Asunto(s)
Músculo Liso , Historia del Siglo XX , Músculo Liso/fisiología , Humanos , Fisiología/historia , Historia del Siglo XXI , AnimalesRESUMEN
Clary sage essential oil (CSEO) is utilized in perfumery, aromatherapy, and skincare. Linalyl acetate (LA), a primary component of CSEO, possesses sedative, anxiolytic, and analgesic properties. However, the mechanism of its analgesic action is not clearly understood. Transient receptor potential ankyrin 1 (TRPA1) channel, a non-selective cation channel, is mainly expressed in sensory neurons and serves as a sensor of various irritants. In this study, we investigated the effects of LA on TRPA1 channel using heterologous expression system and isolated sensory neurons. To detect channel activity, we employed Ca2+ imaging and the whole-cell patch-clamp technique. The analgesic action of LA was measured in a pain-related behavioral mouse model. In cells that heterologously expressed TRPA1, LA diminished [Ca2+]i and current responses to allylisothiocyanate (AITC) and carvacrol: exogenous TRPA1 agonists, and the inhibitory effects were more pronounced for the former than for the latter. Moreover, LA suppressed [Ca2+] i and current responses to PGJ2: an endogenous TRPA1 agonist. Similar inhibitory actions were observed in native TRPA1 channels expressed in mouse sensory neurons. Furthermore, LA diminished PGJ2-induced nociceptive behaviors in mice. These findings suggest that analgesic effects of LA exert through inhibition of nociceptive TRPA1, making it a potential candidate for novel analgesic development.
Asunto(s)
Analgésicos , Monoterpenos , Canal Catiónico TRPA1 , Animales , Canal Catiónico TRPA1/metabolismo , Canal Catiónico TRPA1/genética , Ratones , Analgésicos/farmacología , Monoterpenos/farmacología , Humanos , Masculino , Calcio/metabolismo , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Células HEK293 , Modelos Animales de Enfermedad , Dolor/tratamiento farmacológico , Dolor/metabolismoRESUMEN
Transient receptor potential melastatin 4 (TRPM4) cation channels are expressed in prostate glands. However, the precise role of these channels in prostate contractility remains unclear. In this study, we examined whether TRPM4 channels were involved in adrenergic contractions in the mouse prostate gland. Adrenergic contractile responses elicited by noradrenaline or electrical field stimulation of the sympathetic nerve were isometrically recorded, and the effects of 9-phenanthrol, a specific TRPM4 channel inhibitor, on those contractile responses were investigated in mouse ventral prostate preparations. 9-phenanthrol (10 or 30 µM) inhibited noradrenaline- and sympathetic nerve-evoked contractions in a concentration-dependent manner. A similar inhibitory effect was observed with another TRPM4 channel inhibitor, 4-chloro-2-(2-(naphthalene-1-yloxy) acetamido) benzoic acid (NBA; 10 µM). Inhibition by 9-phenanthrol and NBA were much greater at lower noradrenaline concentrations and lower stimulus frequencies than those of higher concentrations or frequencies. However, 9-phenanthrol did not inhibit the noradrenaline-induced contractile response when the membrane potential was decreased to approximately 0 mV in the 140 mM K+ medium. Moreover, 9-phenanthrol does not affect noradrenaline-induced increases in spontaneous contractions of cardiac atrial preparation. This agent inhibited noradrenaline-induced contractions in the posterior aorta preparation. However, the inhibitory effect was significantly weaker than that observed in the prostate gland. These results suggest that TRPM4 channels are involved in adrenergic contractions in the mouse prostate gland, possibly through membrane depolarization by their opening; therefore, they might be potential candidates for treating benign prostatic hyperplasia.
