RESUMEN
In human atrial myocytes the transient outward current I(to) develops a conspicuous faster inactivation with increasing temperatures. Since ß-subunits are known to modulate I(to) current kinetics, we hypothesized that the temperature sensitivity of I(to) is not only determined by the property of the ion-passing α-subunit Kv4.3 but also by its interaction with accessory ß-subunits. We therefore studied the influence of the transmembrane ß-subunits KCNE1, KCNE2 and DPP6 on Kv4.3/KChIP2 channels in CHO cells at room temperature and at physiological temperature. Exposure to 37°C caused a significant acceleration of the channel kinetics, whereas current densities and voltage dependences remained unaltered at 37°C compared to 23°C. However, Kv4.3/KChIP2 channels without transmembrane ß-subunits showed the strongest temperature sensitivity with considerably increased rates of activation and inactivation at 37°C. KCNE2 significantly slowed the current kinetics at 37°C compared to Kv4.3/KChIP2 channels, whereas KCNE1 did not influence the channel properties at both temperatures. Interestingly, the accelerating effects of DPP6 on current kinetics described at 23°C were diminished at physiological temperature, thus at 37°C current kinetics became remarkably similar for channel complexes Kv4.3/KChIP2 with and without DPP6 isoforms. A Markov state model was developed on the basis of experimental measurements to simulate the influence of ß-subunits on Kv4.3 channel complex at both temperatures. In conclusion, the remarkably fast kinetics of the native I(to) at 37°C could be reproduced by co-expressing Kv4.3, KChIP2, KCNE2 and DPP6 in CHO cells, whereas the high temperature sensitivity of human I(to) could be not mimicked.
Asunto(s)
Subunidades de Proteína/fisiología , Canales de Potasio Shal/metabolismo , Potenciales de Acción , Animales , Células CHO , Cricetinae , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/fisiología , Humanos , Activación del Canal Iónico , Cinética , Cadenas de Markov , Modelos Biológicos , Proteínas del Tejido Nervioso/fisiología , Técnicas de Placa-Clamp , Canales de Potasio/fisiología , Canales de Potasio con Entrada de Voltaje/fisiología , Estabilidad Proteica , TermodinámicaRESUMEN
BACKGROUND AND PURPOSE: The human cardiac transient outward potassium current (Ito) is believed to be composed of the pore-forming Kv4.3 alpha-subunit, coassembled with modulatory beta-subunits as KChIP2, MiRP1 and DPP6 proteins. beta-Subunits can alter the pharmacological response of Ito; therefore, we analysed the effects of flecainide on Kv4.3/KChIP2 channels coassembled with MiRP1 and/or DPP6 beta-subunits. EXPERIMENTAL APPROACH: Currents were recorded in Chinese hamster ovary cells stably expressing K(V)4.3/KChIP2 channels, and transiently transfected with either MiRP1, DPP6 or both, using the whole-cell patch-clamp technique. KEY RESULTS: In control conditions, Kv4.3/KChIP2/MiRP1 channels exhibited the slowest activation and inactivation kinetics and showed an 'overshoot' in the time course of recovery from inactivation. The midpoint values (Vh) of the activation and inactivation curves for Kv4.3/KChIP2/DPP6 and Kv4.3/KChIP2/MiRP1/DPP6 channels were approximately 10 mV more negative than Vh values for Kv4.3/KChIP2 and Kv4.3/KChIP2/MiRP1 channels. Flecainide (0.1-100 microM) produced a similar concentration-dependent blockade of total integrated current flow (IC50 approximately 10 microM) in all the channel complexes. However, the IC50 values for peak current amplitude and inactivated channel block were significantly different. Flecainide shifted the Vh values of both the activation and inactivation curves to more negative potentials and apparently accelerated inactivation kinetics in all channels. Moreover, flecainide slowed recovery from inactivation in all the channel complexes and suppressed the 'overshoot' in Kv4.3/KChIP2/MiRP1 channels. CONCLUSIONS AND IMPLICATIONS: Flecainide directly binds to the Kv4.3 alpha-subunit when the channels are in the open and inactivated state and the presence of the beta-subunits modulates the blockade by altering the gating function.
