RESUMEN
BACKGROUND AND PURPOSE: The regulatory guidelines (ICHS7B) recommending inhibition of the delayed rectifier K(+) current (I(Kr)), carried by human ether-a-go-go-related gene (hERG) channels in cardiac cells (the hERG test), as a 'first line' test for identifying compounds inducing QT prolongation, have limitations, some of which are outlined here. EXPERIMENTAL APPROACH: hERG current was measured in HEK293 cells, stably transfected with hERG channels; action potential duration (APD) and arrhythmogenic effects were measured in isolated Purkinje fibres and perfused hearts from rabbits. KEY RESULTS: 576 compounds were screened in the hERG test: 58% were identified as hERG inhibitors, 39% had no effect and 3% were classified as stimulators. Of the hERG inhibitors, 92 were tested in the APD assay: 55.4% of these prolonged APD, 28.3% had no effect and 16.3% shortened APD. Of the 70 compounds without effect on hERG channels, 54.3% did not affect APD, 25.7% prolonged, while 20% significantly shortened APD. Dofetilide (hERG inhibitor; IC(50), 29 nM) prolonged QT and elicited early after-depolarizations and/or torsade de pointes (TdP) in isolated hearts. Mallotoxin and NS1643 (hERG current stimulators at 3 microM), levcromakalim and nicorandil (no effect on hERG current), all significantly shortened APD and QT, and elicited ventricular fibrillation (VF) in isolated hearts. CONCLUSION AND IMPLICATIONS: The hERG assay alone did not adequately identify drugs inducing QT prolongation. It is also important to detect drug-induced QT shortening, as this effect is associated with a potential risk for ventricular tachycardia and VF, the latter being invariably fatal, whereas TdP has an approximately 15-25% incidence of death.
Asunto(s)
Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Canales de Potasio Éter-A-Go-Go/efectos de los fármacos , Síndrome de QT Prolongado/inducido químicamente , Modelos Biológicos , Potenciales de Acción/efectos de los fármacos , Animales , Línea Celular , Canales de Potasio Éter-A-Go-Go/metabolismo , Femenino , Guías como Asunto , Humanos , Ramos Subendocárdicos/efectos de los fármacos , Conejos , Taquicardia Ventricular/inducido químicamente , Torsades de Pointes/inducido químicamente , Fibrilación Ventricular/inducido químicamenteRESUMEN
INTRODUCTION: Conflicting results associated with the use of I(Ks) blockers on the action potential duration (APD) have raised a question as to whether the variable results arise from the use of different cardiac tissues, beta-adrenergic stimulation, or by the "selectivity" of the chosen I(Ks) blockers. METHODS: We used the highly selective I(Ks) blocker (-)-[3R, 4S] chromanol 293B [(-) chromanol] to mimic drug-induced long QT1 in isolated rabbit Purkinje fibers, papillary muscles, and ventricular trabeculae using the conventional microelectrode technique. RESULTS: I(Ks) block with (-) chromanol at 1 x 10(-5) M did not significantly change the APD at different stimulation rates in all three cardiac tissues. Isoproterenol (Iso:1 x 10(-7) M) shortened APD(90), and (-) chromanol (1 x 10(-5) M) largely prevented this shortening in isolated papillary muscles at 1 Hz [-3% with Iso combined (-) chromanol group versus -16% with iso group; p<0.05] and also at 2 Hz (+7% versus -25% with Iso group; p<0.05), but did not significantly prevent this shortening in isolated Purkinje fibers. In isolated trabeculae, (-) chromanol combined with Iso significantly prolonged the APD(90) by 15% at 1 Hz (versus -10% with Iso group; p<0.05) and by 5% at 2 Hz (versus -11% with Iso group; p<0.05). DISCUSSION: Our study shows that only during beta-adrenoceptor stimulation, pharmacological inhibition of the I(Ks) current plays an important role in the APD recorded from isolated ventricular trabeculae and papillary muscles, but not from Purkinje fibers. These results indicate that the APD prolonging effects of I(Ks)channel blockers during beta-adrenergic receptor stimulation can only be detected from isolated rabbit papillary muscles and ventricular trabeculae, but not Purkinje fibers.
