RESUMEN
Muscarinic agonists induce the activation of the airway smooth muscle (ASM) leading to smooth muscle contraction, important in asthma. This activation is mediated through M2/M3 muscarinic acetylcholine receptors (mAChRs). Muscarinic receptor activity, expressed as [(3)H]QNB binding at plasma membranes from bovine tracheal smooth muscle (BTSM), increased with cGMP and was augmented significantly cGMP plus ATP but diminished with the PKG-II inhibitor, Sp-8-pCPT-cGMPS. The [(3)H]-QNB binding was accelerated by okadaic acid, (OKA), a protein phosphatase (PPase) inhibitor. These two results indicated the involvement of a membrane-bound PPase. Moreover, a cGMP-dependent-[(32)P]γATP phosphorylation of plasma membranes from BTSM was stimulated at low concentrations of muscarinic agonist carbamylcholine (CC). However, higher amounts of CC produced a significant decrement of [(32)P]-labeling. A selective M3mAChR antagonist, 4-DAMP produced a dramatic inhibition of the basal and CC-dependent [(32)P]-labeling. The [(32)P] labeled membrane sediments were detergent solubilized and immunoprecipitated with specific M2/M3mAChR antibodies. The M3mAChR immuno-precipitates exhibited the highest cGMP-dependent [(32)P]-labeling, indicating it is a PKG-II substrate. Experiments using synthetic peptides from the C-terminal of the third intracellular loop (i3) of both M2mAChR (356-369) and M3mAChR (480-493) as external PKG-II substrates resulted in the i3M3-peptide being heavily phosphorylated. These results indicated that PKG-II phosphorylated the M3mAChR at the i3M3 domain ((480)MSLIKEKK(485)), suggesting that Ser(481) may be the target. Finally, this phosphorylation site seems to be regulated by a membrane-bound PPase linked to muscarinic receptor. These findings are important to understand the role of M3mAChR in the patho-physiology of ASM involved in asthma and COPD.
Asunto(s)
Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Agonistas Muscarínicos/farmacología , Antagonistas Muscarínicos/farmacología , Músculo Liso/efectos de los fármacos , Músculo Liso/metabolismo , Receptor Muscarínico M3/metabolismo , Animales , Asma/etiología , Asma/fisiopatología , Bovinos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , GMP Cíclico/farmacología , Proteínas Quinasas Dependientes de GMP Cíclico/antagonistas & inhibidores , Retroalimentación Fisiológica , Humanos , Técnicas In Vitro , Agonistas Muscarínicos/metabolismo , Antagonistas Muscarínicos/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Quinuclidinil Bencilato/metabolismo , Quinuclidinil Bencilato/farmacocinética , Transducción de Señal/efectos de los fármacos , Tionucleótidos/farmacología , Tráquea/efectos de los fármacos , Tráquea/metabolismoRESUMEN
According to the theory of multiple memory systems, specific brain regions interact to determine how the locations of goals are learned when rodents navigate a spatial environment. A number of factors influence the type of strategy used by rodents to remember the location of a given goal in space, including the biological sex of the learner. We recently found that prior to puberty male rats preferred a striatum-dependent stimulus-response strategy over a hippocampus-dependent place strategy when solving a dual-solution task, while age-matched females showed no strategy preference. Because the cholinergic system has been implicated in learning strategy and is known to be sexually dimorphic prior to puberty, we explored the relationship between learning strategy and muscarinic receptor binding in specific brain regions of prepubertal males and female rats. We confirmed our previous finding that at 28 days of age a significantly higher proportion of prepubertal males preferred a stimulus-response learning strategy than a place strategy to solve a dual-solution visible platform water maze task. Equal proportions of prepubertal females preferred stimulus-response or place strategies. Profiles of muscarinic receptor binding as assessed by autoradiography varied according to strategy preference. Regardless of biological sex, prepubertal rats that preferred stimulus-response strategy exhibited lower ratios of muscarinic receptor binding in the hippocampus relative to the dorsolateral striatum compared to rats that preferred place strategy. Importantly, much of the variance in this ratio was related to differences in the ventral hippocampus to a greater extent than the dorsal hippocampus. The ratios of muscarinic receptors in the hippocampus relative to the basolateral amygdala also were lower in rats that preferred stimulus-response strategy over place strategy. Results confirm that learning strategy preference varies with biological sex in prepubertal rats with males biased toward a stimulus-response strategy, and that stimulus-response strategy is associated with lower ratios of muscarinic binding in the hippocampus relative to either the striatum or amygdala.
Asunto(s)
Encéfalo/metabolismo , Receptores Muscarínicos/metabolismo , Caracteres Sexuales , Factores de Edad , Análisis de Varianza , Animales , Animales Recién Nacidos , Encéfalo/anatomía & histología , Encéfalo/efectos de los fármacos , Femenino , Masculino , Aprendizaje por Laberinto , Antagonistas Muscarínicos/farmacocinética , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Quinuclidinil Bencilato/farmacocinética , Ensayo de Unión Radioligante , Ratas , Tritio/farmacocinéticaRESUMEN
Molecular processes regulating cholinergic functions play an important role in the control of respiration under neonatal hypoxia. The present study evaluates neonatal hypoxic insult-mediated cholinergic alterations and the protective role of glucose, oxygen and epinephrine resuscitation. The changes in total muscarinic, muscarinic M1, M2, M3 receptors and the enzymes involved in acetylcholine metabolism--cholineacetyl transferase and acetylcholine easterase in the brain stem were analyzed. Hypoxic stress decreased total muscarinic receptors along with a reduction in muscarinic M1, M2 and M3 receptor genes in the brain stem. The reduction in acetylcholine metabolism is indicated by the down regulated cholineacetyl transferase and up regulated acetylcholine easterase expression. These cholinergic disturbances in the brain stem were reversed by glucose resuscitation to hypoxic neonates. The adverse effects of immediate oxygenation and epinephrine administration were also reported. This has immense clinical significance in establishing a proper resuscitation for the management of neonatal hypoxia.
