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1.
Life Sci ; 272: 119194, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33609541

RESUMO

AIM: The aim of the present study was to investigate the anti-inflammatory response mediated of the M1 muscarinic acetylcholine receptor (mAChR) during experimental colitis. MATERIAL AND METHODS: After the induction of 6% acetic acid colitis, mice were treated with McN-A-343 0.5, 1.0, and 1.5 mg/kg or dexamethasone (DEXA, 2.0 mg/kg) or pirenzepine (PIR, 10 mg/kg; M1 mAChR antagonist). Colonic inflammation was assessed by macroscopic and microscopic lesion scores, colonic wet weight, myeloperoxidase (MPO) activity, interleukin-1 beta (IL1-ß) levels and tumor necrosis factor alpha (TNF-α), glutathione (GSH), malondialdehyde (MDA) and nitrate and nitrite (NO3/NO2), mRNA expression of IKKα, nuclear factor kappa beta (NF-kB) and cyclooxygenase-2 (COX-2), as well protein expression of NF-kB and COX-2. RESULTS: Treatment with McN-A-343 at a concentration of 1.5 mg/kg showed a significant reduction in intestinal damage as well as a decrease in wet weight, MPO activity, pro-inflammatory cytokine concentration, markers of oxidative stress and expression of inflammatory mediators. The action of the M1 agonist by the administration of pirenzepine, which promoted the blocking of the mAChR M1-mediated anti-inflammatory response, has also been proven. CONCLUSION: The results suggest that peripheral colonic M1 mAChR is involved in reversing the pro-inflammatory effect of experimentally induced colitis, which may represent a promising therapeutic alternative for patients with ulcerative colitis.


Assuntos
Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/farmacologia , Colite Ulcerativa/tratamento farmacológico , Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/metabolismo , Animais , Colite/tratamento farmacológico , Colite/metabolismo , Colite Ulcerativa/metabolismo , Colo/metabolismo , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Dexametasona/farmacologia , Modelos Animais de Doenças , Glutationa/metabolismo , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Masculino , Malondialdeído/metabolismo , Camundongos , Agonistas Muscarínicos/farmacologia , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Receptor Muscarínico M1 , Fator de Necrose Tumoral alfa/metabolismo
2.
J Pharmacol Sci ; 142(2): 50-59, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31818640

RESUMO

Cholinergic neurons play an important role in the higher functions of the brain, such as the memory, cognition, and nociception. However, the exact mechanism behind how the stimulation of all the muscarinic M1 receptors in the entire brain results in the alleviation of partial sciatic nerve ligation (PSNL)-induced mechanical hypersensitivity has not been investigated. Thus, we examined which subtype of GABA receptor was involved in the alleviation of PSNL-induce mechanical hypersensitivity produced by an intracerebroventricular administration of a muscarinic M1 receptor agonist, McN-A-343. Administering a GABAA receptor antagonist, bicuculline, resulted in no changes to the McN-A-343-induced anti-hypersensitivity in PSNL mice whereas a GABAB receptor antagonist, CGP35348, dose-dependently inhibited the anti-hypersensitivity. Furthermore, CGP35348 increased mechanical hypersensitivity in naïve mice, and the hypersensitivity was blocked by NMDA receptor antagonists, MK-801 and D-AP5. Additionally, muscarinic M1 receptors colocalized with GABAB1 receptors and an NMDA receptor subunit, GluN2A, in a large region of the brain. Consequently, these results suggest that the activation of muscarinic M1 receptors in the entire brain reduces nerve injury-induced mechanical hypersensitivity via the GABAB receptors, and the activation of the GABAB receptors regulates glutamatergic transmission via NMDA receptors.


Assuntos
Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/administração & dosagem , Agonistas Muscarínicos/administração & dosagem , Antagonistas Muscarínicos/administração & dosagem , Receptor Muscarínico M1/metabolismo , Receptores de GABA-A/metabolismo , Receptores de GABA-B/metabolismo , Nervo Isquiático/lesões , Nervo Isquiático/metabolismo , Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/metabolismo , Animais , Bicuculina/farmacologia , Maleato de Dizocilpina/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Masculino , Camundongos , Antagonistas Muscarínicos/metabolismo , Receptor Muscarínico M1/agonistas , Receptores de N-Metil-D-Aspartato/metabolismo , Nervo Isquiático/efeitos dos fármacos , Estresse Mecânico
3.
Brain Res ; 1704: 187-195, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30339810

RESUMO

Cholinergic systems modulate synaptic transmission across the neuraxis and play an important role in higher brain function including cognition, arousal and nociception. The anterior cingulate cortex (ACC) is a fundamental brain region for nociception and chronic pain, and receives cholinergic projections mainly from basal forebrain. Recently, we found that the activation of muscarinic M1 receptors in the ACC produced antinociceptive behavior in response to mechanical stimulation. However, it has not been tested whether stimulating muscarinic receptors in the ACC can reduce mechanical hypersensitivity in animal models of chronic pain. Here, we tested whether the activation of muscarinic M1 receptors in the ACC can alleviate mechanical hypersensitivity in a nerve injury model. The activation of muscarinic M1/M4 receptors by McN-A-343 injected into the contralateral side of the ACC, but not into the ventral posterolateral nucleus, was found to dose-dependently reduce mechanical hypersensitivity 7 days following partial sciatic nerve ligation in rats. The reduction of mechanical hypersensitivity by McN-A-343, was blocked by a selective muscarinic M1 antagonist, but not a M4 receptor antagonist. Importantly, the nerve injury model did not change the protein expression of muscarinic M1 receptors in the ACC. Additionally, a type A γ-aminobutyric acid (GABAA) receptor agonist injected into the ACC reduced the mechanical hypersensitivity in this injury model. Finally, a GABAA receptor antagonist blocked the reduction of mechanical hypersensitivity by McN-A-343 in the injury model. Collectively, these results suggest that activations of muscarinic M1 receptors in the ACC reduce nerve injury-induced mechanical hypersensitivity through GABAergic transmission via GABAA receptors.


