIns(4,5)P(2) hydrolysis according to generation of Ins(1,4,5)P(3) and diacylglycerol in single cells has significantly advanced our ability to investigate these signalling pathways, particularly in relation to single-cell Ca(2+) signals. This article reviews these new approaches and how they have provided novel insights into mechanisms underlying spatio-temporal Ca(2+) signals and phospholipase C activation in neurons.
Assuntos
Sinalização do Cálcio/fisiologia , Inositol 1,4,5-Trifosfato/fisiologia , Neurônios/fisiologia , Fosfatidilinositol 4,5-Difosfato/fisiologia , Transdução de Sinais/fisiologia , Animais , Técnicas Biossensoriais/métodos , Eletrofisiologia , Imunofluorescência/métodosRESUMO
1. The aim of the current study was to characterize which cannabinoid receptors, if any, are present on rat carotid artery smooth muscle. Additionally, the effects of cannabinoids on carotid artery tone, on cyclic AMP accumulation and on forskolin-induced relaxation were examined in the same tissue. 2. Stimulation of carotid arteries with forskolin (10 microM) significantly increased cyclic AMP accumulation, an effect that was inhibited in a concentration-dependent manner by the cannabinoid receptor agonist, methanandamide. 3. Similar inhibition was seen with the CB1 agonist HU-210 but this inhibition was not mimicked by the CB2 agonist, WIN 55,2212-2. 4. The inhibitory effect of methanandamide on cyclic AMP accumulation was prevented by incubation of the arteries with pertussis toxin and was significantly reduced by LY320135, a selective CB1 antagonist, but not by SR 144528, a CB2-selective antagonist. 5. Methanandamide failed to relax carotid arteries pre-contracted with phenylephrine, but inhibited forskolin-induced relaxation of these arteries. This functional inhibition of relaxation by methanandamide was inhibited by CB1-selective (LY320135 and SR 141716A), but not a CB2-selective antagonist (SR 144528). 6. These data demonstrate the presence of functional G protein-linked cannabinoid receptors of the CB1 subtype in the rat carotid artery, but show that these receptors inhibit cyclic AMP accumulation rather than cause relaxation.
Assuntos
Inibidores de Adenilil Ciclases , Artérias Carótidas/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Receptor CB2 de Canabinoide , Receptores de Droga/fisiologia , Animais , Ácidos Araquidônicos/farmacologia , Artérias Carótidas/enzimologia , Artérias Carótidas/fisiologia , Colforsina/farmacologia , AMP Cíclico/metabolismo , Técnicas In Vitro , Masculino , Relaxamento Muscular/efeitos dos fármacos , Relaxamento Muscular/fisiologia , Tono Muscular/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Músculo Liso Vascular/fisiologia , Ratos , Ratos Wistar , Receptores de Canabinoides , Receptores de Droga/efeitos dos fármacos , Receptores de Droga/metabolismoRESUMO
Muscarinic acetylcholine receptor (mAChR) activation of pancreatic ß-cells elevates intracellular Ca(2+) and potentiates glucose-stimulated insulin secretion. In addition, it activates a number of signaling molecules, including ERK1/2, whose activation has been shown to play an important role in regulating pancreatic ß-cell function and mass. The aim of this work was to determine how mAChR activation elevates intracellular Ca(2+) concentration ([Ca(2+)]( i )) and activates ERK1/2 in the pancreatic ß-cell line MIN6. We demonstrate that agonist-stimulated ERK1/2 activation is dependent on the activation of phospholipase C and an elevation in [Ca(2+)]( i ), but is independent of the activation of diacylglycerol-dependent protein kinase C isoenzymes. Using a pharmacological approach, we provide evidence that agonist-induced increases in [Ca(2+)]( i ) and ERK activity require (1) IP(3) receptor-mediated mobilization of Ca(2+) from the endoplasmic reticulum, (2) influx of extracellular Ca(2+) through store-operated channels, (3) closure of K(ATP) channels, and (4) Ca(2+) entry via L-type voltage-operated Ca(2+) channels. Moreover, this Ca(2+)-dependent activation of ERK is mediated via both Ras-dependent and Ras-independent mechanisms. In summary, this study provides important insights into the multifactorial signaling mechanisms linking mAChR activation to increases in [Ca(2+)]( i ) and ERK activity.
Assuntos
Cálcio/metabolismo , Células Secretoras de Insulina/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Receptores Muscarínicos/fisiologia , Carbacol/farmacologia , Linhagem Celular , Agonistas Colinérgicos/farmacologia , Ativação Enzimática/fisiologia , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Transdução de Sinais , Transfecção , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismoRESUMO
Intracellular Ca2+ store release contributes to activity-dependent synaptic plasticity in the central nervous system by modulating the amplitude, propagation, and temporal dynamics of cytoplasmic Ca2+ changes. However, neuronal Ca2+ stores can be relatively insensitive to increases in the store-mobilizing messenger inositol 1,4,5-trisphosphate (IP3). Using a fluorescent biosensor we have visualized M1 muscarinic acetylcholine (mACh) receptor signaling in individual hippocampal neurons and observed increased IP3 production in the absence of concurrent Ca2+ store release. However, coincident glutamate-mediated synaptic activity elicited enhanced and oscillatory IP3 production that was dependent upon ongoing mACh receptor stimulation and S-alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid receptor activation of Ca2+ entry. Moreover, the enhanced levels of IP3 now mobilized Ca2+ from intracellular stores that were refractory to the activation of mACh receptors alone. We conclude that convergent ionotropic and metabotropic receptor inputs can facilitate Ca2+ signaling by enhancing IP3 production as well as augmenting release by Ca2+-induced Ca2+ release.