Asunto(s)
Canales Catiónicos TRPM , Canales de Potencial de Receptor Transitorio , Masculino , Ratones , Animales , Próstata , Músculo Liso , Canales de Potencial de Receptor Transitorio/farmacología , Adrenérgicos/farmacología , Contracción Muscular , Norepinefrina/farmacologíaRESUMEN
Parasympathetic signalling via muscarinic acetylcholine receptors (mAChRs) regulates gastrointestinal smooth muscle function. In most instances, the mAChR population in smooth muscle consists mainly of M2 and M3 subtypes in a roughly 80% to 20% mixture. Stimulation of these mAChRs triggers a complex array of biochemical and electrical events in the cell via associated G proteins, leading to smooth muscle contraction and facilitating gastrointestinal motility. Major signalling events induced by mAChRs include adenylyl cyclase inhibition, phosphoinositide hydrolysis, intracellular Ca2+ mobilisation, myofilament Ca2+ sensitisation, generation of non-selective cationic and chloride currents, K+ current modulation, inhibition or potentiation of voltage-dependent Ca2+ currents and membrane depolarisation. A lack of ligands with a high degree of receptor subtype selectivity and the frequent contribution of multiple receptor subtypes to responses in the same cell type have hampered studies on the signal transduction mechanisms and functions of individual mAChR subtypes. Therefore, novel strategies such as genetic manipulation are required to elucidate both the contributions of specific AChR subtypes to smooth muscle function and the underlying molecular mechanisms. In this article, we review recent studies on muscarinic function in gastrointestinal smooth muscle using mAChR subtype-knockout mice.
Asunto(s)
Tracto Gastrointestinal/metabolismo , Músculo Liso/metabolismo , Receptor Muscarínico M2/genética , Receptor Muscarínico M3/genética , Animales , Proteínas de Unión al GTP/genética , Tracto Gastrointestinal/crecimiento & desarrollo , Tracto Gastrointestinal/patología , Ratones Noqueados/genética , Contracción Muscular/genética , Músculo Liso/crecimiento & desarrollo , Transducción de Señal/genéticaRESUMEN
In mouse ileal myocytes, muscarinic receptor-mediated cationic current (mIcat) occurs mainly through synergism of M2 and M3 subtypes involving Gi/o-type GTP-binding proteins and phospholipase C (PLC). We have further studied the M2/M3 synergistic pathway. Carbachol-induced mIcat was markedly depressed by YM-254890, a Gq/11 protein inhibitor. However, the mIcat was unaffected by heparin, calphostin C, or chelerythrine, suggesting that mIcat activation does not involve signaling molecules downstream of phosphatidylinositol 4,5-bisphosphate (PIP2) breakdown. M2-knockout (KO) mice displayed a reduced mIcat (~10% of wild-type mIcat) because of the lack of M2-Gi/o signaling. The impaired mIcat was insensitive to neuropeptide Y possessing a Gi/o-stimulating activity. M3-KO mice also displayed a reduced mIcat (~6% of wild-type mIcat) because of the lack of M3-Gq/11 signaling, and the mIcat was insensitive to prostaglandin F2α possessing a Gq/11-stimulating activity. These results suggest the importance of Gq/11/PLC-hydrolyzed PIP2 breakdown itself in mIcat activation and also support the idea that the M2/M3 synergistic pathway represents a signaling complex consisting of M2-Gi/o and M3-Gq/11-PLC systems in which both G proteins are special for this pathway but not general in receptor coupling.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Mucosa Intestinal/metabolismo , Miocitos del Músculo Liso/metabolismo , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M3/metabolismo , Animales , Agonistas Colinérgicos/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/agonistas , Cobayas , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Péptidos Cíclicos/farmacología , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M3/agonistasRESUMEN
Gastrointestinal prokinetic agents function as serotonin-4 receptor (5-HT4R) agonists to activate myenteric plexus neurons to release acetylcholine (ACh), which then induce anti-inflammatory action. Details of this pathway, however, remain unknown. The aim of this study is to clarify the anti-inflammatory mechanism underlying the 5-HT4R agonist, mosapride citrate (MOS)-induced anti-inflammatory action on postoperative ileus (POI). POI models were generated from wild-type C57BL6/J (WT), 5-HT4R knock-out (S4R KO), α7 nicotinic AChR KO (α7 R KO), and M2 muscarinic ACh receptor KO (M2R KO) mice. MOS attenuated leukocyte infiltration in WT. MOS-induced anti-inflammatory action was completely abolished in both S4R KO and S4R KO mice upon wild-type bone marrow transplantation. MOS-induced anti-inflammatory action against macrophage infiltration, but not neutrophil infiltration, was attenuated in α7 R KO mice. Selective α7nAChR agonists (PNU-282987 and AR-R17779) also inhibited only macrophage infiltration in POI. MOS-mediated inhibition of neutrophil infiltration was diminished by atropine, M2AChR antagonist, methoctramine, and in M2R KO mice. Stimulation with 5-HT4R inhibits leukocyte infiltration in POI, possibly through myenteric plexus activation. Released ACh inhibited macrophage and neutrophil infiltration likely by activation of α7nAChR on macrophages and M2AChR. Thus, macrophage and neutrophil recruitment into inflamed sites is regulated by different types of AChR in the small intestine.
