RESUMEN
BACKGROUND AND PURPOSE: We aimed to characterize the pharmacology and electrophysiology of N-[3-(1H-benzimidazol-2-yl)-4-chloro-phenyl]pyridine-3-carboxamide (AZSMO-23), an activator of the human ether-a-go-go-related gene (hERG)-encoded K(+) channel (Kv 11.1). EXPERIMENTAL APPROACH: Automated electrophysiology was used to study the pharmacology of AZSMO-23 on wild-type (WT), Y652A, F656T or G628C/S631C hERG, and on other cardiac ion channels. Its mechanism of action was characterized with conventional electrophysiology. KEY RESULTS: AZSMO-23 activated WT hERG pre-pulse and tail current with EC50 values of 28.6 and 11.2 µM respectively. At 100 µM, pre-pulse current at +40 mV was increased by 952 ± 41% and tail current at -30 mV by 238 ± 13% compared with vehicle values. The primary mechanism for this effect was a 74.5 mV depolarizing shift in the voltage dependence of inactivation, without any shift in the voltage dependence of activation. Structure-activity relationships for this effect were remarkably subtle, with close analogues of AZSMO-23 acting as hERG inhibitors. AZSMO-23 blocked the mutant channel, hERG Y652A, but against another mutant channel, hERG F656T, its activator activity was enhanced. It inhibited activity of the G628C/S631C non-inactivating hERG mutant channel. AZSMO-23 was not hERG selective, as it blocked hKv 4.3-hKChIP2.2, hCav 3.2 and hKv 1.5 and activated hCav 1.2/ß2/α2δ channels. CONCLUSION AND IMPLICATIONS: The activity of AZSMO-23 and those of its close analogues suggest these compounds may be of value to elucidate the mechanism of type 2 hERG activators to better understand the pharmacology of this area from both a safety perspective and in relation to treatment of congenital long QT syndrome.
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Bencimidazoles/farmacología , Fenómenos Electrofisiológicos/efectos de los fármacos , Canales de Potasio Éter-A-Go-Go/efectos de los fármacos , Canales Iónicos/efectos de los fármacos , Niacinamida/análogos & derivados , Animales , Bencimidazoles/química , Células CHO , Línea Celular , Cricetinae , Cricetulus , Canales de Potasio Éter-A-Go-Go/metabolismo , Células HEK293 , Humanos , Canales Iónicos/metabolismo , Niacinamida/química , Niacinamida/farmacología , Relación Estructura-ActividadRESUMEN
Cardiovascular-related adverse drug effects are a major concern for the pharmaceutical industry. Activity of an investigational drug at the L-type calcium channel could manifest in a number of ways, including changes in cardiac contractility. The aim of this study was to define which of the two assay technologies - radioligand-binding or automated electrophysiology - was most predictive of contractility effects in an in vitro myocyte contractility assay. The activity of reference and proprietary compounds at the L-type calcium channel was measured by radioligand-binding assays, conventional patch-clamp, automated electrophysiology, and by measurement of contractility in canine isolated cardiac myocytes. Activity in the radioligand-binding assay at the L-type Ca channel phenylalkylamine binding site was most predictive of an inotropic effect in the canine cardiac myocyte assay. The sensitivity was 73%, specificity 83% and predictivity 78%. The radioligand-binding assay may be run at a single test concentration and potency estimated. The least predictive assay was automated electrophysiology which showed a significant bias when compared with other assay formats. Given the importance of the L-type calcium channel, not just in cardiac function, but also in other organ systems, a screening strategy emerges whereby single concentration ligand-binding can be performed early in the discovery process with sufficient predictivity, throughput and turnaround time to influence chemical design and address a significant safety-related liability, at relatively low cost.
