RESUMEN
The novel quaternary ammonium salt (3R)-3-[[[(3-fluorophenyl)[(3,4,5-trifluorophenyl)methyl]amino]carbonyl]oxy]-1-[2-oxo-2-(2-thienyl)ethyl]-1-azoniabicyclo[2.2.2]octane bromide (CHF5407) showed subnanomolar affinities for human muscarinic M1 (hM1), M2 (hM2), and M3 (hM3) receptors and dissociated very slowly from hM3 receptors (t(½) = 166 min) with a large part of the receptorial complex (54%) remaining undissociated at 32 h from radioligand washout. In contrast, [(3)H]CHF5407 dissociated quickly from hM2 receptors (t(½) = 31 min), whereas [(3)H]tiotropium dissociated slowly from both hM3 (t(½) = 163 min) and hM2 receptor (t(½) = 297 min). In the guinea pig isolated trachea and human isolated bronchus, CHF5407 produced a potent (pIC(50) = 9.0-9.6) and long-lasting (up to 24 h) inhibition of M3 receptor-mediated contractile responses to carbachol. In the guinea pig electrically driven left atrium, the M2 receptor-mediated inhibitory response to carbachol was recovered more quickly in CHF5407-pretreated than in tiotropium-pretreated preparations. CHF5407, administered intratracheally to anesthetized guinea pigs, potently inhibited acetylcholine (Ach)-induced bronchoconstriction with an ED(50) value of 0.15 nmol/kg. The effect was sustained over a period of 24 h, with a residual 57% inhibition 48 h after antagonist administration at 1 nmol/kg. In conscious guinea pigs, inhaled CHF5407 inhibited Ach-induced bronchoconstriction for at least 24 h as did tiotropium at similar dosages. Cardiovascular parameters in anesthetized guinea pigs were not significantly changed by CHF5407, up to 100 nmol/kg i.v. and up to 1000 nmol/kg i.t. In conclusion, CHF5407 shows a prolonged antibronchospastic activity both in vitro and in vivo, caused by a very slow dissociation from M3 receptors. In contrast, CHF5407 is markedly short-acting at M2 receptors, a behavior not shared by tiotropium.
Asunto(s)
Broncoconstricción/efectos de los fármacos , Broncodilatadores/farmacología , Carbamatos/farmacología , Antagonistas Muscarínicos/farmacología , Quinuclidinas/farmacología , Receptor Muscarínico M3/antagonistas & inhibidores , Acetilcolina/farmacología , Anciano , Anestesia , Animales , Presión Sanguínea/efectos de los fármacos , Bronquios/efectos de los fármacos , Espasmo Bronquial/inducido químicamente , Espasmo Bronquial/tratamiento farmacológico , Espasmo Bronquial/prevención & control , Broncoconstrictores/farmacología , Broncodilatadores/administración & dosificación , Broncodilatadores/metabolismo , Células CHO , Carbacol/farmacología , Carbamatos/administración & dosificación , Carbamatos/metabolismo , Cricetinae , Cricetulus , Diaminas/administración & dosificación , Diaminas/farmacología , Cobayas , Atrios Cardíacos/efectos de los fármacos , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Cinética , Masculino , Persona de Mediana Edad , Estructura Molecular , Antagonistas Muscarínicos/administración & dosificación , Antagonistas Muscarínicos/metabolismo , Contracción Miocárdica/efectos de los fármacos , Quinuclidinas/administración & dosificación , Quinuclidinas/metabolismo , Receptor Muscarínico M1/genética , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/antagonistas & inhibidores , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Receptor Muscarínico M3/agonistas , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/metabolismo , Derivados de Escopolamina/administración & dosificación , Derivados de Escopolamina/metabolismo , Derivados de Escopolamina/farmacología , Bromuro de Tiotropio , Tráquea/efectos de los fármacos , Transfección , Función Ventricular Izquierda/efectos de los fármacosRESUMEN
The measure of membrane capacitance (C(m)) in cardiac myocytes is of primary importance as an index of their size in physiological and pathological conditions, and for the understanding of their excitability. Although a plethora of very accurate methods has been developed to access C(m) value in single cells, cardiac electrophysiologists still use, in the majority of laboratories, classical direct current techniques as they have been established in the early days of cardiac cellular electrophysiology. These techniques are based on the assumption that cardiac membrane resistance (R(m)) is constant, or changes negligibly, in a narrow potential range around resting potential. Using patch-clamp whole-cell recordings, both in current-clamp and voltage-clamp conditions, and numerical simulations, we document here the voltage-dependency of R(m), up to -45% of its resting value for 10-mV hyperpolarization, in resting rat ventricular myocytes. We show how this dependency makes classical protocols to misestimate C(m) in a voltage-dependent manner (up to 20% errors), which can dramatically affect C(m)-based calculations on cell size and on intracellular ion dynamics. We develop a simple mechanistic model to fit experimental data and obtain voltage-independent estimates of C(m), and we show that accurate estimates can also be extrapolated from the classical approach.