Assuntos
Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/farmacologia , Neurônios GABAérgicos/efeitos dos fármacos , Giro do Cíngulo/efeitos dos fármacos , Hiperalgesia/tratamento farmacológico , Agonistas Muscarínicos/farmacologia , Traumatismos dos Nervos Periféricos/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/uso terapêutico , Animais , Neurônios GABAérgicos/metabolismo , Giro do Cíngulo/metabolismo , Giro do Cíngulo/fisiopatologia , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatologia , Masculino , Agonistas Muscarínicos/uso terapêutico , Traumatismos dos Nervos Periféricos/fisiopatologia , Ratos , Ratos Wistar , Nervo Isquiático/lesões , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/metabolismo
4.
Pharmacology ; 103(1-2): 38-49, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30380560

RESUMO

BACKGROUND/AIMS: In this study, we evaluated the functional impact of facilitatory presynaptic adenosine A2A and muscarinic M1 receptors in the recovery of neuromuscular tetanic depression caused by the blockage of high-affinity choline transporter (HChT) by hemicholinium-3 (HC-3), a condition that mimics a myasthenia-like condition. METHODS: Rat diaphragm preparations were indirectly stimulated via the phrenic nerve trunk with 50-Hz frequency trains, each consisting of 500-750 supramaximal intensity pulses. The tension at the beginning (A) and at the end (B) of the tetanus was recorded and the ratio (R) B/A calculated. RESULTS: Activation of A2A and M1 receptors with CGS21680 (CGS; 2 nmol/L) and McN-A-343c (McN; 3 µmol/L) increased R values. Similar facilitatory effects were obtained with forskolin (FSK; 3 µmol/L) and phorbol 12-myristate 13-acetate (PMA; 10 µmol/L), which activate adenylate cyclase and protein kinase C respectively. HC-3 (4 µmol/L) decreased transmitter exocytosis measured by real-time videomicroscopy with the FM4-64 fluorescent dye and prevented the facilitation of neuromuscular transmission caused by CGS, McN, and FSK, with a minor effect on PMA. The acetylcholinesterase inhibitor, neostigmine (NEO; 0.5 µmol/L), also decreased transmitter exocytosis. The paradoxical neuromuscular tetanic fade caused by NEO (0.5 µmol/L) was also prevented by HC-3 (4 µmol/L) and might result from the rundown of the positive feedback mechanism operated by neuronal nicotinic receptors (blocked by hexamethonium, 120 µmol/L). CONCLUSION: Data suggest that the recovery of tetanic neuromuscular facilitation by adenosine A2A and M1 receptors is highly dependent on HChT activity and may be weakened in myasthenic patients when HChT is inoperative.


Assuntos
Proteínas de Membrana Transportadoras/fisiologia , Receptor A2A de Adenosina/fisiologia , Receptor Muscarínico M1/fisiologia , Período Refratário Eletrofisiológico/efeitos dos fármacos , Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/farmacologia , Adenosina/análogos & derivados , Adenosina/farmacologia , Animais , Colforsina/farmacologia , Diafragma/efeitos dos fármacos , Diafragma/fisiologia , Hemicolínio 3/farmacologia , Neostigmina/farmacologia , Fenetilaminas/farmacologia , Nervo Frênico/efeitos dos fármacos , Nervo Frênico/fisiologia , Ratos , Ratos Wistar , Transmissão Sináptica , Tétano/tratamento farmacológico , Tétano/fisiopatologia , Acetato de Tetradecanoilforbol/farmacologia
5.
PLoS One ; 13(11): e0206532, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30395575

RESUMO

BACKGROUND: Electrical vagal nerve stimulation (VNS) has been used for years to treat patients with drug-resistant epilepsy. This technique also remains under investigation as a specific treatment of patients with Alzheimer's disease. Recently we discovered that VNS induced hippocampal formation (HPC) type II theta rhythm, which is involved in memory consolidation. In the present study, we have extended our previous observation and addressed the neuronal substrate and pharmacological profile of HPC type II theta rhythm induced by VNS in anesthetized rats. METHODS: Male Wistar rats were implanted with a VNS cuff electrode around the left vagus nerve, a tungsten microelectrode for recording the HPC field activity, and a medial septal (MS) cannula for the injection of a local anesthetic, procaine, and muscarinic agents. A direct, brief effect of VNS on the HPC field potential was evaluated before and after medial-septal drug injection. RESULTS: Medial septal injection of local anesthetic, procaine, reversibly abolished VNS-induced HPC theta rhythm. With the use of cholinergic muscarinic agonist and antagonists, we demonstrated that medial septal M1 receptors are involved in the mediation of the VNS effect on HPC theta field potential. CONCLUSION: The MS cholinergic M1 receptor mechanism integrates not only central inputs from the brainstem synchronizing pathway, which underlies the production of HPC type II theta rhythm, but also the input from the vagal afferents in the brain stem.