Asunto(s)
Glucemia/metabolismo , Tronco Encefálico/metabolismo , Colina O-Acetiltransferasa/metabolismo , Epinefrina/farmacología , Hipoxia Encefálica/patología , Receptores Muscarínicos/metabolismo , Animales , Animales Recién Nacidos , Tronco Encefálico/efectos de los fármacos , Colina O-Acetiltransferasa/genética , Colinesterasas/genética , Colinesterasas/metabolismo , Colorantes , Modelos Animales de Enfermedad , Expresión Génica/efectos de los fármacos , Glutatión/genética , Glutatión/metabolismo , Antagonistas Muscarínicos/farmacocinética , Unión Proteica/efectos de los fármacos , Quinuclidinil Bencilato/farmacocinética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores Muscarínicos/genética , Análisis de Regresión , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Tritio/farmacocinéticaRESUMEN
BACKGROUND: Multifactorial diseases such as Alzheimer's disease (AD) should be more efficiently tackled by drugs which hit multiple biological targets involved in their pathogenesis. We have recently developed a new family of huprine-tacrine heterodimers, rationally designed to hit multiple targets involved upstream and downstream in the neurotoxic cascade of AD, namely ß-amyloid aggregation and formation as well as acetylcholinesterase catalytic activity. OBJECTIVE: In this study, the aim was to expand the pharmacological profiling of huprine-tacrine heterodimers investigating their effect on muscarinic M(1) receptors as well as their neuroprotective effects against an oxidative insult. METHODS: Sprague-Dawley rat hippocampus homogenates were used to assess the specific binding of two selected compounds in competition with 1 nM [(3)H]pirenzepine (for M(1) receptors) or 0.8 nM [(3)H]quinuclidinyl benzilate (for M(2) receptors). For neuroprotection studies, SHSY5Y cell cultures were subjected to 250 µM hydrogen peroxide insult with or without preincubation with some huprine-tacrine heterodimers. RESULTS: A low nanomolar affinity and M(1)/M(2) selectivity has been found for the selected compounds. Huprine-tacrine heterodimers are not neurotoxic to SHSY5Y cells at a range of concentrations from 1 to 0.001 µM, and some of them can protect cells from the oxidative damage produced by hydrogen peroxide at concentrations as low as 0.001 µM. CONCLUSION: Even though it remains to be determined if these compounds act as agonists at M(1) receptors, as it is the case of the parent huprine Y, their low nanomolar M(1) affinity and neuroprotective effects expand their multitarget profile and increase their interest as disease-modifying anti-Alzheimer agents.
Asunto(s)
Aminoquinolinas/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/metabolismo , Multimerización de Proteína/fisiología , Tacrina/metabolismo , Análisis de Varianza , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Antagonistas Muscarínicos/farmacocinética , Neuroblastoma/patología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Pirenzepina/farmacocinética , Unión Proteica/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Quinuclidinil Bencilato/farmacocinética , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Tritio/metabolismoRESUMEN
We recently identified LY2033298 as a novel allosteric potentiator of acetylcholine (ACh) at the M(4) muscarinic acetylcholine receptor (mAChR). This study characterized the molecular mode of action of this modulator in both recombinant and native systems. Radioligand-binding studies revealed that LY2033298 displayed a preference for the active state of the M(4) mAChR, manifested as a potentiation in the binding affinity of ACh (but not antagonists) and an increase in the proportion of high-affinity agonist-receptor complexes. This property accounted for the robust allosteric agonism displayed by the modulator in recombinant cells in assays of [(35)S]GTPgammaS binding, extracellular regulated kinase 1/2 phosphorylation, glycogen synthase kinase 3beta phosphorylation, and receptor internalization. We also found that the extent of modulation by LY2033298 differed depending on the signaling pathway, indicating that LY2033298 engenders functional selectivity in the actions of ACh. This property was retained in NG108-15 cells, which natively express rodent M(4) mAChRs. Functional interaction studies between LY2033298 and various orthosteric and allosteric ligands revealed that its site of action overlaps with the allosteric site used by prototypical mAChR modulators. Importantly, LY2033298 reduced [(3)H]ACh release from rat striatal slices, indicating retention of its ability to allosterically potentiate endogenous ACh in situ. Moreover, its ability to potentiate oxotremorine-mediated inhibition of condition avoidance responding in rodents was significantly attenuated in M(4) mAChR knockout mice, validating the M(4) mAChR as a key target of action of this novel allosteric ligand.