Asunto(s)
Membrana Celular/metabolismo , Células Dendríticas/citología , Miocitos Cardíacos/citología , Animales , Biofisica/métodos , Tamaño de la Célula , Capacidad Eléctrica , Electrofisiología , Ventrículos Cardíacos/patología , Iones , Cinética , Masculino , Potenciales de la Membrana , Membranas/metabolismo , Modelos Estadísticos , Técnicas de Placa-Clamp , Estructura Terciaria de Proteína , Ratas , Ratas Wistar , Programas Informáticos , Factores de TiempoAsunto(s)
Electrocardiografía , Potenciales Evocados/fisiología , Hipertrofia Ventricular Izquierda/diagnóstico , Hipertrofia Ventricular Izquierda/fisiopatología , Miocardio/patología , Coartación Aórtica/diagnóstico , Coartación Aórtica/etiología , Humanos , Hipertrofia Ventricular Izquierda/complicacionesRESUMEN
UNLABELLED: Simulations of cardiac tissue bidomain model indicate that point cathodal stimulation gives rise to a dog-bone depolarized region (virtual cathode) extending across fibers, limited by two symmetric hyperpolarized regions (virtual anode) extending along fibers. These predictions were experimentally confirmed by optical mapping studies of transmembrane potentials while no direct validation is reported at the extracellular level. The present study aims at defining the influence of the virtual cathode on extracellular potentials by means of high-density epicardial mapping. METHODS: Epicardial potentials were measured in seven exposed rat hearts by means of a 11 x 11 electrode array with 360 x 540 microns resolution. Cathodal current pulses, 100-200 microA intensity and 1 ms duration, to avoid superposition of stimulus and activation potentials, were delivered from one of the electrode array and unipolar potentials were measured from all other electrodes. RESULTS AND DISCUSSION: a) During stimulus, negative equipotential lines were elliptic along fibers, as expected, but for a 2 mm circular region at the pacing site. b) During 1-2 ms interval between stimulus offset and start of activation, equipotential lines became elliptic across fibers in the presence of the region directly excited by the stimulus field. Start of activation was either symmetric with isochrones initially circular around the pacing site and then elliptic along fibers, or asymmetric initiating at only one side of the pacing site across fibers with isochrones elliptic along fibers. In the latter case, the wave front was blocked through the refractory region directly excited by the stimulus field, subdivided into two wings which collided and merged at the opposite side, giving rise to a plane wave front propagating across fibers away from the pacing site. CONCLUSIONS: High spatial resolution epicardial potential mapping reveals the existence of the virtual cathode and its influence on impulse initiation and conduction. The unexpected existence of a region of conduction block at the pacing site, due to spatial asymmetry of normal cardiac tissue which enhances activation threshold at one of the two sides of the virtual cathode, is intriguing since it is one of the requirements for reentry of conduction in the presence of a circuit with decreased conduction velocity and short duration of refractory period.