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Canales de Calcio Tipo L/efectos de los fármacos , Descubrimiento de Drogas/métodos , Ensayos Analíticos de Alto Rendimiento , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Pruebas de Toxicidad/métodos , Animales , Automatización , Sitios de Unión , Células CHO , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Cricetinae , Cricetulus , Perros , Femenino , Humanos , Ligandos , Potenciales de la Membrana , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Valor Predictivo de las Pruebas , Unión Proteica , Ensayo de Unión Radioligante , Medición de Riesgo , TransfecciónRESUMEN
Measurement of cardiac contractility is a logical part of pre-clinical safety assessment in a drug discovery project, particularly if a risk has been identified or is suspected based on the primary- or non-target pharmacology. However, there are limited validated assays available that can be used to screen several compounds in order to identify and eliminate inotropic liability from a chemical series. We have therefore sought to develop an in vitro model with sufficient throughput for this purpose. Dog ventricular myocytes were isolated using a collagenase perfusion technique and placed in a perfused recording chamber on the stage of a microscope at ~36 °C. Myocytes were stimulated to contract at a pacing frequency of 1 Hz and a digital, cell geometry measurement system (IonOptix™) was used to measure sarcomere shortening in single myocytes. After perfusion with vehicle (0.1% DMSO), concentration-effect curves were constructed for each compound in 4-30 myocytes taken from 1 or 2 dog hearts. The validation test-set was 22 negative and 8 positive inotropes, and 21 inactive compounds, as defined by their effect in dog, cynolomolgous monkey or humans. By comparing the outcome of the assay to the known in vivo contractility effects, the assay sensitivity was 81%, specificity was 75%, and accuracy was 78%. With a throughput of 6-8 compounds/week from 1 cell isolation, this assay may be of value to drug discovery projects to screen for direct contractility effects and, if a hazard is identified, help identify inactive compounds.
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Contracción Miocárdica/fisiología , Miocitos Cardíacos/fisiología , Animales , Perros , Descubrimiento de Drogas/métodos , Femenino , Ventrículos Cardíacos/efectos de los fármacos , Técnicas In Vitro , Contracción Miocárdica/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Reproducibilidad de los Resultados , Sarcómeros/fisiología , Sensibilidad y Especificidad , Grabación en VideoRESUMEN
Cell lines expressing ion channels (IC) and the advent of plate-based electrophysiology device have enabled a molecular understanding of the action potential (AP) as a means of early QT assessment. We sought to develop an in silico AP (isAP) model that provides an assessment of the effect of a compound on the myocyte AP duration (APD) using concentration-effect curve data from a panel of five ICs (hNav1.5, hCav1.2, hKv4.3/hKChIP2.2, hKv7.1/hminK, hKv11.1). A test set of 53 compounds was selected to cover a range of selective and mixed IC modulators that were tested for their effects on optically measured APD. A threshold of >10% change in APD at 90% repolarization (APD(90)) was used to signify an effect at the top test concentration. To capture the variations observed in left ventricular midmyocardial myocyte APD data from 19 different dogs, the isAP model was calibrated to produce an ensemble of 19 model variants that could capture the shape and form of the APs and also quantitatively replicate dofetilide- and diltiazem-induced APD(90) changes. Provided with IC panel data only, the isAP model was then used, blinded, to predict APD(90) changes greater than 10%. At a simulated concentration of 30 µM and based on a criterion that six of the variants had to agree, isAP prediction was scored as showing greater than 80% predictivity of compound activity. Thus, early in drug discovery, the isAP model allows integrating separate IC data and is amenable to the throughput required for use as a virtual screen.
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Potenciales de Acción/fisiología , Fármacos Cardiovasculares/farmacología , Fármacos Cardiovasculares/toxicidad , Simulación por Computador , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Corazón/fisiología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Calibración , Perros , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Canal de Potasio ERG1 , Electrodos Implantados , Fenómenos Electrofisiológicos , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Femenino , Fluorescencia , Colorantes Fluorescentes , Miocitos Cardíacos/fisiología , Bloqueadores de los Canales de Potasio/farmacología , Medición de Riesgo , Valores Limites del UmbralRESUMEN
BACKGROUND AND PURPOSE: The majority of human ether-a-go-go-related gene (hERG) screens aiming to minimize the risk of drug-induced long QT syndrome have been conducted using heterologous systems expressing the hERG 1a subunit, although both hERG 1a and 1b subunits contribute to the K+ channels producing the repolarizing current I(Kr) . We tested a range of compounds selected for their diversity to determine whether hERG 1a and 1a/1b channels exhibit different sensitivities that may influence safety margins or contribute to a stratified risk analysis. EXPERIMENTAL APPROACH: We used the IonWorks™ plate-based electrophysiology device to compare sensitivity of hERG 1a and 1a/1b channels stably expressed in HEK293 cells to 50 compounds previously shown to target hERG channels. Potency was determined as IC50 values (µM) obtained from non-cumulative, eight-point concentration-effect curves of normalized data, fitted to the Hill equation. To minimize possible sources of variability, compound potency was assessed using test plates arranged in alternating columns of cells expressing hERG 1a and 1a/1b. KEY RESULTS: Although the potency of most compounds was similar for the two targets, some surprising differences were observed. Fluoxetine (Prozac) was more potent at blocking hERG 1a/1b than 1a channels, yielding a corresponding reduction in the safety margin. In contrast, E-4031 was a more potent blocker of hERG 1a compared with 1a/1b channels, as previously reported, as was dofetilide, another high-affinity blocker. CONCLUSIONS AND IMPLICATIONS: The current assays may underestimate the risk of some drugs to cause torsades de pointes arrhythmia, and overestimate the risk of others.