Assuntos
Hipocampo/fisiologia , Ritmo Teta/fisiologia , Estimulação do Nervo Vago , Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/administração & dosagem , Anestésicos Locais/administração & dosagem , Animais , Tronco Encefálico/efeitos dos fármacos , Tronco Encefálico/fisiologia , Colinérgicos/administração & dosagem , Fibras Colinérgicas/efeitos dos fármacos , Fibras Colinérgicas/fisiologia , Hipocampo/efeitos dos fármacos , Masculino , Vias Neurais/efeitos dos fármacos , Vias Neurais/fisiologia , Procaína/administração & dosagem , Ratos , Ratos Wistar , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/fisiologia , Núcleos Septais/efeitos dos fármacos , Núcleos Septais/fisiologia , Ritmo Teta/efeitos dos fármacos
6.
Neuron ; 98(6): 1256-1268.e4, 2018 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-29887340

RESUMO

Acetylcholine release in the prefrontal cortex (PFC), acting through muscarinic receptors, has an essential role in regulating flexible behavior and working memory (WM). General muscarinic receptor blockade disrupts PFC WM representations, while selective stimulation of muscarinic receptor subtypes is of great interest for the treatment of cognitive dysfunction in Alzheimer's disease. Here, we tested selective stimulation and blockade of muscarinic M1 receptors (M1Rs) in macaque PFC, during performance of a cognitive control task in which rules maintained in WM specified saccadic responses. We hypothesized that M1R blockade and stimulation would disrupt and enhance rule representation in WM, respectively. Unexpectedly, M1R blockade did not consistently affect PFC neuronal rule selectivity. Moreover, M1R stimulation suppressed PFC activity, and at higher doses, degraded rule representations. Our results suggest that, in primates, the deleterious effects of general muscarinic blockade on PFC WM activity are not mediated by M1Rs, while their overstimulation deteriorates PFC rule maintenance.


Assuntos
Memória de Curto Prazo/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Neurônios/efeitos dos fármacos , Córtex Pré-Frontal/efeitos dos fármacos , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/antagonistas & inibidores , Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/farmacologia , Regulação Alostérica , Animais , Benzamidas/farmacologia , Iontoforese , Macaca mulatta , Memória de Curto Prazo/fisiologia , Neurônios/metabolismo , Pirenzepina/farmacologia , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/fisiopatologia , Receptor Muscarínico M1/metabolismo
7.
Mol Pain ; 13: 1744806917692330, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-28326934

RESUMO

Background Cholinergic systems regulate the synaptic transmission resulting in the contribution of the nociceptive behaviors. Anterior cingulate cortex is a key cortical area to play roles in nociception and chronic pain. However, the effect of the activation of cholinergic system for nociception is still unknown in the cortical area. Here, we tested whether the activation of cholinergic receptors can regulate nociceptive behaviors in adult rat anterior cingulate cortex by integrative methods including behavior, immunohistochemical, and electrophysiological methods. Results We found that muscarinic M1 receptors were clearly expressed in the anterior cingulate cortex. Using behavioral tests, we identified that microinjection of a selective muscarinic M1 receptors agonist McN-A-343 into the anterior cingulate cortex dose dependently increased the mechanical threshold. In contrast, the local injection of McN-A-343 into the anterior cingulate cortex showed normal motor function. The microinjection of a selective M1 receptors antagonist pirenzepine blocked the McN-A-343-induced antinociceptive effect. Pirenzepine alone into the anterior cingulate cortex decreased the mechanical thresholds. The local injection of the GABAA receptors antagonist bicuculline into the anterior cingulate cortex also inhibited the McN-A-343-induced antinociceptive effect and decreased the mechanical threshold. Finally, we further tested whether the activation of M1 receptors could regulate GABAergic transmission using whole-cell patch-clamp recordings. The activation of M1 receptors enhanced the frequency of spontaneous and miniature inhibitory postsynaptic currents as well as the amplitude of spontaneous inhibitory postsynaptic currents in the anterior cingulate cortex. Conclusions These results suggest that the activation of muscarinic M1 receptors in part increased the mechanical threshold by increasing GABAergic transmitter release and facilitating GABAergic transmission in the anterior cingulate cortex.


Assuntos
Analgésicos/uso terapêutico , Giro do Cíngulo/metabolismo , Hiperalgesia/tratamento farmacológico , Receptor Muscarínico M1/metabolismo , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/metabolismo , Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/farmacologia , Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/uso terapêutico , Analgésicos/farmacologia , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , GABAérgicos/farmacologia , Giro do Cíngulo/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Masculino , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Agonistas Muscarínicos/farmacologia , Agonistas Muscarínicos/uso terapêutico , Antagonistas Muscarínicos/farmacologia , Pirenzepina/farmacologia , Ratos , Ratos Wistar , Transmissão Sináptica/efeitos dos fármacos , Ácido gama-Aminobutírico/farmacologia
8.
Neurosci Lett ; 621: 22-27, 2016 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-27063283

RESUMO

The mammalian circadian clock in the suprachiasmatic nucleus (SCN) can be reset by the cholinergic agonist carbachol. In hamsters, intraSCN carbachol produces phase advances during the day. This phenomenon has previously been attributed to the muscarinic receptors, as carbachol-induced phase shifts are blocked by pretreatment with the muscarinic antagonist atropine. The SCN contains all five muscarinic receptors, leaving open the question as to which muscarinic receptors mediate these shifts. Here we test two selective muscarinic agonists, the M1/4 agonist McN-A-343 and the M2/3 agonist bethanechol, in addition to the non-selective cholinergic agonist carbachol. Consistent with previous reports, carbachol produced significant phase advances when injected to the SCN during the mid-subjective day. At the doses used here, McN-A-343, but not bethanechol, also produced significant phase shifts when injected to the SCN during the mid-subjective day. Phase shifts to McN-A-343 were as large as those produced by carbachol, suggesting that activation of the M1/4 receptors alone can fully account for the daytime phase advances produced by cholinergic agonists. Given acetylcholine's role in arousal, and the similarity between phase advances to carbachol/McN-A-343 and to exercise and arousal manipulations, it is possible that acetylcholine may contribute to non-photic resetting of the circadian clock.