Asunto(s)
Acetilcolina/metabolismo , Antipsicóticos/farmacología , Unión Competitiva/efectos de los fármacos , Receptor Muscarínico M4/fisiología , Acetilcolina/farmacología , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/fisiología , Sitio Alostérico/efectos de los fármacos , Sitio Alostérico/fisiología , Animales , Antipsicóticos/química , Reacción de Prevención/efectos de los fármacos , Reacción de Prevención/fisiología , Línea Celular , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Cricetinae , Cricetulus , Relación Dosis-Respuesta a Droga , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Técnicas In Vitro , Ratones , Ratones Noqueados , Modelos Moleculares , Análisis Multivariante , Antagonistas Muscarínicos/farmacocinética , N-Metilescopolamina/farmacocinética , Ácidos Nicotínicos/química , Ácidos Nicotínicos/farmacología , Parasimpatolíticos/farmacocinética , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Quinuclidinil Bencilato/farmacocinética , Ensayo de Unión Radioligante/métodos , Ratas , Receptor Muscarínico M4/química , Receptor Muscarínico M4/deficiencia , Receptor Muscarínico M4/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Tiofenos/química , Tiofenos/farmacología , Tritio/metabolismo , Tritio/farmacocinéticaRESUMEN
BACKGROUND/AIMS: Xanomeline has been shown to bind in a unique manner at M1 and M3 muscarinic receptors, with interactions at both the orthosteric site and an allosteric site. We have previously shown that brief exposure of Chinese hamster ovary cells that express the M3 receptor to xanomeline followed by removal of free agonist results in a delayed decrease in radioligand binding and receptor response to agonists. In the current study, we were interested in determining the mechanisms of this effect. METHODS: Cells were treated with carbachol, pilocarpine or xanomeline for 1 h followed by washing and either used immediately or after waiting for 23 h. Control groups included cells that were not exposed to agonists and cells that were treated with agonists for 24 h. Radioligand binding and functional assays were conducted to determine the effects of agonist treatments. RESULTS: The above treatment protocol with xanomeline resulted in similar effects of the binding of [(3)H]NMS and [(3)H]QNB. When receptor function is blocked using a variety of methods, the long-term effects of xanomeline binding were absent. CONCLUSION: Our data indicate that xanomeline wash-resistant binding at the receptor allosteric site leads to receptor downregulation and that receptor activation is necessary for these effects.
Asunto(s)
Agonistas Muscarínicos/farmacología , Agonistas Muscarínicos/farmacocinética , Piridinas/farmacología , Piridinas/farmacocinética , Receptor Muscarínico M3/agonistas , Receptor Muscarínico M3/fisiología , Tiadiazoles/farmacología , Tiadiazoles/farmacocinética , Animales , Atropina/farmacología , Sitios de Unión/efectos de los fármacos , Carbacol/farmacología , Línea Celular Transformada , Cricetinae , Interacciones Farmacológicas , Femenino , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Humanos , N-Metilescopolamina/farmacocinética , Fosfatidilinositoles/metabolismo , Pilocarpina/farmacología , Quinuclidinil Bencilato/farmacocinética , Factores de Tiempo , TransfecciónRESUMEN
OBJETIVO: Investigar la posible participación de la transmisión muscarínica en la depresión miocárdicainducida por propofol, anestésico intravenoso no relacionadoquímicamente con otras drogas. A pesar de que los efectos adversos son raros, se ha reportado la aparición de bradicardia, que puede llevar incluso a paro cardíaco en algunos pacientes. En la actualidad se desconoce elmecanismo de este efecto, pero se ha sugerido la posible participación de los receptores colinérgicos muscarínicos(RCMs).MATERIAL Y MÉTODOS: La interacción del propofol con RCMs de aurícula humana se determinó por ensayos deinhibición usando benzilato de [3H]-quinuclidinilo ([3H]- QNB).RESULTADOS: El propofol desplazó la unión de [3H]- QNB de los RCMs de aurícula humana, de maneradependiente de la concentración; no obstante, el efecto observado no fue consistente con un modelo de simplecompetición entre propofol y [3H]-QNB.CONCLUSIÓN: El propofol parece tener la capacidad de modificar la actividad de los RCMs de aurícula humana,siendo éste un efecto que podría estar relacionado con su capacidad de inducir bradicardia (AU)
OBJECTIVE: To investigate the possible role of muscarinic cholinergic receptors (MCRs) in the depression ofmyocardial function induced by propofol, an intravenous anesthetic chemically unrelated to other drugs. Althoughadverse effects are rare, bradycardia has been reported and this can lead to cardiac arrest in some patients. Themechanism behind this effect is still unknown but a possible role for MCRs has been suggested.MATERIAL AND METHODS: The interaction of propofol with human atrial MCRs was determined by means ofinhibition tests using [3H] quinuclidinyl benzilate ([3H] QNB).RESULTS: The displacement of [3H] QNB binding to human atrial MCRs by propofol was concentrationdependent but the observed effect was not consistent with a model of simple competition between propofol and [3H]QNB.CONCLUSION: Propofol appears to have the ability to modify the activity of human atrial MCRs and this effectmay be related to its ability to induce bradycardia (AU)
Asunto(s)
Humanos , Propofol/farmacocinética , Contracción Miocárdica , Receptores Muscarínicos , Atrios Cardíacos , Quinuclidinil Bencilato/farmacocinética , Anestésicos/farmacocinéticaRESUMEN
Sodium valproate (VPA) has been used clinically for treatment of not only epilepsy but also mood disorder. Although VPA is effective for treatment of epilepsy via inhibition of gamma-aminobutyric acid transaminase, it remains unknown why VPA is effective for the treatment of mood disorder. The authors examined the effect of VPA at therapeutic concentrations (300 and 600 microM) on the elevation of intracellular free calcium concentration ([Ca(2+)](i)) induced by carbachol, a muscarinic receptor agonist, in 1321N1 human astrocytoma cells. Treatment of the cells with 300 and 600 microM VPA for 2 min did not change the carbachol-induced [Ca(2+)](i) elevation. Treatment with 300 and 600 microM VPA for 48 h, however, reduced the elevation. Since we have shown that Li(+) reduced carbachol-induced [Ca(2+)](i) elevation in protein kinase C (PKC)-downregulated 1321N1 cells [Kurita, M., Mashiko, H., Rai, M., Kumasaka, T., Kouno, S., Niwa, S., Nakahata, N., 2002. Lithium chloride at a therapeutic concentration reduces Ca(2+)response in protein kinase C down-regulated human astrocytoma cells, Eur. J. Pharmacol. 442, 17-22.], the activity of PKC was examined. Treatment with VPA at the same concentrations for 24 or 48 h weakly reduced protein kinase C activity in membrane and cytosol fractions from the cells. On the other hand, the treatment of the cells with 600 microM VPA for 24 or 48 h slightly increased the B(max) value, but not the K(d) value, in the binding of [(3)H]quinuclidinyl benzylate, a muscarinic receptor ligand, to the membranes, suggesting that the number or affinity of muscarinic receptor did not decrease after VPA treatment. These results indicate that VPA at therapeutic concentrations slightly decreases the PKC activity and inhibits muscarinic receptor-mediated [Ca(2+)](i) elevation probably through change in the intracellular signaling pathway. VPA-induced reduction of PKC activity and [Ca(2+)](i) elevation may play a role in the treatment of mood disorder.