Asunto(s)
Cromanos/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Receptores Adrenérgicos beta/efectos de los fármacos , Función Ventricular , Potenciales de Acción/efectos de los fármacos , Agonistas Adrenérgicos beta/farmacología , Animales , Combinación de Medicamentos , Femenino , Ventrículos Cardíacos/citología , Ventrículos Cardíacos/efectos de los fármacos , Técnicas In Vitro , Isoproterenol/farmacología , Síndrome de QT Prolongado/fisiopatología , Microelectrodos , Músculos Papilares/citología , Músculos Papilares/efectos de los fármacos , Músculos Papilares/fisiología , Ramos Subendocárdicos/citología , Ramos Subendocárdicos/fisiología , Conejos , Receptores Adrenérgicos beta/fisiologíaRESUMEN
INTRODUCTION: QT dispersion (QTd) can be measured from three leads of the ECG in patients with myocardial ischemia. However, whether QT and JT dispersion (QTd, JTd) can be calculated from a three-lead of the ECG in drug-induced long QT syndrome (LQTS) in animals remains elusive. Therefore, we determined to what extent a three-lead measurement of the surface ECG accurately detects dispersion of QT and JT in comparison with multi-lead assessments in anaesthetized rabbits, challenged with methoxamine and additionally infused intravenously with solvent or dofetilide. METHODS: Using several ECG leads in anaesthetized rabbits challenged intravenously with an alpha(1)-adrenoceptor agonist methoxamine, we assessed the QT and JT interval, as well as QT and JT dispersion, at baseline and in response to solvent or dofetilide (0.02 or 0.04 mg/kg/min iv for 60 min), an I(Kr) blocker. For that purpose, we recorded and analyzed the surface ECG and assessed QT and JT dispersion by four methods: (1) 12-lead ECG; (2) six precordial leads (V1-V6); (3) three leads most likely to contribute to the dispersion (aVF, V1, and V4); (4) three quasi-orthogonal leads (aVF, I, and V2). QT and JT dispersion were significantly lower in 6- and 3-lead measurements than in 12-lead measurement, both at baseline and during infusion of solvent or dofetilide. At 5 and 10 min of infusion, dofetilide at 0.02 or 0.04 mg/kg/min iv markedly increased QT and JT dispersion by 100% to 500% in all four ECG lead combinations. This dose regimen of dofetilide markedly prolonged QT and JT intervals in lead II, and was associated with high incidences of polymorphous ventricular tachycardia (PVT: 30% at 0.02 mg/kg/min; 100% at 0.04 mg/kg/min) and of ventricular fibrillation (VF: 17% with 0.02 mg/kg/min; 58% with 0.04 mg/kg/min). CONCLUSIONS: Our present study shows that the measurement of QT and JT dispersion in three surface ECG leads only (aVF, I, V2 or aVF, V1 V4), instead of 12 ECG leads, is an appropriate approach to assess drug-induced heterogeneity or dispersion of ventricular repolarization in anaesthetized rabbits, both at baseline and during arrhythmogenic sensitization with methoxamine and challenged with dofetilide.
Asunto(s)
Electrocardiografía/instrumentación , Electrocardiografía/métodos , Síndrome de QT Prolongado/fisiopatología , Taquicardia Ventricular/fisiopatología , Agonistas alfa-Adrenérgicos/administración & dosificación , Agonistas alfa-Adrenérgicos/farmacología , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Electrocardiografía/efectos de los fármacos , Femenino , Inyecciones Intravenosas , Síndrome de QT Prolongado/inducido químicamente , Metoxamina/administración & dosificación , Metoxamina/farmacología , Fenetilaminas/administración & dosificación , Fenetilaminas/farmacología , Bloqueadores de los Canales de Potasio/administración & dosificación , Bloqueadores de los Canales de Potasio/farmacología , Conejos , Sulfonamidas/administración & dosificación , Sulfonamidas/farmacología , Taquicardia Ventricular/inducido químicamenteRESUMEN
A buffered solution was perfused at a constant flow rate (2 ml/min) through both iliac arteries in rat hindquarters. Perfusion pressures were measured in normal and collateralized vascular beds of the left and right hind-leg, respectively. Bolus injections of various agonists produced concentration-dependent increases in perfusion pressure in both collateralized and normal circulatory beds. Serotonin, in particular, and noradrenaline, to a lesser extent, produced more pronounced vasoconstriction on the collateral side than on the normal side. The difference in vasoreactivity to serotonin was related to a difference in both vascular structure and sensitivity of both types of vascular bed. Vasoconstriction induced by serotonin was inhibited by 5-HT2 antagonists. Selective blockade of alpha 1,alpha 2,beta 1-beta 2 adrenoceptors and amine uptake blockade were ineffective. This study indicates that, in rat hind-legs, the collateralized vascular bed is superreactive to serotonin in comparison with the normal bed. This resetting of reactivity to serotonin is due to the specific vascular structure as well as to an increased 5-HT2 receptor-mediated sensitivity.