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Evaluación Preclínica de Medicamentos/métodos , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Fluoxetina/farmacología , Potenciales de Acción/efectos de los fármacos , Arritmias Cardíacas/inducido químicamente , Arritmias Cardíacas/genética , Línea Celular Transformada , Canales de Potasio Éter-A-Go-Go/genética , Células HEK293 , Humanos , Concentración 50 Inhibidora , Síndrome de QT Prolongado/inducido químicamente , Síndrome de QT Prolongado/genética , Piperidinas/farmacología , Subunidades de Proteína , Piridinas/farmacología , Sensibilidad y Especificidad , Torsades de Pointes/inducido químicamente , Torsades de Pointes/genéticaRESUMEN
Owing to its association with Torsades de Pointes, drug-induced QT interval prolongation has been and remains a significant hurdle to the development of safe, effective medicines. Genetic and pharmacological evidence highlighting the pivotal role the human ether-a-go-go-related gene (hERG) channel was a critical step in understanding how to start addressing this issue. It led to the development of hERG assays with the rapid throughput needed for the short timescales required in early drug discovery. The resulting volume of hERG data has fostered in silico models to help chemists design compounds with reduced hERG potency. In early drug discovery, a pragmatic approach based on exceeding a given potency value has been required to decide when a compound is likely to carry a low QT risk, to support its progression to late-stage discovery. At this point, the in vivo efficacy and metabolism characteristics of the potential drug are generally defined, as well its safety profile, which includes usually a dog study to assess QT interval prolongation risk. The hERG and in vivo QT data, combined with the likely indication and the estimated free drug level for efficacy, are put together to assess the risk that the potential drug will prolong QT in man. Further data may be required to refine the risk assessment before making the major investment decisions for full development. The non-clinical data are essential to inform decisions about compound progression and to optimize the design of clinical QT studies.
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Diseño de Fármacos , Canales de Potasio Éter-A-Go-Go/metabolismo , Síndrome de QT Prolongado/inducido químicamente , Animales , Perros , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Humanos , Medición de Riesgo/métodos , Torsades de Pointes/inducido químicamenteRESUMEN
INTRODUCTION: The safety implications of blocking the human cardiac Na(+) channel (hNav1.5) make it prudent to test for this activity early in the drug discovery process and design-out any potential liability. This needs a method with adequate throughput and a demonstrable predictive value to effects in native cardiac tissues. Here we describe the validation of a method that combines the ability to screen tens of compounds a day, with direct assessment of channel function. METHODS: The electrophysiological and pharmacological properties of hNav1.5 were compared using two methods: conventional, low-throughput electrophysiology and planar-array-based, medium-throughput electrophysiology (IonWorks HT). A pharmacological comparison was also made between IonWorks HT and canine cardiac Purkinje Fibre action potential upstroke data. RESULTS: Activation curve parameters for hNav1.5 in IonWorks HT were not statistically different (p>0.05) from those generated using conventional electrophysiology. IonWorks HT V(1/2)=-22+/-0.8 mV, slope=6.9+/-0.2 (n=11); conventional electrophysiology V(1/2)=-20+/-1.6 mV, slope=6.4+/-0.3 (n=11). Potency values for a range of hNav1.5 blockers determined using IonWorks HT correlated closely with those obtained using conventional electrophysiology (R=0.967, p<0.001). The assay was able to distinguish between highly use-dependent blockers (e.g. tetracaine) and blockers that do not display strong use-dependence (e.g. quinidine). Comparison of the degree of hNav1.5 inhibition and decrease in canine Purkinje fibre action potential upstroke velocity (V(max)) showed that the IonWorks HT assay would have predicted the outcome in Purkinje fibres in the majority of cases, with false negative and positive rates estimated at 8 and 7%, respectively. Finally, hNav1.5 pharmacology was similar when determined using either IonWorks HT or IonWorks Quattro, although the latter yielded more consistent data. DISCUSSION: The assay described combines a functional assessment of hNav1.5 with medium-throughput. Furthermore the assay was able to reveal information on the use-dependency of compound block, as well as predicting Na(+) channel effects in more integrated systems such as the cardiac Purkinje fibre action potential. This makes it possible to determine quantitative potency data, and mechanistic information about use-dependence, in a timeframe short enough to influence medicinal chemistry.