Asunto(s)
Astrocitoma/metabolismo , Calcio/metabolismo , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Quinasa C/metabolismo , Ácido Valproico/farmacología , Unión Competitiva/efectos de los fármacos , Carbacol/farmacología , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Agonistas Colinérgicos/farmacología , Citosol/efectos de los fármacos , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Antagonistas Muscarínicos/farmacocinética , Quinuclidinil Bencilato/farmacocinética , Factores de TiempoRESUMEN
To investigate changes in muscarinic receptor-stimulated phospholipase C-beta (PLC-beta) activity during brain development, we examined the functional coupling of each of the three major protein components of the phosphoinositide system (M1, M3, and M5 muscarinic receptor subtypes; Gq/11 proteins; PLC-beta1-4 isoforms) in membrane preparations from post-mortem human prefrontal cerebral cortex collected at several stages of prenatal and postnatal development. In human prenatal brain membranes, PLC was found to be present and could be activated by calcium, but the ability of guanosine-5'-o-3 thiotriphosphate (GTPgammaS) or carbachol (in the presence of GTPgammaS) to modulate prenatal PLC-beta was significantly weaker than that associated with postnatal PLC-beta. Western blot analysis revealed that the levels of Galphaq/11 did not change significantly during development. In contrast, dramatically higher levels of expression of PLC-beta1-4 isoforms and of M1, M3, and M5 muscarinic receptors were detected in the child vs. the fetal brain, a finding that might underlie the observed increased activity of PLC. Thus, inositol phosphate production may be more efficiently regulated by altering the amount of effectors (PLC-beta1-4) and receptors (M1,3,5 subtypes) than by altering the level of Galphaq/11 subunits. These results demonstrate that different PLC isoforms are expressed in the prefrontal cortex of the developing human brain in an age-specific manner, suggesting specific roles not only in synaptic transmission but also in the differentiation and maturation of neurons in the developing brain.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Corteza Prefrontal/enzimología , Transducción de Señal/fisiología , Fosfolipasas de Tipo C/metabolismo , Actinas/metabolismo , Adolescente , Adulto , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Niño , Preescolar , Relación Dosis-Respuesta a Droga , Femenino , Feto , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Humanos , Masculino , Corteza Prefrontal/embriología , Corteza Prefrontal/crecimiento & desarrollo , Unión Proteica/fisiología , Quinuclidinil Bencilato/análogos & derivados , Quinuclidinil Bencilato/farmacocinética , Ensayo de Unión Radioligante/métodos , Receptores Muscarínicos/metabolismo , Tritio/farmacocinéticaRESUMEN
The developing nervous system is thought to be particularly sensitive to polychlorinated biphenyls (PCBs) present as food contaminants together with methylmercury (MeHg). Effects of perinatal co-exposure to PCB153 and MeHg on brain cholinergic muscarinic receptors (MRs) were investigated by saturation binding studies in mature and immature rats. MeHg alone (1mg/kg/day, GD7-PND7) enhanced cerebral MRs more in dams (87% and 60% in cerebellum and cerebral cortex, respectively) than in PND21 pups (0-50%) in accordance with the higher Hg levels detected in the adult brain (7-9 microg/g) than in the male and female offspring's brain (1.5-2.8 microg/g). Prenatal administration of PCB153 (20mg/kg/day, GD10-GD16), leading to higher contaminant levels in the offspring brain than in that of adults (25-66 microg/g versus 3 microg/g), induced cerebral MR changes of similar extent at both ages, namely decreased cerebellar (20-30%) and increased cortical MR density (40-50%). Co-exposure to PCB and MeHg had no more effect than exposure to either compound alone on cerebral cortex MRs, whereas, in the cerebellum, the combined treatment induced a PCB-like lowering of the MR density that masked the MeHg-induced receptor increase. None of the treatments affected the striatal and hippocampal MRs. A lower MeHg dose (0.5 mg/kg/day) was without any effect on cerebral MRs. These results show that MRs are one of the sensitive biochemical endpoints of the central nervous system altered by developmental exposure to MeHg and PCB153. Cerebral cortex and cerebellum were the most susceptible targets in the response to these neurotoxicants. MR changes were detected in both immature and adult animals and the interaction of MeHg and PCB153 at the level of these receptors occurred in a non-additive manner.