Assuntos
Relógios Circadianos , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M4/agonistas , Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/farmacologia , Animais , Betanecol/farmacologia , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Relógios Circadianos/efeitos dos fármacos , Masculino , Mesocricetus , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M3/agonistas , Núcleo Supraquiasmático/efeitos dos fármacos , Núcleo Supraquiasmático/fisiologia
9.
PLoS One ; 9(10): e109272, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25295619

RESUMO

BACKGROUND: The cholinergic anti-inflammatory pathway (CAP) is based on vagus nerve (VN) activity that regulates macrophage and dendritic cell responses in the spleen through alpha-7 nicotinic acetylcholine receptor (a7nAChR) signaling. Inflammatory bowel disease (IBD) patients present dysautonomia with decreased vagus nerve activity, dendritic cell and T cell over-activation. The aim of this study was to investigate whether central activation of the CAP alters the function of dendritic cells (DCs) and sequential CD4+/CD25-T cell activation in the context of experimental colitis. METHODS: The dinitrobenzene sulfonic acid model of experimental colitis in C57BL/6 mice was used. Central, intracerebroventricular infusion of the M1 muscarinic acetylcholine receptor agonist McN-A-343 was used to activate CAP and vagus nerve and/or splenic nerve transection were performed. In addition, the role of α7nAChR signaling and the NF-kB pathway was studied. Serum amyloid protein (SAP)-A, colonic tissue cytokines, IL-12p70 and IL-23 in isolated splenic DCs, and cytokines levels in DC-CD4+CD25-T cell co-culture were determined. RESULTS: McN-A-343 treatment reduced colonic inflammation associated with decreased pro-inflammatory Th1/Th17 colonic and splenic cytokine secretion. Splenic DCs cytokine release was modulated through α7nAChR and the NF-kB signaling pathways. Cholinergic activation resulted in decreased CD4+CD25-T cell priming. The anti-inflammatory efficacy of central cholinergic activation was abolished in mice with vagotomy or splenic neurectomy. CONCLUSIONS: Suppression of splenic immune cell activation and altered interaction between DCs and T cells are important aspects of the beneficial effect of brain activation of the CAP in experimental colitis. These findings may lead to improved therapeutic strategies in the treatment of IBD.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Colite/imunologia , Colite/metabolismo , Células Dendríticas/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Baço/citologia , Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/farmacologia , Animais , Antígeno CD11c/metabolismo , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nervo Vago/efeitos dos fármacos , Nervo Vago/metabolismo
10.
Eur J Pharmacol ; 740: 436-41, 2014 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-25008070

RESUMO

Endotoxin tolerance is a mechanism in which cells receiving low doses of endotoxin, enter a transient phase with less inflammatory response to the next endotoxin challenges. Central nervous system is known to modulate systemic inflammation through activation of the cholinergic system; however, the role of central anti-inflammatory pathway in pathophysiology of hepatic endotoxin tolerance is unknown. Our study was designed to assess the effect central muscarinic type 1 receptor (M1) activation on development of endotoxin tolerance in rat liver. Endotoxin tolerance was induced by daily intraperitoneal injection of endotoxin (1 mg/kg) for 5 days. Animals were randomly divided into two groups which received intracerebroventricular injection of either MCNA-343 (an M1 agonist, 5 ng/kg) or saline 1h after intraperitoneal injection of saline or endotoxin. The responsiveness to endotoxin was assessed by measuring hepatic MCP-1 (monocyte chemotactic protein-1), iNOS (inducible nitric oxide synthase) and TNF-α (tumor necrosis-α) mRNA expression 3h after intraperitoneal administration of endotoxin using quantitative RT-PCR. A significant reduction in hepatic expression of MCP-1, iNOS and TNF-α was observed in rats with 5 days endotoxin challenge in comparison with rats given a single dose of endotoxin. There was no significant difference in hepatic expression of MCP-1, iNOS or TNF-α between acute and chronic LPS-treated groups in rats given MCNA-343. Central MCNA-343 stimulation could prevent the induction of hepatic endotoxin tolerance in animals receiving repeated doses of endotoxin. This indicates that M1 cholinergic receptor activation in the central nervous system can modulate endotoxin tolerance in rat liver.


Assuntos
Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/farmacologia , Tolerância a Medicamentos/fisiologia , Lipopolissacarídeos/farmacologia , Fígado/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Receptor Muscarínico M1/agonistas , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Quimiocina CCL2/genética , Expressão Gênica , Fígado/metabolismo , Masculino , Óxido Nítrico Sintase Tipo II/genética , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Receptor Muscarínico M1/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Necrose Tumoral alfa/genética
11.
Learn Mem ; 21(5): 263-71, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24737918