Asunto(s)
Antiinflamatorios/farmacología , Intestino Delgado/efectos de los fármacos , Receptores Colinérgicos/metabolismo , Acetilcolina/metabolismo , Animales , Antiinflamatorios/uso terapéutico , Benzamidas/farmacología , Benzamidas/uso terapéutico , Compuestos Bicíclicos con Puentes/farmacología , Hidrocarburos Aromáticos con Puentes/farmacología , Diaminas/farmacología , Ileus/tratamiento farmacológico , Ileus/patología , Intestino Delgado/metabolismo , Leucocitos/citología , Leucocitos/inmunología , Leucocitos/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Morfolinas/farmacología , Morfolinas/uso terapéutico , Receptores Colinérgicos/química , Receptores Colinérgicos/genética , Receptores de Serotonina 5-HT4/química , Receptores de Serotonina 5-HT4/genética , Receptores de Serotonina 5-HT4/metabolismo , Compuestos de Espiro/farmacología , Receptor Nicotínico de Acetilcolina alfa 7/agonistas , Receptor Nicotínico de Acetilcolina alfa 7/genética , Receptor Nicotínico de Acetilcolina alfa 7/metabolismoRESUMEN
Here, we investigated the effects of 9-hydroxyphenanthrene (9-phenanthrol), a potent and selective transient receptor potential melastatin 4 (TRPM4) channel blocker, on the resting membrane potential and cholinergic contractile responses to elucidate the functional role of TRPM4 channels in the contractile activities of mouse detrusor and ileal longitudinal smooth muscles. We observed that, 9-phenanthrol (3-30 µM) did not significantly inhibit high K+-induced contractions in both preparations; however, 9-phenanthrol (10 µM) strongly inhibited cholinergic contractions evoked by electrical field stimulation in detrusor preparations compared to inhibitions in ileal preparations. 9-Phenanthrol (10 µM) significantly inhibited the muscarinic agonist, carbachol-induced contractile responses and slowed the maximum upstroke velocities of the contraction in detrusor preparations. However, the agent (10 µM) did not inhibit the contractions due to intracellular Ca2+ release evoked by carbachol, suggesting that the inhibitory effect of 9-phenanthrol may primarily be due to the inhibition of the membrane depolarization process incurred by TRPM4 channels. On the other hand, 9-phenanthrol (10 µM) did not affect carbachol-induced contractile responses in ileal preparations. Further, 9-phenanthrol (10 µM) significantly hyperpolarized the resting membrane potential and decreased the basal tone in both detrusor and ileal muscle preparations. Taken together, our results suggest that TRPM4 channels are constitutively active and are involved in setting of the resting membrane potential, thereby regulating the basal tone in detrusor and ileal smooth muscles. Thus, TRPM4 channels play a significant role in cholinergic signaling in detrusor, but not ileal, smooth muscles.
Asunto(s)
Potenciales de la Membrana/fisiología , Contracción Muscular/fisiología , Músculo Liso/fisiología , Canales Catiónicos TRPM/fisiología , Vejiga Urinaria/fisiología , Animales , Carbacol/farmacología , Colinérgicos/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Íleon , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Fenantrenos/farmacología , Canales Catiónicos TRPM/antagonistas & inhibidores , Canales Catiónicos TRPM/efectos de los fármacos , Vejiga Urinaria/efectos de los fármacosRESUMEN
ATP-sensitive K+ (KATP) channels are expressed in gastrointestinal smooth muscles, and their activity is regulated by muscarinic receptor stimulation. However, the physiological significance and mechanisms of muscarinic regulation of KATP channels are not fully understood. We examined the effects of the KATP channel opener cromakalim and the KATP channel blocker glibenclamide on electrical activity of single mouse ileal myocytes and on mechanical activity in ileal segment preparations. To explore muscarinic regulation of KATP channel activity and its underlying mechanisms, the effect of carbachol (CCh) on cromakalim-induced KATP channel currents ( IKATP) was studied in myocytes of M2 or M3 muscarinic receptor-knockout (KO) and wild-type (WT) mice. Cromakalim (10 µM) induced membrane hyperpolarization in single myocytes and relaxation in segment preparations from WT mice, whereas glibenclamide (10 µM) caused membrane depolarization and contraction. CCh (100 µM) induced sustained suppression of IKATP in cells from both WT and M2KO mice. However, CCh had a minimal effect on IKATP in M3KO and M2/M3 double-KO cells. The Gq/11 inhibitor YM-254890 (10 µM) and PLC inhibitor U73122 (1 µM), but not the PKC inhibitor calphostin C (1 µM), markedly decreased CCh-induced suppression of IKATP in WT cells. These results indicated that KATP channels are constitutively active and contribute to the setting of resting membrane potential in mouse ileal smooth muscles. M3 receptors inhibit the activity of these channels via a Gq/11/PLC-dependent but PKC-independent pathways, thereby contributing to membrane depolarization and contraction of smooth muscles. NEW & NOTEWORTHY We systematically investigated the regulation of ATP-sensitive K+ channels by muscarinic receptors expressed on mouse ileal smooth muscles. We found that M3 receptors inhibit the activity of ATP-sensitive K+ channels via a Gq/11/PLC-dependent, but PKC-independent, pathway. This muscarinic suppression of ATP-sensitive K+ channels contributes to membrane depolarization and contraction of smooth muscles.