Asunto(s)
Circulación Colateral/efectos de los fármacos , Serotonina/farmacología , Vasoconstricción/efectos de los fármacos , Animales , Circulación Colateral/fisiología , Miembro Posterior/irrigación sanguínea , Masculino , Perfusión , Ratas , Ratas Endogámicas , Antagonistas de la Serotonina , Vasoconstricción/fisiologíaRESUMEN
The ultrastructural morphology of the different endogenous stages of Eimeria maxima and E. brunetti was evaluated after oral treatment of inoculated chickens with a single dose of 5 mg/kg diclazuril. The drug induced no ultrastructural change in the growth or differentiation of the various schizont stages of both Eimeria spp. In E. maxima, the micromorphological appearance of micro- and macrogamonts developing from the blast from to maturation also remained unaffected by drug treatment. However, in all fertilized macrogamonts the normal pattern of oocyst wall establishment was completely disturbed, resulting in the formation of an abnormally thickened, incomplete oocyst wall and the necrosis of the zygote. In E. brunetti, the growth and nuclear division during microgametogenesis were not affected but differentiation was clearly abnormal. In comparison with the controls, this abnormal differentiation was characterized by a less extensive enlargement of the parasite surface area, aberrant morphological configurations of condensed heterochromatin, intracytoplasmic flagella formation, and glycogen accumulation. Finally, the complete degeneration of all microgamonts ensued. The growth and differentiation leading to mature macrogamonts was not disturbed; however, subsequent oocyst wall formation was largely precluded and the macrogamonts proceeded to degenerate completely. We conclude that diclazuril treatment primarily affected particular stages in the sexual development of both Eimeria spp., resulting in the complete eradication of these coccidian species.
Asunto(s)
Pollos/parasitología , Coccidiostáticos/farmacología , Eimeria/efectos de los fármacos , Nitrilos/farmacología , Triazinas/farmacología , Animales , Pared Celular/efectos de los fármacos , Pared Celular/ultraestructura , Coccidiosis/tratamiento farmacológico , Coccidiosis/veterinaria , Eimeria/crecimiento & desarrollo , Eimeria/ultraestructura , Enfermedades de las Aves de Corral/tratamiento farmacológico , Enfermedades de las Aves de Corral/parasitología , Especificidad de la EspecieRESUMEN
In situ perfusion of rat hindquarters was performed through both iliac arteries. Perfusion pressures were measured concomitantly in the normal vascular bed and in the ligation-induced collateral bed of the left and right hindleg, respectively. An increased vaso-constrictive response of the collateral versus normal blood vessels was found after bolus injections of increasing concentrations of the thromboxane A2 (TXA2) mimic U-46619. This increased reactivity was related to a difference in vascular structure and in receptor-mediated sensitivity. The vasoconstrictive effect of U-46619 was dose-dependently inhibited by the combined TXA2 synthetase inhibitor-TXA2/prostaglandin endoperoxide (PGH2) receptor antagonist ridogrel and the single TXA2/PGH2 receptor antagonist sulotroban. Selective blockade of alpha 1-, alpha 2-, beta 1-beta 2- and S2-receptors, cyclooxygenase inhibition, TXA2 synthesis inhibition, amine depletion and amine uptake blockade had no influence on the vasoconstrictive action of U-46619. Ca2+ entry blockade by flunarizine and nifedipine significantly reduced the vasoconstriction. This study shows that (1) rat peripheral collaterals in relation to normal arterial blood vessels are significantly more reactive to TXA2; (2) the vasoconstrictive effect of U-46619 is mediated predominantly by TXA2/PGH2 receptors; (3) the activation of these receptors elicits indirectly transmembrane Ca2+ entry to trigger vasoconstriction.