RESUMO

The hippocampal formation receives strong cholinergic input from the septal/diagonal band complex. Although the functional effects of cholinergic activation have been extensively studied in pyramidal neurons within the hippocampus and entorhinal cortex, less is known about the role of cholinergic receptors on dentate gyrus neurons. Using intracellular recordings from rat dentate hilar neurons, we find that activation of m1-type muscarinic receptors selectively increases the excitability of glutamatergic mossy cells but not of hilar interneurons. Following brief stimuli, cholinergic modulation reveals a latent afterdepolarization response in mossy cells that can extend the duration of stimulus-evoked depolarization by >100 msec. Depolarizing stimuli also could trigger persistent firing in mossy cells exposed to carbachol or an m1 receptor agonist. Evoked IPSPs attenuated the ADP response in mossy cells. The functional effect of IPSPs was amplified during ADP responses triggered in the presence of cholinergic receptor agonists but not during slowly decaying simulated ADPs, suggesting that modulation of ADP responses by IPSPs arises from destabilization of the intrinsic currents underlying the ADP. Evoked IPSPs also could halt persistent firing triggered by depolarizing stimuli. These results show that through intrinsic properties modulated by muscarinic receptors, mossy cells can prolong depolarizing responses to excitatory input and extend the time window where multiple synaptic inputs can summate. By actively regulating the intrinsic response to synaptic input, inhibitory synaptic input can dynamically control the integration window that enables detection of coincident inputs and shape the spatial pattern of hilar cell activity.


Assuntos
Hipocampo/citologia , Potenciais Pós-Sinápticos Inibidores/fisiologia , Fibras Nervosas/fisiologia , Células Piramidais/fisiologia , Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/farmacologia , Animais , Animais Recém-Nascidos , Carbacol/farmacologia , Agonistas Colinérgicos/farmacologia , Estimulação Elétrica , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Técnicas In Vitro , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Pirenzepina/análogos & derivados , Pirenzepina/farmacologia , Células Piramidais/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley
12.
J Physiol ; 590(16): 3953-64, 2012 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-22674722

RESUMO

Previous studies have suggested that muscarinic receptor activation modulates glutamatergic transmission. M-type potassium channels mediate the effects of muscarinic activation in the hippocampus, and it has been proposed that they modulate glutamatergic synaptic transmission. We tested whether M1 muscarinic receptor activation enhances glutamatergic synaptic transmission via the inhibition of the M-type potassium channels that are present in Schaffer collateral axons and terminals. Miniature excitatory postsynaptic currents (mEPSCs) were recorded from CA1 pyramidal neurons. The M1 receptor agonist, NcN-A-343, increased the frequency of mEPSCs, but did not alter their amplitude. The M-channel blocker XE991 and its analogue linopirdine also increased the frequency of mEPSCs. Flupirtine, which opens M-channels, had the opposite effect. XE991 did not enhance mEPSCs frequency in a calcium-free external medium. Blocking P/Q- and N-type calcium channels abolished the effect of XE991 on mEPSCs. These data suggested that the inhibition of M-channels increases presynaptic calcium-dependent glutamate release in CA1 pyramidal neurons. The effects of these agents on the membrane potentials of presynaptic CA3 pyramidal neurons were studied using current clamp recordings; activation of M1 receptors and blocking M-channels depolarized neurons and increased burst firing. The input resistance of CA3 neurons was increased by the application of McN-A-343 and XE991; these effects were consistent with the closure of M-channels. Muscarinic activation inhibits M-channels in CA3 pyramidal neurons and its efferents ­ Schaffer collateral, which causes the depolarization, activates voltage-gated calcium channels, and ultimately elevates the intracellular calcium concentration to increase the release of glutamate on CA1 pyramidal neurons.


Assuntos
Potenciais de Ação/fisiologia , Ácido Glutâmico/metabolismo , Receptores Muscarínicos/classificação , Receptores Muscarínicos/metabolismo , Transmissão Sináptica/fisiologia , Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/farmacologia , Aminopiridinas/farmacologia , Animais , Antracenos/farmacologia , Cálcio/metabolismo , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Indóis/farmacologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Bloqueadores dos Canais de Potássio/farmacologia , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley
13.
Urologia ; 79(2): 102-8, 2012.
Artigo em Italiano | MEDLINE | ID: mdl-22610841

RESUMO

INTRODUCTION: The purpose of the study is to understand whether the cholinergic stimulation is important, not only in inducing contraction of the detrusor muscle, but also in modulating the proliferation of smooth muscle cells. These results could help to better understand the role of antimuscarinic drugs, which are currently used for the treatment of many urological diseases. PATIENTS AND METHODS: Primary cultures were prepared from biopsies of human detrusor muscle of subjects >65 years. From the cell culture set-up for each patient, mRNA was extracted and both the gene expression and the influence of increasing passages on the expression of muscarinic receptor subtypes were evaluated by semi-quantitative and quantitative PCR (RT-PCR and Q-RT-PCR). The rate of cell proliferation induced by cholinergic drugs was assessed by the evaluation of the [3H]-thymidine incorporation. RESULTS: The gene expression analysis demonstrated that the range of expression of muscarinic subtypes in human detrusor smooth muscle cells (HDSMCs) is M2 > M3 > M1 > M4 >> M5. The exposure to the cholinergic agonist carbachol induced a concentration-dependent increase in cell proliferation rate. The pharmacological characterization indicated that this effect was mainly mediated by the receptor subtypes M3 and M2. DISCUSSION: The cholinergic stimulation led to an increase in HDSMC proliferation, suggesting that this phenomenon might be involved in the pathogenic mechanism through which the cervico-urethral obstruction causes a detrusor hypertrophy, followed by a loss of function. These results could then provide an indication of the use of antimuscarinic drugs in the treatment of lower urinary tract disorders.