Asunto(s)
Íleon/fisiología , Canales KATP/metabolismo , Contracción Muscular , Miocitos del Músculo Liso/metabolismo , Receptores Muscarínicos/metabolismo , Transducción de Señal , Potenciales de Acción , Animales , Carbacol/farmacología , Cromakalim/farmacología , Estrenos/farmacología , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/antagonistas & inhibidores , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Íleon/metabolismo , Canales KATP/genética , Masculino , Ratones , Agonistas Muscarínicos/farmacología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/fisiología , Péptidos Cíclicos/farmacología , Pirrolidinonas/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismoRESUMEN
ML204, a potent transient receptor potential canonical 4 (TRPC4) channel blocker, is often used to elucidate the involvement of TRPC4 channels in receptor-operated signaling processes in visceral smooth muscles. In the present study, we investigated the possible antagonistic actions of ML204 on M2 and M3 muscarinic receptors, which mediate contractions in mouse ileal and detrusor smooth muscles. In ileal and detrusor smooth muscle preparations, ML204 (3 or 10 µM) significantly inhibited electrical field stimulation (EFS)-evoked cholinergic contractions. However, it did not significantly inhibit high K+-induced and EFS-evoked non-cholinergic contractions in the ileal preparations. When the muscarinic agonist, carbachol was cumulatively applied, ML204 (1, 3 and 10 µM) caused a rightward parallel shift of the concentration-response curves of carbachol. Additionally, ML204 (1, 3 and 10 µM) inhibited carbachol-induced negative chronotropic response in atrial preparations, which is mediated by M2 muscarinic receptors. Furthermore, ML204 significantly inhibited the contractions evoked by carbachol-induced intracellular Ca2+ release, which is mediated by M3 muscarinic receptors. These results suggested that ML204 might exhibit antagonistic actions on M2 and M3 muscarinic receptors; in addition, the inhibitory effects of ML204 against EFS-induced cholinergic contractions might be attributed to this receptor antagonism rather than inhibition of TRPC4 channel activity. Therefore, these effects should be considered when ML204 is used as a TRPC4 channel blocker.
Asunto(s)
Antagonistas Muscarínicos/farmacología , Contracción Muscular/fisiología , Receptores Muscarínicos/fisiología , Canales Catiónicos TRPC/fisiología , Animales , Fibrilación Atrial , Carbacol , Japón , Masculino , Ratones , Músculo Liso/fisiología , Miocardio , Canales Catiónicos TRPC/antagonistas & inhibidoresRESUMEN
Muscarinic acetylcholine receptors have been suggested to be implicated in arginine-vasopressin secretion because intracerebroventricular muscarinic agonist administration induces arginine-vasopressin release into the circulation. Although which subtype is involved in the regulation of arginine-vasopressin secretion is unclear, M2 receptors have been reported to be highly expressed in the hypothalamus. In the present study, M2 receptor-knockout mice were used to elucidate whether M2 receptor regulates arginine-vasopressin synthesis in the paraventricular nuclei and supraoptic nuclei of the hypothalamus. The number of arginine-vasopressin-immunoreactive neurons in M2 receptor-knockout mice was significantly decreased in the supraoptic nuclei, but not in the paraventricular nuclei compared with wild-type mice. Plasma arginine-vasopressin level in M2 receptor-knockout mice was also significantly lower than in the wild-type mice. Urinary volume and frequency as well as water intake in M2 receptor-knockout mice were significantly higher than those in wild-type mice. The V2 vasopressin receptor expression in kidneys of M2 receptor-knockout mice was comparable with that of wild-type mice, and increased urination in M2 receptor-knockout mice was significantly decreased by administration of desmopressin, a specific V2 receptor agonist, suggesting that V2 receptors in the kidneys of M2 receptor-knockout mice are intact. These results suggest that M2 receptors promote arginine-vasopressin synthesis in the supraoptic nuclei and play a role in the regulation and maintenance of body fluid.