Asunto(s)
Antiarrítmicos/farmacología , Flecainida/farmacología , Subunidades de Proteína , Canales de Potasio Shal/efectos de los fármacos , Animales , Antiarrítmicos/administración & dosificación , Células CHO , Cricetinae , Cricetulus , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas , Relación Dosis-Respuesta a Droga , Femenino , Flecainida/administración & dosificación , Humanos , Concentración 50 Inhibidora , Proteínas de Interacción con los Canales Kv/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Técnicas de Placa-Clamp , Péptido Hidrolasas/metabolismo , Canales de Potasio/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Canales de Potasio Shal/metabolismo , TransfecciónRESUMEN
BACKGROUND AND PURPOSE: This study was designed to establish the pathology-specific inhibitory effects of the IKur/Ito/IK,ACh blocker AVE0118 on atrium-selective channels and its corresponding effects on action potential shape and effective refractory period in patients with chronic AF (cAF). EXPERIMENTAL APPROACH: Outward K+-currents of right atrial myocytes and action potentials of atrial trabeculae were measured with whole-cell voltage clamp and microelectrode techniques, respectively. Outward currents were dissected by curve fitting. KEY RESULTS: Four components of outward K+-currents and AF-specific alterations in their properties were identified. Ito was smaller in cAF than in SR, and AVE0118 (10 microM) apparently accelerated its inactivation in both groups without reducing its amplitude. Amplitudes of rapidly and slowly inactivating components of IKur were lower in cAF than in SR. The former was abolished by AVE0118 in both groups, the latter was partially blocked in SR, but not in cAF, even though its inactivation was apparently accelerated in cAF. The large non-inactivating current component was similar in magnitude in both groups, but decreased by AVE0118 only in SR. AVE0118 strongly suppressed AF-related constitutively active IK,ACh and prolonged atrial action potential and effective refractory period exclusively in cAF. CONCLUSIONS AND IMPLICATIONS: In atrial myocytes of cAF patients, we detected reduced function of distinct IKur components that possessed decreased component-specific sensitivity to AVE0118 most likely as a consequence of AF-induced electrical remodelling. Inhibition of profibrillatory constitutively active IK,ACh may lead to pathology-specific efficacy of AVE0118 that is likely to contribute to its ability to convert AF into SR.
Asunto(s)
Fibrilación Atrial/tratamiento farmacológico , Compuestos de Bifenilo/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Anciano , Fibrilación Atrial/fisiopatología , Enfermedad Crónica , Electrofisiología , Femenino , Atrios Cardíacos/citología , Atrios Cardíacos/patología , Humanos , Técnicas In Vitro , Masculino , Microelectrodos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Técnicas de Placa-Clamp , Canales de Potasio/metabolismoRESUMEN
Structure of starch in feed and chyme of horses (7 with a cannula at the caudal end of the jejunum and 2 with a cecal fistula) after feeding high starch diets (maize: whole, broken, ground, expanded and as silage, oats: whole, rolled or ground, rolled barley, raw potatoes, and tapioca) was investigated by light and electron microscopy. Structure of feed starch and morphology or starch degradation in the chyme corresponded to data on preileal starch digestibility which was investigated in a parallel study. Barriers for starch digestion in the gastrointestinal tract of the horse were structure of the plant storage organ, as for example, a tight connection between starch granules in maize gains as well as the structure of the starch granules itself. The highly digestible oat starch was degraded by exocorrosion around the grains, whereas in other, less digestible, starch types degradation occurred by endocorrosion via pin holes. The number and size of the pin holes increased with increasing preileal starch digestibility. The effect of various ways of decomposition on preileal digestibility increased with advanced destruction of the original starch structure. Expanding was most effective. The granules were destroyed completely and the starch became soluble. Simple examination by light microscopy is a fast method to evaluate the degree of starch decomposition in the feed.