Assuntos
Acetilcolina/farmacologia , Agonistas Muscarínicos/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Bexiga Urinária/efeitos dos fármacos , Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/farmacologia , Idoso , Atropina/farmacologia , Carbacol/farmacologia , Divisão Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Antagonistas Muscarínicos/farmacologia , Contração Muscular/efeitos dos fármacos , Piperidinas/farmacologia , Pirenzepina/análogos & derivados , Pirenzepina/farmacologia , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/biossíntese , Receptores Muscarínicos/biossíntese , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/genética
14.
Pharmacol Ther ; 135(2): 216-45, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22643681

RESUMO

The unusual pharmacology of McN-A-343 was first described by Roszowski in 1961. The agonist appeared to be a selective stimulant of muscarinic receptors in sympathetic ganglia, now known to be the muscarinic M1 receptor subtype. However, subsequent research demonstrated that McN-A-343 is a partial agonist with similar affinity at all five muscarinic acetylcholine receptor subtypes and its relative selectivity depends on a higher efficacy at the M1 (and M4) subtypes. Being a partial agonist its action is also dependent on factors, such as receptor density and coupling efficacy between receptor activation and tissue response. Nevertheless, the relatively high efficacy at M1 receptors led to its widespread use as an aid to distinguish responses mediated through M1 receptors from those utilizing M2 or M3 muscarinic receptor subtypes, especially in the CNS. There is also evidence that it has an allosteric action at some receptor subtypes. Recently, it was demonstrated that McN-A-343 can bind to an allosteric site on the M2 receptor as well as to the orthosteric site and has thus been termed a "bitopic agonist". This allosteric site differs from that occupied by allosteric modulators, such as gallamine. Comparison of comparable mutagenic changes in M2 and M4 receptors also suggests that McN-A-343 utilizes different regions of the two receptors for ERK1/2 activation. McN-A-343 has a number of non-muscarinic actions. These include activation of some types of nicotinic acetylcholine receptors, antagonism of serotonin 5-HT3 and 5-HT4 receptor subtypes, inhibition of the uptake mechanism and a local anesthetic action.


Assuntos
Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/farmacologia , Agonistas Muscarínicos/farmacologia , Animais , Sistema Cardiovascular/efeitos dos fármacos , Sistema Nervoso Central/efeitos dos fármacos , Glândulas Endócrinas/efeitos dos fármacos , Humanos , Músculo Liso/efeitos dos fármacos , Sistema Nervoso Periférico/efeitos dos fármacos
15.
Eur J Pharmacol ; 670(1): 236-43, 2011 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-21924260

RESUMO

Although muscarinic M(2) and M(3) receptors are known to be important for regulation of gastric and small intestinal motility, muscarinic receptor subtypes regulating colonic function remain to be investigated. The aim of this study was to characterize muscarinic receptors involved in regulation of colonic contractility. M(2) and/or M(3) receptor knockout (KO) and wild-type mice were used in in vivo (defecation, colonic propulsion) and in vitro (contraction) experiments. Amount of feces was significantly decreased in M(3)R-KO and M(2)/M(3)R-KO mice but not in M(2)R-KO mice. Ranking of colonic propulsion was wild-type=M(2)R-KO>M(3)R-KO>M(2)/M(3)R-KO. In vitro, the amplitude of migrating motor complexes in M(2)R-KO, M(3)R-KO and M(2)/M(3)R-KO mice was significantly lower than that in wild-type mice. Carbachol caused concentration-dependent contraction of the proximal colon and distal colon from wild-type mice. In M(2)R-KO mice, the concentration-contraction curves shifted to the right and downward. In contrast, carbachol caused non-sustained contraction and relaxation in M(3)R-KO mice depending on its concentration. Carbachol did not cause contraction but instead caused relaxation of colonic strips from M(2)/M(3)R-KO mice. 4-[[[(3-chlorophenyl)amino]carbonyl]oxy]-N,N,N-trimethyl-2-butyn-1-aminium chloride (McN-A-343) caused a non-sustained contraction of colonic strips from wild-type mice, and this contraction was changed to a sustained contraction by tetrodotoxin, pirenzepine and L-nitroarginine methylester (L-NAME). In the colon of M(2)/M(3)R-KO mice, McN-A-343 caused only relaxation, which was decreased by tetrodotoxin, pirenzepine and L-NAME. In conclusion, M(1), M(2) and M(3) receptors regulate colonic motility of the mouse. M(2) and M(3) receptors mediate cholinergic contraction, but M(1) receptors on inhibitory nitrergic nerves counteract muscarinic contraction.


Assuntos
Colo/efeitos dos fármacos , Colo/fisiologia , Motilidade Gastrointestinal , Receptores Muscarínicos/deficiência , Receptores Muscarínicos/metabolismo , Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/farmacologia , Animais , Fenômenos Biomecânicos , Carbacol/farmacologia , Colo/metabolismo , Defecação/efeitos dos fármacos , Feminino , Motilidade Gastrointestinal/efeitos dos fármacos , Técnicas de Inativação de Genes , Técnicas In Vitro , Masculino , Camundongos , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Contração Muscular/efeitos dos fármacos , Pirenzepina/farmacologia , Receptores Muscarínicos/genética
16.
Peptides ; 32(2): 286-92, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21126550