Asunto(s)
Arginina Vasopresina/biosíntesis , Receptor Muscarínico M2/fisiología , Núcleo Supraóptico/metabolismo , Animales , Fármacos Antidiuréticos/metabolismo , Líquidos Corporales/metabolismo , Femenino , Ratones , Ratones Noqueados , Neuronas/metabolismo , Núcleo Hipotalámico Paraventricular/metabolismo , Receptor Muscarínico M2/genética , Equilibrio Hidroelectrolítico/genéticaRESUMEN
Cognitive impairment often occurs in Parkinson's disease (PD), but the mechanism of onset remains unknown. Recently, we reported that PD model mice produced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) show facilitation of hippocampal memory extinction, which may be the cause of cognitive impairment in PD. When we examined the cAMP/CREB signaling in the hippocampus, decreased levels of cAMP and phosphorylated CREB were observed in the dentate gyrus (DG) of MPTP-treated mice. Administration of rolipram improved the memory deficits with concomitant recovery of cAMP and phosphorylated CREB levels, suggesting that reduced cAMP/CREB signaling in the DG leads to cognitive impairment in MPTP-treated mice.
Asunto(s)
Miedo , Hipocampo/metabolismo , Intoxicación por MPTP/tratamiento farmacológico , Memoria/efectos de los fármacos , Enfermedad de Parkinson/tratamiento farmacológico , Rolipram/farmacología , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Animales , Conducta Animal/efectos de los fármacos , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Extinción Psicológica , Hipocampo/efectos de los fármacos , Intoxicación por MPTP/metabolismo , Intoxicación por MPTP/psicología , Masculino , Ratones , Ratones Endogámicos C57BL , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/psicologíaRESUMEN
Isolated rat thoracic aortic strips undergoing noradrenaline-induced contraction were treated with an adult heartworm (HW) crude extract and then examined for isometric changes in tension. HW extract caused relaxation of endothelium-intact strips, but not endothelium-denuded strips. This effect was inhibited by treatment with NG-nitro-L-arginine methyl ester hydrochloride (L-NAME) and could be reversed by additional treatment with L-arginine. However, HW extract at a high concentration caused slight relaxation of endothelium-denuded strips, and relaxation persisted after L-NAME treatment in endothelium intact-strips. These data suggested that the relaxation induced by HW extract was mainly endothelium-dependent, nitric oxide-mediated, but in part, also endothelium-independent. In addition, a bioassay using isolated rat thoracic aortas may be a useful tool for investigating vasoactive substances in the HW extract.