Asunto(s)
Arteria Ilíaca/fisiología , Endoperóxidos de Prostaglandinas Sintéticos/farmacología , Tromboxano A2/farmacología , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico , Animales , Circulación Colateral/efectos de los fármacos , Miembro Posterior/irrigación sanguínea , Masculino , Prostaglandina H2 , Prostaglandinas H/farmacología , RatasRESUMEN
A single 5-mg/kg oral dose of diclazuril affected both the asexual and sexual development of Eimeria tenella in experimentally inoculated chickens. In second-generation schizonts, early growth and nuclear divisions progressed normally, but a marked inhibition of merozoite formation was observed. Exogenesis of merozoites was largely prevented, whereas production of micronemes, amylopectin granules, and dense bodies and the formation of rhoptries, conoid, and pellicle continued. All these subcellular organelles accumulated, together with differentiated nuclei, within the main cytoplasmic mass. In the end, complete necrosis of the schizonts occurred. In macrogamonts, dilation of the rough endoplasmic reticulum around type II wall-forming bodies, fusion of type II wall-forming body contents, disturbance of the normal parallel arrangement of rough endoplasmic reticulum, and disruption of row formation of amylopectin granules became evident. In the microgamonts, normal evagination of microgametes was prevented; the flagellar complex formed within the main cytoplasmic mass and the differentiated nuclei remained present within the parasite body. The macro- and microgamonts also ended up in a stage of complete necrosis. These data indicate that diclazuril treatment primarily affects the normal differentiation of the respective endogenous stages during parasite development. This leads to complete degeneration of schizonts and gamonts indicating the lethal effect of this new anticoccidial compound.
Asunto(s)
Pollos/parasitología , Coccidiosis/veterinaria , Coccidiostáticos/farmacología , Eimeria/efectos de los fármacos , Nitrilos/farmacología , Enfermedades de las Aves de Corral/parasitología , Triazinas/farmacología , Animales , Núcleo Celular/ultraestructura , Coccidiosis/tratamiento farmacológico , Coccidiosis/parasitología , Coccidiostáticos/uso terapéutico , Citoplasma/ultraestructura , Eimeria/crecimiento & desarrollo , Eimeria/ultraestructura , Microscopía Electrónica , Nitrilos/uso terapéutico , Enfermedades de las Aves de Corral/tratamiento farmacológico , Triazinas/uso terapéuticoRESUMEN
Complete desquamation of the endothelium of the rat carotid artery by balloon catheter stripping resulted within 2 weeks in the formation of a large intimal thickening. After an enzyme cytochemical technique was applied to localize cytosolic purine nucleoside phosphorylase (PNP), light microscopical evaluation indicated that this intimal thickening in normocholesterolemic rats was composed of 5.8% to 11.8% (mean 8.8%) PNP-positive cells. At the electron microscopic level, all these PNP-positive cells were identified as macrophages by the absence of a basement membrane and plasmalemmal vesicles and by the occurrence of specific intracytoplasmic granules. The nearly nonreactive intimal cells were classified as modified smooth muscle cells. Additional evidence of the macrophage nature of the PNP-stained intimal cells was obtained by differential immunogold labeling of these cells with a monoclonal antibody against rat macrophages. Moreover, in hypercholesterolemic rats, only the cells stained for PNP transformed into foam cells (between 8.5% and 11.4% of all nucleated intimal cells; mean 9.6%). This study shows that PNP cytochemistry discriminates macrophages from modified smooth muscle cells in the rat carotid intimal thickening. It further suggests that the intimal thickening in normocholesterolemic rats originates not only from migration and proliferation of smooth muscle cells, but also from a considerable number of leukocyte-derived macrophages. Whether the latter cells are actively involved in the establishment of the intimal thickening as has been suggested in dietary hypercholesterolemia, remains to be verified.