Asunto(s)
Encéfalo/efectos de los fármacos , Compuestos de Metilmercurio/toxicidad , Bifenilos Policlorados/toxicidad , Efectos Tardíos de la Exposición Prenatal , Receptores Muscarínicos/metabolismo , Factores de Edad , Animales , Animales Recién Nacidos , Encéfalo/crecimiento & desarrollo , Encéfalo/patología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Femenino , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Masculino , Compuestos de Metilmercurio/metabolismo , Antagonistas Muscarínicos/farmacocinética , Bifenilos Policlorados/metabolismo , Embarazo , Unión Proteica/efectos de los fármacos , Quinuclidinil Bencilato/farmacocinética , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Factores Sexuales , Tritio/farmacocinéticaRESUMEN
In this paper we demonstrate that, circulating antibodies from schizophrenic patients interacting with cerebral M1 muscarinic acetylcholine receptors (M1 mAChRs), can act as an inducer of m1 mAChR-mRNA, and neuronal nitric oxide synthase (nNOS) mRNA gene expression of rat frontal cortex. The different signaling pathways involved in the autoantibody's actions, were characterized. As previously reported serum autoantibodies from schizophrenic patients reacted against neural cells surface inhibiting the binding of the specific mAChR radioligand to rat cerebral frontal cortex membrane. Moreover, by ELISA using M1 synthetic peptide (with identical aminoacid sequence to human M1 mAChR) as coating antigen we demonstrated the reactivity against the second extracellular loop of human cerebral M1 mAChR. The corresponding affinity-purified anti M1 peptide IgG (anti M1 peptide IgG) from schizophrenic patients by stimulation of M1 mAChR exerted an increase in m1 mAChR-mRNA and nNOS-mRNA levels, that significantly correlated with the accumulation of phosphoinositides (IPs) and activation of NOS (alpha = 0.05). All these effects were blunted by pirenzepine and mimicked the action of the authentic agonist. Concurrent analysis of the effects of nNOS, phospholipase C (PLC) and calcium/calmodulin (CaM) inhibition on both, m1 mAChR-mRNA and nNOS-mRNA levels, showing that antibody up-regulation mRNA level is under the control of endogenous nitric oxide (NO) signaling system. On the basis of our results, the activation of M1 mAChR by schizophrenic autoantibody appears to induce nNOS-mRNA expression and reciprocally, the activation of NOS up-regulates m1 mAChR gene expression. These results gave support to the participation of an autoimmune process in a particular group of chronic schizophrenic patients.
Asunto(s)
Autoanticuerpos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Óxido Nítrico Sintasa de Tipo I/metabolismo , Receptor Muscarínico M1/metabolismo , Esquizofrenia/inmunología , Adulto , Análisis de Varianza , Animales , Autoanticuerpos/química , Northern Blotting/métodos , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , Cromatografía de Afinidad/métodos , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Regulación de la Expresión Génica/fisiología , Humanos , Fosfatos de Inositol/metabolismo , Masculino , Persona de Mediana Edad , Antagonistas Muscarínicos/farmacocinética , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo I/genética , Quinuclidinil Bencilato/farmacocinética , Ensayo de Unión Radioligante/métodos , Ratas , Ratas Wistar , Receptor Muscarínico M1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Tritio/farmacocinéticaRESUMEN
The cholesterol-lowering drug lovastatin enhances the secretion of the alpha-secretase cleavage product of amyloid precursor protein (APP). To investigate whether this effect is mediated via activation of alpha7 nicotinic acetylcholine receptors (nAChRs), we treated SH-SY5Y cells and PC12 cells with lovastatin and measured the levels of alpha7 nAChRs, the alpha-form of secreted APP (alphaAPPs), and lovastatin-related lipids, including cholesterol and ubiquinone. The results showed that low concentrations of lovastatin significantly induced up-regulation of alpha7 nAChRs. No effects of lovastatin were observed on alpha3-containing nAChRs, muscarinic receptors, or N-methyl-D-aspartate receptors. alphaAPPs levels increased in the culture medium of cells treated with lovastatin, whereas no change in whole APP was observed. The increase in alphaAPPs was inhibited by prior exposure of these cells to alpha-bungarotoxin, an antagonist of alpha7 nAChRs. The concentrations of lovastatin used in the study did not change the cholesterol content, but high doses can decrease the levels of ubiquinone and cell viability. These results indicate that lovastatin may play a neuronal role that is cholesterol independent. We also show that the up-regulation of alpha7 nAChRs stimulated by lovastatin is involved in a mechanism that enhances production of alphaAPPs during APP processing.
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Precursor de Proteína beta-Amiloide/biosíntesis , Colesterol/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Lovastatina/farmacología , Neuronas/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Animales , Northern Blotting/métodos , Western Blotting/métodos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacocinética , Bungarotoxinas/farmacología , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión/métodos , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Humanos , Isótopos/farmacocinética , Neuroblastoma , Neuronas/metabolismo , Nicotina/farmacología , Agonistas Nicotínicos/farmacocinética , Células PC12 , Unión Proteica/efectos de los fármacos , Piridinas/farmacocinética , Quinuclidinil Bencilato/farmacocinética , ARN Mensajero/metabolismo , Ensayo de Unión Radioligante/métodos , Ratas , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Nicotínicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Ubiquinona/metabolismoRESUMEN
Loss of cholinergic neurons along with muscarinic acetylcholine receptors (mAChRs) in cerebral cortex and hippocampus is closely associated with Alzheimer's disease (AD). Recent drug development for AD treatment focuses heavily on identifying M(1) receptor agonists. However, mAChRs undergo down-regulation in response to agonist-induced sustained activation. Therefore, therapeutic effectiveness wanes during continuous use. Thus, another potentially effective approach, which overcomes this drawback is to develop compounds, which instead up-regulate M(1) receptor expression. In the present study, we took this alternative approach and contrasted in Chinese hamster ovary cells transfected with human m(1) subtype gene (CHOm(1) cells) changes of M(1) receptor expression levels caused by muscarinic agonists and upregulators of its expression. The muscarinic agonists carbachol and pilocarpine reduced M(1) receptor number in CHOm(1) cells by 29 and 46%, respectively, at 100muM, whereas panaxynol, a polyacetylene compound isolated from the lipophilic fraction of Panax notoginseng, concentration-dependently up-regulated the M(1) receptor number after pre-incubation with CHOm(1) cells for 48 h, reaching a plateau at 1 microM, and was accompanied by enhanced M(1) mRNA levels. Moreover, the protein kinase A (PKA) inhibitor RP-adenosine-3',5'-cyclic mono-phosphoro-thioate triethylamine salt (RP-cAMPs) 5 microM completely prevented panaxynol-induced up-regulation of M(1) receptors. Panaxynol (1muM) caused a significant and consistent stimulation of cAMP accumulation (27% increase above basal at 40 min). These results suggest that in CHOm(1) cells panaxynol up-regulates M(1) receptor number through cAMP pathway-mediated stimulation of gene transcription.