Asunto(s)
Aorta Torácica/efectos de los fármacos , Dirofilaria immitis/química , Relajación Muscular/efectos de los fármacos , Músculo Liso Vascular/efectos de los fármacos , Extractos de Tejidos/farmacología , Animales , Perros , Endotelio Vascular/efectos de los fármacos , Femenino , Masculino , Ratas , Ratas Wistar , Técnicas de Cultivo de TejidosRESUMEN
In order to investigate the effects of SKF96365 (SKF), which is a non-selective cationic channel blocker, on K(+) channel currents, we recorded currents through ATP sensitive K(+) (IKATP), voltage-gated K(+) (IKv) and Ca(2+) activated K(+) channels (IBK) in the absence and presence of SKF in single small intestinal myocytes of mice with patch-clamp techniques. SKF (10 µM) reversibly abolished IKATP that was induced by cromakalim (10 µM), which is a selective ATP sensitive K(+) channel opener. These inhibitory effects were induced in a concentration-dependent and voltage-independent manner. The 50% inhibitory concentration (IC50) was 0.85 µM, which was obviously lower than that reported for the muscarinic cationic current. In addition, SKF (1 µM ≈ the IC50 value in IKATP suppression) reversibly inhibited the IKv that was induced by repetitive depolarizing pulses from -80 to 20 mV. However, the extent of the inhibitory effects was only ~30%. In contrast, SKF (1 µM) had no significant effects on spontaneous transient IBK and caffeine-induced IBK. These results indicated that SKF inhibited ATP sensitive K(+) channels and voltage-gated K(+) channels, with the ATP sensitive K(+) channels being more sensitive than the voltage-gated K(+) channels. These inhibitory effects on K(+) channels should be considered when SKF is used as a cationic channel blocker.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Imidazoles/farmacología , Intestino Delgado/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/metabolismo , Animales , Femenino , Técnicas In Vitro , Intestino Delgado/citología , Masculino , Ratones , Canales Catiónicos TRPC/metabolismoRESUMEN
AIMS: Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive loss of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc). In PD, thinking and retrieval deficits often arise from cognitive impairments. However, the mechanism of cognitive disorders in PD remains unknown. Therefore, we investigated cognitive function in PD model mice produced by intraperitoneal administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which specifically destroys the DAergic neurons in the SNpc. MAIN METHODS: We evaluated the cognitive function of MPTP-treated mice (PD mice) using the contextual fear conditioning test. In the test, each experiment consists of three phases: training, re-exposure, and testing. Mice were trained with a foot shock (a weak unconditioned stimulus: 1mA/2s duration, once, or an intense unconditioned stimulus: 2mA/2s duration, twice), and 24h later, mice were re-exposed to the training context for 3min to determine reconsolidation or 30min to determine extinction. The percentage of time spent freezing was measured during the test session as indexes of memory consolidation, reconsolidation, and extinction. KEY FINDINGS: Reconsolidation of PD mice occurred normally but memory extinction was facilitated in PD mice compared to control mice. Moreover, memory retention in PD mice was attenuated earlier than in controls following repeated conditioned stimuli every day. SIGNIFICANCE: PD mice with selective loss of DAergic neurons in the SNpc showed attenuated memory retention, probably via facilitated extinction learning.
Asunto(s)
1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/administración & dosificación , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Neuronas Dopaminérgicas/efectos de los fármacos , Neuronas Dopaminérgicas/patología , Extinción Psicológica/efectos de los fármacos , Porción Compacta de la Sustancia Negra/patología , Animales , Conducta Animal/efectos de los fármacos , Núcleo Caudado/efectos de los fármacos , Núcleo Caudado/patología , Condicionamiento Clásico/efectos de los fármacos , Electrochoque , Masculino , Memoria/efectos de los fármacos , Ratones , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/patología , Porción Compacta de la Sustancia Negra/efectos de los fármacos , Porción Compacta de la Sustancia Negra/metabolismo , Putamen/efectos de los fármacos , Putamen/patología , Prueba de Desempeño de Rotación con Aceleración Constante , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
To elucidate the roles played by the interstitial cells of Cajal in the myenteric layer (ICC-MY) in cholinergic neuromuscular transmission, we recorded mechanical and electrical activities in response to electrical field stimulation (EFS) of the ileal longitudinal muscle strips from WBB6F1-W/W(V) (W/W(V)) mutant mice, that lacked ICC-MY and compared with those in WBB6F1-+/+ (+/+) control mice. In +/+ muscle strips, EFS induced phasic contractions, which were abolished or strongly attenuated by atropine or tetrodotoxin. In W/W(V) preparations, EFS induced similar phasic contractions, but the cholinergic component was smaller than that in +/+ strips. This was despite of the fact that the contractions because of exogenous applications of carbachol and high K(+) solution in W/W(V) strips were comparable to or rather greater than those in the +/+ preparations. EFS induced atropine-sensitive excitatory junction potentials (EJPs) in the +/+ longitudinal smooth muscle cells but not in W/W(V) cells. In the presence of eserine, EFS induced atropine-sensitive EJPs in W/W(V) cells. These results suggest that ICC-MY mediate the cholinergic neuromuscular transmission in mouse ileal longitudinal smooth muscles. In addition, the other pathway in which ICC-MY are not involved can operate concomitantly.