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Evaluación Preclínica de Medicamentos/métodos , Electrofisiología , Proteínas Musculares/antagonistas & inhibidores , Proteínas Musculares/metabolismo , Canales de Sodio/metabolismo , Animales , Fenómenos Biofísicos , Biofisica , Células CHO , Cricetinae , Cricetulus , Perros , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Concentración 50 Inhibidora , Masculino , Potenciales de la Membrana , Canal de Sodio Activado por Voltaje NAV1.5 , Valor Predictivo de las Pruebas , Ramos Subendocárdicos/efectos de los fármacos , Reproducibilidad de los Resultados , Bloqueadores de los Canales de Sodio/farmacologíaRESUMEN
INTRODUCTION: Regulatory and competitive pressure to reduce the QT interval prolongation risk of potential new drugs has led to focus on methods to test for inhibition of the human ether-a-go-go-related gene (hERG)-encoded K+ channel, the primary molecular target underlying this safety issue. Here we describe the validation of a method that combines medium-throughput with direct assessment of channel function. METHODS: The electrophysiological and pharmacological properties of hERG were compared using two methods: conventional, low-throughput electrophysiology and planar-array-based, medium-throughput electrophysiology (IonWorks HT). A pharmacological comparison was also made between IonWorks HT and an indirect assay (Rb+ efflux). RESULTS: Basic electrophysiological properties of hERG were similar whether recorded conventionally (HEK cells) or using IonWorks HT (CHO cells): for example, tail current V1/2 -12.1+/-5.0 mV (32) for conventional and -9.5+/-6.0 mV (46) for IonWorks HT (mean+/-S.D. (n)). A key finding was that as the number of cells per well was increased in IonWorks HT, the potency reported for a given compound decreased. Using the lowest possible cell concentration (250,000 cells/ml) and 89 compounds spanning a broad potency range, the pIC50 values from IonWorks HT (CHO-hERG) were found to correlate well with those obtained using conventional methodology (HEK-hERG)(r=0.90; p<0.001). Further validation using CHO-hERG cells with both methods confirmed the correlation (r=0.94; p<0.001). In contrast, a comparison of IonWorks HT and Rb+ efflux data with 649 compounds using CHO-hERG cells showed that the indirect assay consistently reported compounds as being, on average, 6-fold less potent, though the differences varied depending on chemical series. DISCUSSION: The main finding of this work is that providing a relatively low cell concentration is used in IonWorks HT, the potency information generated correlates well with that determined using conventional electrophysiology. The effect on potency of increasing cell concentration may relate to a reduced free concentration of test compound owing to partitioning into cell membranes. In summary, the IonWorks HT hERG assay can generate pIC50 values based on a direct assessment of channel function in a timeframe short enough to influence chemical design.