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AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , Alcoholes Grasos/farmacología , Receptor Muscarínico M1/metabolismo , Transducción de Señal/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Alquinos , Animales , Northern Blotting/métodos , Células CHO , Carbacol/farmacología , Agonistas Colinérgicos/farmacología , Cricetinae , Cricetulus , AMP Cíclico/farmacología , Diinos , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Medicamentos Herbarios Chinos/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Inhibidores de Fosfodiesterasa/farmacología , Pilocarpina/farmacología , Unión Proteica/efectos de los fármacos , Quinuclidinil Bencilato/farmacocinética , ARN Mensajero/biosíntesis , Radioinmunoensayo/métodos , Receptor Muscarínico M1/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Tionucleótidos/farmacología , Factores de Tiempo , Transfección/métodos , Tritio/farmacocinética , Regulación hacia Arriba/efectos de los fármacosRESUMEN
The topography of distribution of 3H-dihydroalprenolol, 3H-quinucledinyl benzilate, 3H-dopamine, and 3H-DAGO binding sites in the central part of the sinoatrial node in rat heart was studied by autoradiography after electrophysiological identification of the dominant pacemaker region location. Receptor asymmetry between the lateral and median regions of the central part of the sinoatrial node was shown. The dominant pacemaker region lay in the lateral area of the sinoatrial node; the number of binding sites for all four ligands was minimum in it. The number of binding sites gradually increased in the cranial and caudal directions from the dominant pacemaker region along the sinoatrial node artery (more smoothly in the caudal direction). The relative densities of bindings sites for 3H-dihydroalprenolol and 3H-dopamine were higher in the lateral region compared to the perinodal working myocardium, while the densities for 3H-quinucledinyl benzilate and 3H-DAGO were virtually the same. The distribution of binding sites along the artery in the median region of the sinoatrial node was even for 3H-quinucledinyl benzilate and 3H-DAGO. For 3H-DAGO these parameters were close to those in the perinodal atrial myocardium, for 3H-quinucledinyl benzilate somewhat lower. Curves presenting the distribution of binding site densities for 3H-dihydroalprenolol and 3H-dopamine in the median region of the sinoatrial node were similar, with a pronounced peak in the region contralateral to the dominant pacemaker region, and significantly higher binding parameters compared to those for the perinodal atrial myocardium. The difference consisted in higher density of 3H-dopamine binding sites in the median region of the sinoatrial node in comparison with the lateral region. Binding activity was maximum in the wall of the sinoatrial node artery. The distribution of binding sites for ligands to the main autonomic nervous system neurotransmitters in the rat heart sinoatrial node is heterogeneous.
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Dihidroalprenolol/análisis , Dopamina/análisis , Encefalina Ala(2)-MeFe(4)-Gli(5)/análisis , Quinuclidinil Bencilato/análisis , Nodo Sinoatrial/química , Animales , Dihidroalprenolol/farmacocinética , Dopamina/farmacocinética , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacocinética , Ligandos , Masculino , Quinuclidinil Bencilato/farmacocinética , Ratas , Ratas Wistar , Receptores de Superficie Celular/efectos de los fármacos , Receptores de Superficie Celular/metabolismo , Nodo Sinoatrial/fisiología , TritioRESUMEN
To determine changes in signal transduction from the muscarinic acetylcholine receptor (mAChR) to G protein in brain tissue of dementia with Lewy bodies (DLB) and Alzheimer's disease (AD), we investigated GTP-sensitive agonist high affinity binding, which is considered an index of the formation of the mAChR-G protein complex. Brain tissue was obtained at necropsy from eight patients with DLB, nine patients with Alzheimer's disease and seven patients as controls. Membrane fractions were prepared from frontal and temporal cerebral tissues. Displacement curves of [(3)H]l-quinuclidinyl benzilate (QNB) binding by carbamylcholine were analyzed by the nonlinear least-squares methods. The proportion of and affinity for the agonist in GTP-sensitive agonist high affinity binding were estimated. The percentages GTP-sensitive agonist high affinity bindings were significantly decreased in DLB (P<0.01) and Alzheimer's disease (P<0.05) only in the frontal lobe. There were no significant differences in the temporal lobe. The ratio of agonist affinity (Kd value of low affinity component/Kd value of high affinity component) did not significantly differ among groups in either the frontal lobe or temporal lobe. The concentration of mAChR-G protein complex is considered reduced in the frontal lobe of brains with DLB and Alzheimer's disease. Therefore, signal transduction from mAChR to G protein was disturbed in the frontal lobe in these diseases.