Asunto(s)
Íleon/fisiología , Células Intersticiales de Cajal/fisiología , Músculo Liso/fisiología , Plexo Mientérico/fisiología , Adrenérgicos/farmacología , Animales , Atropina/farmacología , Colinérgicos/farmacología , Estimulación Eléctrica , Guanetidina/farmacología , Íleon/inervación , Técnicas In Vitro , Masculino , Potenciales de la Membrana , Ratones , Ratones Mutantes , Contracción Muscular , Músculo Liso/inervación , NG-Nitroarginina Metil Éster/farmacología , Unión Neuromuscular/fisiología , Fisostigmina/farmacología , Transmisión SinápticaRESUMEN
Cholinergic nerve-mediated excitatory junction potentials (EJPs) in the longitudinal muscle of mouse ileum were characterized by using M2 or M3 muscarinic receptor-knockout (KO) mice and 1-[ß-[3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SK&F 96365) and pertussis toxin (PTX). EJPs evoked by electrical field stimulation (EFS) in wild-type preparations, initially determined to be cholinergic in origin using tetrodotoxin, atropine, and eserine, were profoundly depressed after SK&F 96365 treatment known to block muscarinic receptor-operated cation channels. A similar depression of the EJPs was also observed by PTX treatment, which is predicted to disrupt M2-mediated pathways linked to cation channel activation. In M2-KO mouse preparations, cholinergic EJPs were evoked by EFS with their relative amplitude of 20%-30% to the wild-type EJP and strongly inhibited by SK&F 96365. No cholinergic EJP was seen in M3-KO as well as M2/M3 double-KO preparations. The results suggest that the wild-type cholinergic EJP is not a simple mixture of M2 and M3 responses, but due to synergistic activation of cation channels by both M2 and M3 receptors in the murine ileal longitudinal muscle.
Asunto(s)
Potenciales de Acción , Canales de Cloruro/metabolismo , Neuronas Colinérgicas/fisiología , Íleon/citología , Músculo Liso/citología , Miocitos del Músculo Liso/metabolismo , Unión Neuromuscular/fisiología , Receptor Muscarínico M2/fisiología , Receptor Muscarínico M3/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Células Cultivadas , Canales de Cloruro/fisiología , Estimulación Eléctrica , Femenino , Masculino , Ratones , Ratones Noqueados , Toxina del Pertussis/farmacologíaRESUMEN
An isolated atrial preparation of the mouse is useful for analyzing the actions of drugs on the myocardium, autonomic neurons and endocardial endothelium. The aim of the present study was to examine the functions of intrinsic neurons of the atrium using a ganglionic stimulant, 1,1-dimethyl-4-phenylpiperazinium (DMPP). DMPP (1-100 µM) caused a negative chronotropic action followed by a positive chronotropic action in spontaneously beating right atria and also caused biphasic inotropic actions consisting of initial inhibition followed by potentiation of electrical field stimulation (EFS)-induced contraction in the left atria. Inotropic actions in the left atria induced by DMPP were characterized using some autonomic drugs and M2 and/or M3 muscarinic receptor knockout (M2R-KO, M3R-KO and M2M3R-KO) mice. Atropine and hexamethonium decreased the initial negative inotropic actions of DMPP. In the atria from pertussis toxin-treated, M2R-KO and M2/M3R-KO mice, the negative inotropic actions were abolished. On the other hand, the following positive inotropic actions were decreased by hexamethonium, atropine and atenolol. In the atria from reserpine-treated mice, positive inotropic actions were also decreased. The positive inotropic action induced by DMPP was almost the same in M2R-KO mice but was reduced in both M3R-KO mice and M2/M3R-KO mice. In conclusion, DMPP caused biphasic inotropic/chronotropic actions in the mouse atrium through activation of intrinsic cholinergic and adrenergic neurons. M2 and M3 muscarinic receptors and ß1-adrenoceptor are thought to be involved in these actions.
Asunto(s)
Yoduro de Dimetilfenilpiperazina/farmacología , Estimulantes Ganglionares/farmacología , Atrios Cardíacos/efectos de los fármacos , Receptor Muscarínico M2/fisiología , Receptor Muscarínico M3/fisiología , Receptores Adrenérgicos beta 1/fisiología , Antagonistas de Receptores Adrenérgicos beta 1/farmacología , Animales , Atenolol/farmacología , Atropina/farmacología , Inhibidores de la Colinesterasa/farmacología , Estimulación Eléctrica , Femenino , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Antagonistas Muscarínicos/farmacología , Contracción Miocárdica/efectos de los fármacos , Fisostigmina/farmacología , Receptor Muscarínico M2/antagonistas & inhibidores , Receptor Muscarínico M3/antagonistas & inhibidoresRESUMEN
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.