RESUMO

Gastrin-releasing peptide (GRP) has been implicated in the itch-scratch cycle. We investigated if this gut-brain-skin peptide plays a role in the compulsive, hindleg scratching of the neck of mice by 5'-guanidinonaltrindole (GNTI), the kappa opioid receptor antagonist, and in the antipruritic activity of nalfurafine, the kappa opioid agonist. Previously, we showed that GNTI (0.03-1mg/kg, s.c.) elicits dose-related scratching and that nalfurafine (0.001-0.02mg/kg, s.c.) inhibits this behavior in mice. Utilizing immunohistochemistry, GRP positive nerve fibers were detected in mouse skin and superficial layer of the dorsal horn of the spinal cord as well as GRP positive cells in the dorsal root ganglion. Pretreating mice with either a pseudopeptide GRP receptor antagonist, RC-3095 (10-30mg/kg, s.c. at -15min), or a peptide GRP receptor antagonist, [d-Phe(6)]bombesin(6-13) methyl ester (2-100nmol, i.t. at -10min), did not suppress GNTI-induced scratching. However, pretreating mice with either antagonist inhibited scratching precipitated by the GRP receptor agonist, GRP(18-27) (2nmol, i.t.). Pretreating mice with a muscarinic M(1) receptor agonist, McN-A-343 (1.5-15µg/5µl, i.t. at -10min) antagonized GNTI-induced scratching. Norbinaltorphimine (20mg/kg, i.p. at -18 to -20h), a kappa opioid antagonist, countered the antiscratch activity of nalfurafine. We conclude that (a) the GRP receptor system does not mediate GNTI-induced scratching and (b) the kappa opioid system is involved, at least in part, in the scratch suppressing activity of nalfurafine.


Assuntos
Comportamento Compulsivo/metabolismo , Peptídeo Liberador de Gastrina/metabolismo , Naltrexona/análogos & derivados , Prurido/metabolismo , Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/farmacologia , Animais , Bombesina/análogos & derivados , Bombesina/antagonistas & inibidores , Bombesina/farmacologia , Comportamento Compulsivo/induzido quimicamente , Comportamento Compulsivo/prevenção & controle , Gânglios Espinais/metabolismo , Asseio Animal/efeitos dos fármacos , Guanidinas , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Morfinanos/farmacologia , Naltrexona/farmacologia , Fibras Nervosas/metabolismo , Fragmentos de Peptídeos/farmacologia , Pirenzepina/análogos & derivados , Pirenzepina/farmacologia , Prurido/induzido quimicamente , Prurido/prevenção & controle , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/antagonistas & inibidores , Receptores da Bombesina/antagonistas & inibidores , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/antagonistas & inibidores , Pele/inervação , Pele/metabolismo , Medula Espinal/metabolismo , Compostos de Espiro/farmacologia
17.
Br J Pharmacol ; 160(6): 1534-49, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20590642

RESUMO

BACKGROUND AND PURPOSE: We investigated how McN-A-343 inhibited the alkylation of the M(1) muscarinic receptor by its nitrogen mustard derivative and that of ACh to identify whether it interacts allosterically or orthosterically. EXPERIMENTAL APPROACH: We incubated the M(1) muscarinic receptor expressed in Chinese hamster ovary cells with ACh mustard for various periods of time in the presence of McN-A-343 or known allosteric and orthosteric ligands. After stopping the reaction and removing unreacted ligands, unalkylated receptors were measured using [(3)H]N-methylscopolamine. Analogous experiments were done using a nitrogen mustard analog of McN-A-343. Affinity constants, cooperativity values for allosteric interactions and rate constants for receptor alkylation were estimated using a mathematical model. KEY RESULTS: The kinetics of receptor alkylation by the nitrogen mustard derivatives of ACh and McN-A-343 were consistent with a two-step model in which the aziridinium ion rapidly forms a reversible receptor complex, which converts to a covalent complex at a slower rate. The inhibition of receptor alkylation by acetycholine, N-methylscopolamine and McN-A-343 was consistent with competitive inhibition, whereas that caused by gallamine was consistent with allosterism. Affinity constants estimated from alkylation kinetics agreed with those measured by displacement of [(3)H]N-methylscopolamine binding. CONCLUSIONS AND IMPLICATIONS: Our results suggest that McN-A-343 and its nitrogen mustard derivative interact competitively with ACh and N-methylscopolamine at the orthosteric site on the M(1) muscarinic receptor. Measuring how drugs modulate the kinetics of receptor alkylation by an irreversible ligand is a powerful approach for distinguishing between negative allosteric modulators and competitive inhibitors.


Assuntos
Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/farmacologia , Acetilcolina/análogos & derivados , Compostos de Mostarda Nitrogenada/farmacologia , Receptor Muscarínico M1/efeitos dos fármacos , Acetilcolina/farmacologia , Alquilação , Animais , Células CHO , Cricetinae , Cricetulus , Masculino , N-Metilescopolamina/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Muscarínico M1/metabolismo
18.
Mol Pharmacol ; 78(4): 745-55, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20643905

RESUMO

Investigating how a test drug alters the reaction of a site-directed electrophile with a receptor is a powerful method for determining whether the drug acts competitively or allosterically, provided that the binding site of the electrophile is known. In this study, therefore, we mutated nucleophilic residues near and within the orthosteric pockets of M(1) and M(2) muscarinic receptors to identify where acetylcholine mustard and 4-[(2-bromoethyl)methyl-amino]-2-butynyl-N-(3-chlorophenyl)carbamate (BR384) bind covalently. BR384 is the nitrogen mustard analog of [4-[[N-(3-chlorophenyl)carbamoyl]oxy]-2-butynyl]trimethylammonium chloride (McN-A-343). Mutation of the highly conserved aspartic acid in M(1) (Asp105) and M(2) (Asp103) receptors to asparagine largely prevented receptor alkylation by acetylcholine mustard, although modest alkylation still occurred at M(2) D103N at high concentrations of the mustard. Receptor alkylation by BR384 was also greatly inhibited in the M(1) D105N mutant, but some alkylation still occurred at high concentrations of the compound. In contrast, BR384 rapidly alkylated the M(2) D103N mutant. Its affinity was reduced to one tenth, however. The alkylation of M(2) D103N by BR384 was competitively inhibited by N-methylscopolamine and allosterically inhibited by gallamine. Mutation of a variety of other nucleophilic residues, some in combination with D103N, had little effect on M(2) receptor alkylation by BR384. Our results suggest that BR384 alkylates at least one residue other than the conserved aspartic acid at the ligand-binding site of M(1) and M(2) receptors. This additional residue seems to be located within or near the orthosteric-binding pocket and is not part of the allosteric site for gallamine.