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Electrofisiología/instrumentación , Canales de Potasio Éter-A-Go-Go/efectos de los fármacos , Técnicas de Placa-Clamp/instrumentación , Bloqueadores de los Canales de Potasio/farmacología , Animales , Células CHO , Línea Celular , Cricetinae , Canal de Potasio ERG1 , Humanos , Reproducibilidad de los Resultados , Rubidio/metabolismoRESUMEN
1. The role of the cGMP pathway in the modulation of the cardiac L-type Ca2+ current (ICa,L) by nitric oxide (NO) was examined in rat ventricular myocytes. 2. The NO donors DEANO, SIN-1, SNP, SNAP and GSNO had no significant effects on basal ICa,L. However, DEANO (100 microM) inhibited ICa,L after the current had been previously stimulated by either isoprenaline (Iso, 1-10 nM), a beta-adrenergic agonist, or isobutylmethyl-xanthine (IBMX, 10-80 microM), a wide spectrum phosphodiesterase (PDE) inhibitor. 3. The anti-adrenergic effect of DEANO on ICa,L was not mimicked by other NO donors (SIN-1, SNAP and SPNO). 4. The NO-sensitive guanylyl cyclase inhibitor ODQ (10 microM), antagonized the inhibitory effect of DEANO on ICa,L. Likewise, inhibitors of the cGMP-dependent protein kinase (cG-PK), Rp-8-chloro-phenylthio-cGMP (10 microM) and KT5823 (0.1 and 0.3 microM), also abolished the inhibitory effect of DEANO on Iso (1-10 nM)-stimulated ICa,L. 5. Intracellular dialysis with exogenous cAMP (10-100 microM) blunted the inhibitory effect of DEANO (10 and 100 microM) on ICa,L. SNAP and SNP also had no effect on the cAMP-stimulated ICa,L. 6. Pre-treatment of the myocytes with pertussis toxin (0.5 microg ml-1, 4-6 h at 37 degrees C) eliminated the inhibitory effect of DEANO (100 microM) on ICa,L, in the presence of either Iso (0.01 and 1 nM) or IBMX (10-80 microM). 7. These results demonstrate that DEANO produces anti-adrenergic effects in rat ventricular myocytes. This effect of DEANO occurs in a cGMP-dependent manner, and involves activation of cG-PK and regulation of a pertussis toxin-sensitive G protein.
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Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Proteínas de Unión al GTP/fisiología , Corazón/efectos de los fármacos , Donantes de Óxido Nítrico/farmacología , Animales , Electrofisiología , Guanilato Ciclasa/antagonistas & inhibidores , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/efectos de los fármacos , Técnicas In Vitro , Masculino , Potenciales de la Membrana/fisiología , Microdiálisis , Miocardio/citología , Técnicas de Placa-Clamp , Ratas , Ratas WistarRESUMEN
1. The biophysical and pharmacological characteristics of the hyperpolarization activated non- selective cation current (If) were recorded using whole-cell voltage clamp in embryonic stem (ES) cell-derived cardiomyocytes at different stages of development. 2. The cation current was detected in a large percentage (65 %) of early stage (EDS, differentiated for 7 + 3-4 days) cells at a current density of 11.4 +/- 0.6 pA pF-1 (n = 47). In late stage (LDS, differentiated for 7 + 9-12 days) cells the percentage of cells expressing If decreased (45 %), but If densities (15.5 +/- 0.9 pA pF-1, n = 20) were increased. 3. The muscarinic agonist carbachol (CCh, 1 microM) depressed basal If in EDS cells by 45.7 +/- 6.5 %, n = 5) and was without effect in LDS cardiomyocytes (n = 4). The beta-adrenoceptor agonist isoprenaline (ISO, 1 microM) stimulated If in LDS cells by 33 +/- 5.2 % (n = 6) but not in EDS cells (n = 5). 4. Cell infusion with the catalytic subunit of the cAMP-dependent protein kinase (PKA, 7 microM) stimulated If in EDS cells by 37.0 +/- 2.9 %, (n = 4), but subsequent superfusion of 8-bromo-cAMP (200 microM) was without effect. Intracellular perfusion of LDS cardiomyocytes with the highly selective peptide inhibitor of PKA (PKI, 20 microM) completely inhibited the stimulation of the L-type Ca2+ current (ICa,L) as well as of If by ISO (1 microM). 5. Extracellular superfusion with phosphodiesterase (PDE) inhibitors - IBMX, a non-selective antagonist, Erythro-9-(2-hydoxy-3-nonyl)adenine (EHNA), a PDE2 antagonist and rolipram, a PDE4 antagonist - resulted in stimulation of ICa,L and If in EDS cells. By contrast, milrinone and cilostamide, two PDE3 antagonists, stimulated ICa,L, but not If. 6. The present work demonstrates that If is functionally expressed during early cardiomyogenesis. Similar to ICa,L, If is regulated during embryonic development by phosphorylation via PKA. In contrast to ICa,L, If is not regulated by PDE3 suggesting different localization of these ion channels with respect to PDE3.