Asunto(s)
Potenciales Evocados/fisiología , Corazón/fisiología , Animales , Electrodos Implantados , Fibras Musculares Esqueléticas/fisiología , Pericardio/fisiología , RatasRESUMEN
In rat models of cardiac hypertrophy (moderate aortic coarctation: ACm, n=18; severe aortic coarctation: ACs, n=27; aging: OLD, n=25; spontaneous chronic hypertension: SHR, n=18) and properly matched control animals (C(ACm), n=17; C(ACs), n=19; C(OLD), n=24; C(SHR), n=22), we investigated the relative contribution of intense autonomic activity and cardiac structural damage to ventricular arrhythmogenesis. We used an "in vivo" to tissue level approach, by correlating in the same animal: (i) social stress-induced ventricular arrhythmias, telemetrically recorded, and (ii) left ventricular weights (LVW) and amount and geometrical properties of myocardial fibrosis (MF). Arterial blood pressure was significantly higher in ACm (+11%), ACs (+28%) and SHR (+34%) than in controls. LVW were approximately 20% greater in ACm, ACs and OLD and 50% greater in SHR. MF was about twice as great and characterized by more frequent occurrence of microscopic scarring in ACm and ACs, and eight times greater and associated with both a higher number and a larger size of fibrotic foci in OLD and SHR compared to controls. Social stress increased ventricular arrhythmia vulnerability in all models of cardiac hypertrophy, as well as in controls. The arrhythmogenic action of stress was facilitated in ACs, OLD and SHR. A correlation between structural cardiac remodeling and ventricular arrhythmias was found only in SHR and OLD, which exhibited the greatest increase in LVW and/or MF. Social stress proved to be a valuable tool for analyzing the combined effects of autonomic stimulation and altered myocardial substrate on the genesis of potentially life-threatening arrhythmias in social animals.
Asunto(s)
Arritmias Cardíacas/patología , Cardiomegalia/patología , Estrés Psicológico/psicología , Envejecimiento/psicología , Animales , Coartación Aórtica/patología , Arritmias Cardíacas/etiología , Peso Corporal/fisiología , Cardiomegalia/complicaciones , Electrocardiografía , Fibrosis/patología , Relaciones Interpersonales , Miocardio , Tamaño de los Órganos/fisiología , Ratas , Ratas Wistar , Estrés Psicológico/complicaciones , Estrés Psicológico/patología , TelemetríaRESUMEN
Spread and modulation of electrical activity in cardiac tissue requires intercellular transfer of current via gap junctions, specialised regions of densely packed ionic channels. Electrotonic interaction is determined not merely by intercellular electrical resistance (Rj) but rather by the interplay of Rj and sarcolemmal passive and active electrical properties (Zaniboni et al., Spitzer et al.). In this work we combined a well established protocol to measure Rj in cell pairs (Weingart e Maurer) with a stimulation protocol which allowed to simultaneously study parameters relative to action potential transfer during sequential stimulation. Current clamp experiments, performed on cardiomyocyte pairs held in double-patch configuration, allowed to simultaneously monitor, at a relatively high frequency (1 Hz), membrane resistance (Rm), resting potential (Vm), maximum depolarization rate (dv/dtmax) and time to peak of dv/dtmax in both cells as well as Rj. Spontaneous electrical uncoupling was observed in guinea pig cell pairs with little or no effect on action potential transfer. Pharmacological uncoupling with 40 microM beta-glycyrrhetinic acid reached, in one case, a much higher level of Rj and dramatically increased time delay for action potential appearance. When only Rj was measured over a short time interval after approximately two minutes from cell-attachments, values of Rj approximately 40 M omega in rat cell pairs (n = 20) and Rj approximately 15 M omega in guinea pig cell pairs (n = 24) were obtained. The possibility of monitoring simultaneously active and intercellular/cellular passive electrical properties makes this protocol particularly suitable to study dynamic changes in Rj during action potential transfer.