Asunto(s)
Colon/efectos de los fármacos , Colon/fisiología , Motilidad Gastrointestinal , Receptores Muscarínicos/deficiencia , Receptores Muscarínicos/metabolismo , Cloruro de (4-(m-Clorofenilcarbamoiloxi)-2-butinil)trimetilamonio/farmacología , Animales , Fenómenos Biomecánicos , Carbacol/farmacología , Colon/metabolismo , Defecación/efectos de los fármacos , Femenino , Motilidad Gastrointestinal/efectos de los fármacos , Técnicas de Inactivación de Genes , Técnicas In Vitro , Masculino , Ratones , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/farmacología , Contracción Muscular/efectos de los fármacos , Pirenzepina/farmacología , Receptores Muscarínicos/genéticaRESUMEN
The present study was designed to explore the inhibitory mechanism by nitric oxide (NO) of the tachykininergic neuro-muscular transmissions in the hamster ileum. In the presence of guanethidine (1 µM), atropine (0.5 µM), nifedipine (0.1 µM) and apamin (100 nM), electrical field stimuli (EFS; 0.5 ms duration, 15 V) evoked non-adrenergic, non-cholinergic excitatory junction potentials (EJPs) in circular smooth muscle cells. The EJPs were markedly inhibited by the tachykinin NK1 receptor antagonists [D-Pro(4), D-Trp(7,9)]-SP(4-11) (3 µM). Both the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 200 µM) and the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ, 10 µM), did not affect on the resting membrane potentials, but enhanced the tachykininergic EJPs. In the presence of L-NAME (200 µM), exogenously applied NO (10 µM) and the membrane permeable analogue of guanosine 3',5'-cyclic monophosphate (cGMP), 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP, 3 mM), significantly inhibited the tachykininergic EJPs. Application of EFS (0.5 msec duration, 15 V) with trains of 20 pulses at 20 Hz increased amount of released substance P (SP). The release of SP was further increased by the treatment of L-NAME or ODQ, but markedly reduced by exogenously applied NO and 8-Br-cGMP. These results suggest that the endogenous NO may inhibit the tachykininergic neuro-muscular transmissions by the decrease of SP release from the tachykininergic neurons, possibly through a guanylate cyclase-cGMP-dependent mechanism in the hamster ileum.
Asunto(s)
GMP Cíclico/metabolismo , Íleon/metabolismo , Unión Neuromuscular/fisiología , Óxido Nítrico/metabolismo , Taquicininas/metabolismo , Animales , Cricetinae , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacología , Masculino , Potenciales de la Membrana , Mesocricetus , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , NG-Nitroarginina Metil Éster/farmacología , Oxadiazoles/farmacología , Quinoxalinas/farmacología , Sustancia P/metabolismoRESUMEN
Botulinum neurotoxin serotype A (BoNT/A) inhibits acetylcholine release at the neuromuscular junction in isolated muscles, and ouabain can partially block its effect. However, it is not clear whether ouabain attenuates BoNT/A-induced neuromuscular paralysis in vivo. In this work, we investigated the effects of ouabain on BoNT/A-induced neuromuscular paralysis in mice. Ouabain was administered to mice intraperitoneally immediately after a single injection of BoNT/A into skeletal muscle. The effects of ouabain on BoNT/A-induced muscle paralysis were assessed by quantitative monitoring of muscle tension and digit abduction via the digit abduction scoring (DAS) assay. A single administration of ouabain significantly prolonged BoNT/A-induced neuromuscular paralysis. Moreover, consecutive daily injection of ouabain exacerbated BoNT/A-induced neuromuscular paralysis, and led to a significant decrease in both twitch and tetanic forces as assayed in isolated BoNT/A-injected muscles. We next looked at the effects of ouabain on BoNT/A-induced muscle atrophy. Administration of ouabain led to a decrease in the myofibrillar cross-sectional area (CSAs) by 14 post-BoNT/A injection. In addition, repeated administration of ouabain increased mRNA expression levels of ubiquitin ligases, which are markers of muscle atrophy, in BoNT/A-injected muscle. These results suggest that ouabain exacerbates BoNT/A-induced neuromuscular paralysis via a marked progression of BoNT/A-induced muscle atrophy.