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Enfermedad de Alzheimer/metabolismo , Encéfalo/efectos de los fármacos , Agonistas Colinérgicos/farmacocinética , Guanosina Trifosfato/farmacología , Enfermedad por Cuerpos de Lewy/metabolismo , Receptores Muscarínicos/metabolismo , Anciano , Anciano de 80 o más Años , Análisis de Varianza , Autopsia/métodos , Unión Competitiva/efectos de los fármacos , Unión Competitiva/fisiología , Encéfalo/citología , Encéfalo/metabolismo , Carbacol/farmacocinética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Antagonistas Muscarínicos/farmacocinética , Cambios Post Mortem , Quinuclidinil Bencilato/farmacocinéticaRESUMEN
It has been proposed that anti-myocardial antibodies (Ab) against neurotransmitter (NT) receptors are involved in the immunopathology of chronic Chagas' heart disease. We demonstrated that an anti-Trypanosoma cruzi monoclonal Ab (mAb), CAK20.12, binds to murine cardiac beta-adrenergic and muscarinic acetyl choline (mACh) receptors eliciting abnormal physiological responses on normal heart. No cross-linking requirement for mAb actions was demonstrated using Fab fragment derived from CAK20.12. mAb binding to synthetic peptides from the second extracellular loop of both beta1-adrenergic and mACh receptors, demonstrated by ELISA, identified the region of NT receptors involved. Cross-reactivity between these peptides and T. cruzi antigen was confirmed by binding inhibition assays. These results support the existence of cross-reactivity due to molecular mimicry between a parasite antigen and the major antigenic epitopes present on both beta1-adrenergic and M2-ACh receptors. Its possible relationship with cardiac dysfunction during chronic stage of Chagas' disease is also discussed.
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Anticuerpos Monoclonales/farmacología , Anticuerpos Antiprotozoarios/farmacología , Contracción Miocárdica/efectos de los fármacos , Pindolol/análogos & derivados , Receptor Muscarínico M2/inmunología , Receptores Adrenérgicos beta 1/inmunología , Trypanosoma cruzi/inmunología , Antagonistas Adrenérgicos beta , Análisis de Varianza , Animales , Unión Competitiva/efectos de los fármacos , Unión Competitiva/fisiología , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos/metabolismo , Epítopos/farmacología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Técnicas In Vitro , Isótopos de Yodo/farmacocinética , Ratones , Ratones Endogámicos BALB C , Antagonistas Muscarínicos/farmacocinética , Contracción Miocárdica/fisiología , Pindolol/farmacocinética , Quinuclidinil Bencilato/farmacocinética , Radioinmunoensayo/métodos , Ensayo de Unión Radioligante/métodos , Receptor Muscarínico M2/química , Receptores Adrenérgicos beta 1/química , Volumetría/métodos , Trypanosoma cruzi/químicaRESUMEN
Investigations on the effect of normal healthy ageing on the muscarinic system have shown conflicting results. Also, in vivo determination of muscarinic receptor binding has been hampered by a lack of subtype selective ligands and differences in methods used for quantification of receptor densities. Recent in vitro and in vivo work with the muscarinic antagonist (R,R)-I-QNB indicates this ligand has selectivity for m(1) and m(4) muscarinic receptor subtypes. Therefore, we used (R,R)[(123)I]-I-QNB and single photon emission tomography to study brain m(1) and m(4) muscarinic receptors in 25 healthy female subjects (11 younger subjects, age range 26-32 years and 14 older subjects, age range 57-82 years). Our aims were to ascertain the viability of tracer administration and imaging within the same day, and to evaluate whether normalization to whole brain, compared to normalization to cerebellum, could alter the clinical interpretation of results. Images were analyzed using the simplified reference tissue model and by two ratio methods: normalization to whole brain and normalization to cerebellum. Significant correlations were observed between kinetic analysis and normalization to cerebellum, but not to whole brain. Both the kinetic analysis and normalization to cerebellum showed age-related reductions in muscarinic binding in frontal, orbitofrontal, and parietal regions. Normalization to whole brain, however, failed to detect age-related changes in any region. Here we show that, for this radiotracer, normalizing to a region of negligible specific binding (cerebellum) significantly improves sensitivity when compared to global normalization.
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Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Quinuclidinil Bencilato/análogos & derivados , Quinuclidinil Bencilato/farmacocinética , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M4/metabolismo , Tomografía Computarizada de Emisión de Fotón Único/métodos , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Envejecimiento/metabolismo , Femenino , Humanos , Interpretación de Imagen Asistida por Computador/métodos , Persona de Mediana Edad , Radiofármacos/farmacocinética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Distribución TisularRESUMEN
OBJECTIVE: To determine the effects of cholinergic treatment on the muscarinic receptor in patients with Alzheimer's disease. METHODS: 12 patients with mild to moderate Alzheimer's disease and six controls were studied. The patients underwent ADAS-COG psychometric assessment and SPECT brain imaging with (123)I quinuclidinyl benzilate (QNB), to demonstrate the postsynaptic muscarinic M1 receptor, before being randomised in a double blind study to receive either an acetylcholinesterase inhibitor (donepezil) or placebo for four months. Following this, the ADAS-COG and the (123)I-QNB receptor scan were repeated. The controls were imaged on one occasion only. All image analyses were undertaken using SPM99. RESULTS: (123)I-QNB imaging showed a significant relation between baseline psychometric impairment and deficits on scanning. Both placebo and actively treated groups had reductions in (123)I-QNB uptake. Greater reductions in receptor binding were demonstrated in the placebo group than in those receiving active treatment. Intraindividual reproducibility of the (123)I-QNB imaging technique appeared highly robust. CONCLUSIONS: The results suggest that (123)I-QNB uptake is better preserved in Alzheimer's disease patients on cholinergic treatment than on placebo. Cholinergic treatment may play a neuroprotective role. Sequential (123)I-QNB imaging seems to be a powerful tool in monitoring the response of these receptors to disease modifying treatments.