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Cationes/metabolismo , Corazón/embriología , Canales Iónicos/biosíntesis , Miocardio/metabolismo , Proteínas del Tejido Nervioso , Células Madre/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Agonistas Adrenérgicos beta/farmacología , Animales , Canales de Calcio Tipo L/efectos de los fármacos , Canales de Calcio Tipo L/metabolismo , Diferenciación Celular/fisiología , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Canales Iónicos/agonistas , Canales Iónicos/antagonistas & inhibidores , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Agonistas Muscarínicos/farmacología , Miocardio/citología , Técnicas de Placa-Clamp , Inhibidores de Fosfodiesterasa/farmacología , Fosforilación/efectos de los fármacos , Canales de Potasio , Células Madre/citologíaRESUMEN
We report that in vivo injection of endotoxin (EDTX, 6 mg. kg(-)(1)) induces cardiovascular alterations in rats that closely mimic the clinical situation, as assessed by in vivo hemodynamic measurements in anesthetized and conscious, chronically instrumented animals. The patch-clamp technique was used to characterize the L-type calcium current (I(Ca)) and its autonomic regulation in isolated cardiac myocytes. The density of I(Ca) progressively decreased at 12 and 36 h after EDTX injection. However, the dihydropyridine (+/-)Bay K 8644 (100 nM) enhanced I(Ca) to levels similar to those in control and EDTX-treated myocytes. In addition, the net stimulatory effect of a beta-adrenergic agonist (isoproterenol) on I(Ca) was increased 12 h after EDTX injection. This change in the beta-adrenergic effect declined 24 h later. The potentiation in the beta-adrenergic stimulation of I(Ca) was mimicked by L858051 (10 microM), a direct activator of adenylyl cyclase, but not by IBMX (200 microM), a phosphodiesterase inhibitor. Besides, the antiadrenergic effect of acetylcholine on I(Ca) was unchanged 12 h after EDTX injection, but increased 36 h after EDTX injection. These results support the hypothesis that time-dependent changes in the adenylyl cyclase pathway in cardiac myocytes may contribute, via the autonomic regulation of I(Ca), to the severity of myocardial dysfunction during sepsis.
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Sistema Nervioso Autónomo/fisiología , Endotoxinas/farmacología , Corazón/inervación , Miocardio/citología , 1-Metil-3-Isobutilxantina/farmacología , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Acetilcolina/farmacología , Acetilcolina/fisiología , Adenilil Ciclasas/metabolismo , Antagonistas Adrenérgicos/farmacología , Agonistas Adrenérgicos beta/farmacología , Animales , Agonistas de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/metabolismo , Colforsina/análogos & derivados , Colforsina/farmacología , Diterpenos , Electrofisiología , Endotoxemia/fisiopatología , Endotoxinas/administración & dosificación , Activadores de Enzimas/farmacología , Escherichia coli , Corazón/fisiología , Hemodinámica , Técnicas In Vitro , Inyecciones Intravenosas , Isoproterenol/farmacología , Masculino , Miocardio/metabolismo , Técnicas de Placa-Clamp , Inhibidores de Fosfodiesterasa/farmacología , Ratas , Sepsis/fisiopatologíaRESUMEN
We studied the mechanism of action of methylene blue (Mblue), a putative guanylyl cyclase inhibitor, on the L-type calcium current (ICa) and the muscarinic activated K+ current (IK,ACh) in rat ventricular and atrial myocytes, respectively, and on the binding of [3H]quinuclidinyl benzylate in rat ventricular membranes. Superfusion, but not internal dialysis, with 30 microM Mblue antagonized the inhibitory effect of acetylcholine (ACh, 1 microM) on beta-adrenergic stimulation of ICa with isoprenaline (Iso, 10 nM or 1 microM). However, Mblue had no effect on the basal ICa or on the stimulation of ICa by Iso in the absence of ACh. The activation of IK,ACh by 3 microM ACh was also antagonized by Mblue in a dose-dependent manner. In contrast, Mblue had no effect on the activation of IK,ACh by either guanosine-5'-O-(3-thio)triphosphate or guanosine-5'-(beta,gamma-imido)triphosphate. Chlorpromazine (CPZ), a piperazine derivative like Mblue, also inhibited the muscarinic activation of IK,ACh in a dose-dependent manner. The specific binding of [3H]QNB, a muscarinic ligand, to rat ventricular membranes was displaced in a dose-dependent manner by Mblue and CPZ. The piperazine derivatives behaved like competitive antagonists of [3H]QNB binding, exhibiting equilibrium dissociation constant (Ki) values of 187 nM for Mblue and 366 nM for CPZ. In conclusion, Mblue exerts antimuscarinic effects on ICa and IK,ACh in rat cardiac myocytes that are best explained by the binding of Mblue to the M2 subtype of muscarinic receptors. This property probably contributes to the antimuscarinic effect of the putative guanylyl cyclase inhibitor reported in previous studies.