Assuntos
Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/metabolismo , Acetilcolina/análogos & derivados , Mutagênese Sítio-Dirigida , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M2/metabolismo , Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/análogos & derivados , Acetilcolina/química , Acetilcolina/metabolismo , Sítio Alostérico/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , Células CHO , Cricetinae , Cricetulus , Humanos , Mecloretamina/química , Mecloretamina/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida/métodos , Ligação Proteica/genética , Receptor Muscarínico M1/genética , Receptor Muscarínico M2/genética
19.
J Biol Chem ; 285(25): 19012-21, 2010 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-20406819

RESUMO

The recently identified small molecule, 3-amino-5-chloro-6-methoxy-4-methylthieno[2,3-b]pyridine-2-carboxylic acid cyclopropylamide (LY2033298), is the first selective allosteric modulator of the muscarinic acetylcholine receptors (mAChRs) that mediates both receptor activation and positive modulation of the endogenous agonist, acetylcholine (ACh), via the same allosteric site on the M(4) mAChR. We thus utilized this novel chemical tool, as well as ACh, the bitopic (orthosteric/allosteric) agonist, McN-A-343, and the clinically efficacious M(1)/M(4) mAChR-preferring agonist, xanomeline, in conjunction with site-directed mutagenesis of four different regions of the M(4) mAChR (extracellular loops 1, 2, and 3, and transmembrane domain 7), to identify regions that govern ligand-specific modes of binding, signaling, and allosteric modulation. In the first extracellular loop (E1), we identified Ile(93) and Lys(95) as key residues that specifically govern the signaling efficacy of LY2033298 and its binding cooperativity with ACh, whereas Phe(186) in the E2 loop was identified as a key contributor to the binding affinity of the modulator for the allosteric site, and Asp(432) in the E3 loop appears to be involved in the functional (activation) cooperativity between the modulator and the endogenous agonist. In contrast, the highly conserved transmembrane domain 7 residues, Tyr(439) and Tyr(443), were identified as contributing to a key activation switch utilized by all classes of agonists. These results provide new insights into the existence of multiple activation switches in G protein-coupled receptors (GPCRs), some of which can be selectively exploited by allosteric agonists, whereas others represent global activation mechanisms for all classes of ligand.


Assuntos
Acetilcolina/química , Sítio Alostérico , Receptor Muscarínico M4/química , Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/farmacologia , Animais , Cricetinae , Humanos , Ligantes , Modelos Biológicos , Mutagênese , Mutagênese Sítio-Dirigida , Ligação Proteica , Piridinas/química , Piridinas/farmacologia , Receptor Muscarínico M4/agonistas , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Tiadiazóis/química , Tiadiazóis/farmacologia
20.
Biochem Pharmacol ; 79(7): 1025-35, 2010 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-19913516

RESUMO

We investigated whether the aziridinium ion formed from a nitrogen mustard derivative (4-[(2-bromoethyl)methyl-amino]-2-butynyl N-(3-chlorophenyl)carbamate; BR384) structurally related to McN-A-343 (4-(trimethyl-amino)-2-butynyl N-(3-chlorophenyl)carbamate) interacts allosterically or orthosterically with the M(2) muscarinic receptor. Chinese hamster ovary cells expressing the human M(2) muscarinic receptor were incubated with the aziridinium ion of BR384 in combination with McN-A-343 or other known orthosteric and allosteric ligands for various incubation times. After removing unreacted ligands, we measured the binding of [(3)H]N-methylscopolamine to residual unalkylated receptors. Affinity constants, rate constants for alkylation, and cooperativity constants were estimated for the interacting ligands using a mathematical model. Receptor alkylation by BR384 was consistent with a two-step process. After rapidly equilibrating with the receptor (step one), the aziridinium ion-receptor complex became covalently linked with a first order rate constant of about 0.95min(-1) (step two). McN-A-343, acetylcholine and N-methylscopolamine competitively protected the M(2) receptor from irreversible alkylation by BR384. In contrast, the allosteric modulators, gallamine and WIN 51,708 (17-beta-hydroxy-17-alpha-ethynyl-5-alpha-androstano[3,2-beta]pyrimido[1,2-alpha]benzimidazole), allosterically inhibited or had no effect on, respectively, receptor alkylation by BR384. There was good agreement between affinity constants estimated from the kinetics of receptor alkylation and by displacement of [(3)H]N-methylscopolamine binding. Our results suggest that BR384 covalently binds to the orthosteric site of the M(2) receptor and that McN-A-343 binds reversibly at the same locus. Our method of analyzing allosteric interactions does not suffer from the limitations of more conventional approaches and can be adapted to detect allosteric interactions at receptors other than the muscarinic subtypes.


Assuntos
Cloreto de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamônio/farmacologia , Alquinos/farmacologia , Carbamatos/farmacologia , Compostos de Mostarda Nitrogenada/farmacologia , Receptor Muscarínico M2/metabolismo , Acetilcolina/farmacologia , Alquilação , Androstanos/farmacologia , Animais , Benzimidazóis/farmacologia , Células CHO , Cricetinae , Cricetulus , Trietiodeto de Galamina/farmacologia , Masculino , N-Metilescopolamina/metabolismo , Ratos , Ratos Sprague-Dawley
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