Asunto(s)
Digestión/fisiología , Caballos/fisiología , Almidón/metabolismo , Animales , Avena/química , Avena/metabolismo , Ciego/metabolismo , Ciego/fisiología , Hordeum/química , Hordeum/metabolismo , Yeyuno/metabolismo , Yeyuno/fisiología , Microscopía Electrónica de Rastreo/veterinaria , Tamaño de la Partícula , Solanum tuberosum/química , Solanum tuberosum/metabolismo , Almidón/análisis , Zea mays/química , Zea mays/metabolismoRESUMEN
Three horses (approximately 190 kg BW) fitted with a permanent fistula at the end of the jejunum were used. To a control diet (1/3 hay, 2/3 mixed feed) one of two fat types (coconut fat or soybean oil) were added at 2 levels resulting in fat intakes of 0.1 g (control diet) to 0.5 or 1 g/kg BW 0.5 d, respectively. Each experimental period consisted of 2 weeks adaptation, 2 days of breath tests (before and hourly after the morning meal) and 5 days sampling of chyme. Crude fat, crude protein, concentrations of organic acids (SCFA, lactic acid), pH, and the minerals calcium, magnesium and phosphorus were determined in the chyme; H2 and CH4 in the expired air. The following results were obtained: 1) Fat feeding significantly (P < 0.01) stimulated (independent of amount or kind of fat) the jejunoileal flow of chyme. 2) Preileal fat digestibility increased significantly (P < 0.01) from 30-38% during the control periods to 73-80% (moderate fat intake) and 82-86% (high fat intake). Differences between the fat sources were not significant. 3) Fat addition resulted dose dependent in a reduction (P < 0.05) of lactic acid as well as SCFA concentrations of chyme (at 5th h postprandial). 4) Fat intake caused a reduction in the H2-concentration of the exhaled air (P < 0.05). Such effect was not found with the CH4-concentration, except the high soybean oil level which tended to reduce the concentration. 5) The addition of fat had no significant effects on preileal net absorption of magnesium and calcium, whilst the net secretion of phosphorus significantly increased (P < 0.01). 6) The preileal protein digestibility (control periods 48-53%) was slightly decreased (P < 0.05), due to the fat inclusion.
Asunto(s)
Grasas de la Dieta/metabolismo , Digestión/fisiología , Caballos/fisiología , Íleon/metabolismo , Aceites de Plantas/metabolismo , Aceite de Soja/metabolismo , Animales , Calcio/análisis , Calcio/metabolismo , Aceite de Coco , Cocos , Ácidos Grasos Volátiles/análisis , Ácidos Grasos Volátiles/metabolismo , Mucosa Gástrica/metabolismo , Tránsito Gastrointestinal/fisiología , Caballos/metabolismo , Hidrógeno/análisis , Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , Íleon/microbiología , Íleon/fisiología , Yeyuno/metabolismo , Yeyuno/microbiología , Yeyuno/fisiología , Ácido Láctico/análisis , Ácido Láctico/metabolismo , Magnesio/análisis , Magnesio/metabolismo , Metano/análisis , Metano/metabolismo , Fósforo/análisis , Fósforo/metabolismo , Estómago/microbiología , Estómago/fisiologíaRESUMEN
In this study preileal starch digestibility of starchy feeds (oats, corn, barley, potatoes, manioc) was determined in seven jejunofistulated horses. The grains were fed whole (oats, corn), rolled (oats, barley), crushed, ground and expanded (corn); the potatoes were fresh, the manioc rolled. Ground corn was also fed in combination with amylase. The feeds were fed partly isolated or in combination with alfalfa meal or hay (Table 1). At least four horses with a cannula in the terminal jejunum were used for each diet. Two meals per day were offered at 12 h intervals. The starch intake was mostly about 2 g/kg bw/meal, except one period with oats (3.9 g starch/kg bw) and with expanded corn (1.4 g/kg bw). Jejunal chyme was postprandially collected 11 times (from 1st to the 11th h after the morning meal for 15 min). Starch was determined polarimetrically. The preileal digestibility of starch was calculated by the marker method (chronic oxide 0.25% DM) and by estimating the total jejunoileal chyme flow during 12 h postprandially extrapolating the sample volume from the 15 min sampling periods. The results of both methods agreed quite well. Preileal digestibility of oat starch (80-90%) was (independent of doses or preparation or of the combination with hay, Table 4) significantly higher than that of whole or crushed corn (30%) or barley (26%). Grinding of corn significantly increased preileal digestibility to 51%, expanding to 90%. The addition of amylase improved digestion of ground corn by 10% (absolute). The preileal digestibility of potato or manioc was less than 10%. Individual factors in the horse (chewing intensity, amylase activity) had also considerable influence on preileal starch digestibility.