Asunto(s)
Corazón/efectos de los fármacos , Azul de Metileno/farmacología , Antagonistas Muscarínicos/farmacología , Miocardio/ultraestructura , Acetilcolina/metabolismo , Acetilcolina/farmacología , Agonistas Adrenérgicos beta/farmacología , Animales , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Membranas/metabolismo , Miocardio/citología , Miocardio/metabolismo , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiología , Quinuclidinil Bencilato/metabolismo , Ratas , Ratas Wistar , Receptores Muscarínicos/metabolismo , TritioRESUMEN
1. To investigate the participation of guanylyl cyclase in the muscarinic regulation of the cardiac L-type calcium current (ICa), we examined the effects of three guanylyl cyclase inhibitors, 1H-[1,2,4]oxidiazo-lo[4,3-a]quinoxaline-1-one (ODQ), 6-anilino-5,8-quinolinedione (LY 83583), and methylene blue (MBlue), on the beta-adrenoceptor; muscarinic receptor and nitric oxide (NO) regulation of ICa and on the muscarinic activated potassium current I(K,ACh), in frog atrial and ventricular myocytes. 2. ODQ (10 microM) and LY 83583 (30 microM) antagonized the inhibitory effect of an NO-donor (S-nitroso-N-acetylpenicillamine, SNAP, 1 microM) on the isoprenaline (Iso)-stimulated ICa which was consistent with their inhibitory action on guanylyl cyclase. However, MBlue (30 microM) had no effect under similar conditions. 3. In the absence of SNAP, LY 83583 (30 microM) potentiated the stimulations of ICa by either Iso (20 nM), forskolin (0.2 microM) or intracellular cyclic AMP (5-10 microM). ODQ (10 microM) had no effect under these conditions, while MBlue (30 microM) inhibited the Iso-stimulated ICa. 4. LY 83583 and MBlue, but not ODQ, reduced the inhibitory effect of up to 10 microM acetylcholine (ACh) on ICa. 5. MBlue, but not LY 83583 and ODQ, antagonized the activation of I(K,ACh) by ACh in the presence of intracellular GTP, and this inhibition was weakened when I(K,ACh) was activated by intracellular GTPgammaS. 6. The potentiating effect of LY 83583 on Iso-stimulated ICa was absent in the presence of either DL-dithiothreitol (DTT, 100 microM) or a combination of superoxide dismutase (150 u ml(-1)) and catalase (100 u ml(-1)). 7. All together, our data demonstrate that, among the three compounds tested, only ODQ acts in a manner which is consistent with its inhibitory action on the NO-sensitive guanylyl cyclase. The two other compounds produced severe side effects which may involve superoxide anion generation in the case of LY 83583 and alteration of beta-adrenoceptor and muscarinic receptor-coupling mechanisms in the case of M Blue.
Asunto(s)
Canales de Calcio/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Guanilato Ciclasa/antagonistas & inhibidores , Corazón/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , Aminoquinolinas/farmacología , Animales , Canales de Calcio Tipo L , AMP Cíclico/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Azul de Metileno/farmacología , Óxido Nítrico/fisiología , Nitroprusiato/farmacología , Rana esculenta , Receptores Adrenérgicos beta/fisiología , Receptores Muscarínicos/fisiología , Superóxidos/metabolismoRESUMEN
Muscarinic agonists regulate the L-type calcium current in isolated cardiac myocytes. The second messengers pathways involved in this regulation are discussed briefly, with particular emphasis on the involvement of cAMP and cGMP pathways.