Asunto(s)
Arterias Carótidas/patología , Macrófagos/patología , Pentosiltransferasa/análisis , Purina-Nucleósido Fosforilasa/análisis , Animales , Biomarcadores/análisis , Arterias Carótidas/enzimología , Arterias Carótidas/ultraestructura , Colesterol/sangre , Histocitoquímica , Hipercolesterolemia/sangre , Hipercolesterolemia/enzimología , Hipercolesterolemia/patología , Inmunohistoquímica , Macrófagos/enzimología , Macrófagos/ultraestructura , Masculino , Ratas , Ratas EndogámicasRESUMEN
Since previously described simple methods were too insensitive to quantify the extent of microvascular permeability increase in the rat cremaster muscle, we applied a new quantitative technique using radioactive colloidal gold (198Au) as the macromolecular tracer. Dose-response effects on microvascular permeability were found with increasing doses of subscrotally injected serotonin or histamine. The presence of colloidal gold particles in the subendothelial space of the postcapillary venules, as verified ultrastructurally, indicated endothelial permeation of the tracer macromolecules. The increased permeability elicited by serotonin was inhibited in a dose-dependent way by serotonin receptor antagonists (potency: methysergide greater than or equal to ketanserin = ritanserin greater than cinanserin), suggesting the presence of functional S2-serotonergic receptors on postcapillary venular endothelial cells. A dose-related inhibition of the histamine-induced extravasation was seen with the H1 antagonists astemizole and azatidine (astemizole greater than azatidine); the H2 antagonists cimetidine and ranitidine had no inhibitory effect. The radioactive colloidal gold technique is a sensitive and reliable tool for pharmacological experiments aimed at investigating the extravasation phenomenon in small tissue samples such as the rat cremaster muscle.
Asunto(s)
Permeabilidad Capilar , Oro Coloidal Radiactivo , Histamina/farmacología , Músculos/irrigación sanguínea , Serotonina/farmacología , Animales , Permeabilidad Capilar/efectos de los fármacos , Antagonistas de los Receptores Histamínicos H1/farmacología , Masculino , Ratas , Ratas Endogámicas , Antagonistas de la Serotonina/farmacologíaRESUMEN
Collateral arteries can clearly be visualized in corrosion cast material after ligation of the femoral artery in rats. To characterize the influence of serotonin on the blood circulation under the experimental condition of unilateral ligation, we performed intermuscular thermoflow measurements. The data showed that intraperitoneal serotonin (8.7 and 20 mg.kg-1) injection markedly reduced the muscle temperature in both hindlegs. However, the effect was significantly more pronounced on the ligated side, where the blood was supplied through a collateral circulation, than on the non-ligated side with its normal arterial vasculature. Almost identical changes were obtained in rats with an early as well as in animals with a more advanced stage of collateral development (challenge with 8.7 mg.kg-1 serotonin). Evans blue dye experiments and angiographic data further revealed that, after challenge with 20 mg.kg-1 serotonin, the severe temperature decrease at the ligated side probably reflected a nearly complete blockage of blood supply. Pretreatment with the S2 receptor antagonist ketanserin (2.5 mg.kg-1) resulted in a significant reduction in the extent and duration of the serotonin-induced temperature decrease as revealed by the temperature measurements; such a treatment inhibited also the blockage in blood flow as seen with the other techniques. This study shows that serotonin, through its vasoconstrictive properties, can restrict the blood flow to the lower extremities of the rat, particularly when the blood is supplied through collateral circulation. This probably results in moderate to severe skeletal muscle ischaemia. It also suggests that the vasoconstriction is not only important in the inhibition of collateral blood flow in recently established collaterals but also in collateral vessels in a more advanced stage of development. The in vivo vasoconstriction and subsequent reduction of the blood supply caused by serotonin can be effectively counteracted by pretreatment with ketanserin.