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Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/fisiopatología , Inhibidores de la Colinesterasa/uso terapéutico , Indanos/uso terapéutico , Antagonistas Muscarínicos/farmacocinética , Piperidinas/uso terapéutico , Quinuclidinil Bencilato/farmacocinética , Anciano , Anciano de 80 o más Años , Inhibidores de la Colinesterasa/farmacología , Donepezilo , Método Doble Ciego , Femenino , Humanos , Indanos/farmacología , Radioisótopos de Yodo , Masculino , Persona de Mediana Edad , Piperidinas/farmacología , Placebos , Psicometría , Sensibilidad y Especificidad , Índice de Severidad de la Enfermedad , Tomografía Computarizada de Emisión de Fotón Único , Resultado del TratamientoRESUMEN
Sabcomeline [(R-(Z)-(+)-alpha-(methoxyiamino)-1-azabicyclo[2.2.2]octane-3-acetonitrile)] is a potent and functionally selective muscarinic M1 receptor partial agonist. However, little is known of the binding properties of sabcomeline under in vivo conditions. In this study, muscarinic receptor occupancy by sabcomeline in mouse brain regions and heart was estimated using [3H]quinuclidinyl benzilate (QNB) and [3H]N-methylpiperidyl benzilate (NMPB) as radioligands. In the cerebral cortex, hippocampus, and striatum, the estimated IC50 value of sabcomeline for [3H]NMPB binding was almost 0.2 mg/kg. Sabcomeline was not a selective ligand to M1 receptors as compared with biperiden in vivo. In the cerebral cortex, maximum receptor occupancy was observed about 1 hr after intravenous injection of sabcomeline (0.3 mg/kg), and the binding availability of mACh receptors had almost returned to the control level by 3-4 hr. These findings indicated that the binding kinetics of sabcomeline is rather rapid in mouse brain. Examination of dopamine D2 receptor binding revealed that sabcomeline affected the kinetics of both [3H]raclopride and [3H]N-methylspiperone (NMSP) binding in the striatum. It significantly decreased the k3 and k4 of [3H]raclopride binding resulting in an increase in binding potential (BP = k3/k4 = Bmax/Kd) in sabcomeline-treated mice, and an approximately 15% decrease in k3 of [3H]NMSP binding was also observed. Although the mechanism is still unclear, sabcomeline altered dopamine D2 receptor affinity or availability by modulations via neural networks.
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Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Piperidinas/farmacocinética , Quinuclidinil Bencilato/farmacocinética , Receptores Dopaminérgicos/metabolismo , Animales , Encéfalo/diagnóstico por imagen , Cerebelo/diagnóstico por imagen , Cerebelo/metabolismo , Corteza Cerebral/diagnóstico por imagen , Corteza Cerebral/metabolismo , Cuerpo Estriado/diagnóstico por imagen , Cuerpo Estriado/metabolismo , Técnicas de Diagnóstico por Radioisótopo , Relación Dosis-Respuesta a Droga , Iminas , Masculino , Tasa de Depuración Metabólica , Ratones , Ratones Endogámicos , Especificidad de Órganos , Unión Proteica , Quinuclidinas , Racloprida/farmacocinética , Cintigrafía , Radiofármacos/farmacocinética , Receptores Dopaminérgicos/efectos de los fármacos , Espiperona/farmacocinética , Distribución Tisular , Tritio/farmacocinéticaRESUMEN
Derivatives of the muscarinic antagonist 3-quinuclidinyl-4-iodobenzilate (QNB), particularly [123I]-(R,R)-I-QNB, are currently being assessed as in vivo ligands to monitor muscarinic receptors in Alzheimer's disease (AD) and dementia with Lewy bodies (DLB), relating changes to disease symptoms and to treatment response with cholinergic medication. To assist in the evaluation of in vivo binding, muscarinic receptor density in post-mortem human brain was measured by autoradiography with [125I]-(R,R)-I-QNB and [125I]-(R,S)-I-QNB and compared to M1 ([3H]pirenzepine) and M2 and M4 ([3H]AF-DX 384) receptor binding. Binding was calculated in tissue containing striatum, globus pallidus (GPe), claustrum, and cingulate and insula cortex, in cases of AD, DLB, Parkinson's disease (PD) and normal elderly controls. Pirenzepine, AF-DX 384 and (R,S)-I-QNB binding in the striatum correlated positively with increased Alzheimer-type pathology, and AF-DX 384 and (R,R)-I-QNB cortical binding correlated positively with increased Lewy body (LB) pathology; however, striatal pirenzepine binding correlated negatively with cortical LB pathology. M1 receptors were significantly reduced in striatum in DLB compared to AD, PD, and controls and there was a significant correlation between M1 and dopamine D2 receptor densities. [3H]AF-DX 384 binding was higher in the striatum and GPe in AD. Binding of [125I]-(R,R)-I-QNB, which may reflect increased muscarinic M4 receptors, was higher in cortex and claustrum in DLB and AD. [125I]-(R,S)-I-QNB binding was higher in the GPe in AD. Low M1 and D2 receptors in DLB imply altered regulation of the striatal projection neurons which express these receptors. Low density of striatal M1 receptors may relate to the extent of movement disorder in DLB, and to a reduced risk of parkinsonism with acetylcholinesterase inhibition.