RESUMEN
O objetivo deste trabalho foi realizar uma análise direta da expressão de RNAm de KATP por RT-PCR em rim e aorta isolados de ratos com cirrose (induzida por tetracloreto de carbono) e de controles. O presente trabalho também estudou as relações entre a cirrose induzida e a excreção urinária de sódio e a atividade simpática em ratos cirróticos. Os ratos foram colocados em gaiolas metabólicas com acesso livre a comida e água. A cirrose foi induzida por repetidas doses de tetracloreto de carbono por gavage gástrica. Depois de algumas semanas, o rim e a aorta foram dissecados e foi feita a extração de RNA. A dosagem de letrólitos foi feita no sangue e na urina. A função renal foi estimada elo clearance de creatinina e a excreção urinária de sódio. As catecolaminas séricas foram medidas por análise de HPLC. Em primeiro lugar, a análise do RNAm de KATP expressou-se em fígados com cirrose e fibrose vigorosa, mas não com fibrose moderada. Posteriormente, a análise de RT-PCR revelou que a expressão de RNAm e KATP foi detectada somente na aorta dissecada de ratos com cirrose. Finalmente, uma reabsorção aumentada de sódio, sem falência renal, sugeriu que um potencial mediador aumente a atividade do sistema simpático. Conclusão: Estes resultados sugerem que o RNAm de KATP seja expresso em ratos cirróticos com ativação simpática e disfunção renal. Este canal pode estar envolvido em outra via, onde o tônus vascular pode ser modulado na cirrose.
Asunto(s)
Ratas , Adenosina Trifosfato , Tetracloruro de Carbono , Canales de Potasio/análisis , Cirrosis Hepática Experimental , Riñón/fisiología , Sistema Nervioso Simpático/fisiopatología , Sodio/orinaRESUMEN
OBJECTIVE: To identify K+ channels of smooth muscle of human umbilical artery using the patch-clamp technique and to study their effect on resting tone of umbilical artery rings. METHODS: Whole-cell and single-channel patch-clamp recordings in enzymatically isolated smooth muscle cells were made. Measurements of developed isometric force were performed on intact tissue. RESULTS: Delayed rectifier K+ channels (KDR) and large-conductance Ca2+-activated K+ channels (BKCa) contribute to the whole-cell voltage- and time-dependent outward K+ current, as it was specifically inhibited by 5 mM 4-aminopyridine (4-AP; KDR blocker) (92 +/- 4% at 0 mV, n = 7), by 1 mM tetraethylammonium (TEA; BKCa blocker) (71 +/- 4% at +60 mV, n = 4), and by 200 nM iberiotoxin (BKCa blocker) (64 +/- 7% at +60 mV, n = 4). In outside-out patches, BKCa channels had a single-channel conductance of 132 +/- 4 pS (n = 24) in asymmetric K+ conditions and 216 +/- 4 pS (n = 4) in a symmetric K+ gradient. The activity of the BKCa channels was significantly augmented by 1 microM Ca2+ in the inside-out configuration. 4-AP had no effect on resting tone of intact arterial rings. TEA produced contraction of arterial rings whereas phloretin, an activator of BKCa, relaxed them, which means that BKCa channels are functional in intact tissue and are involved in the maintenance of resting tone in this human vessel. CONCLUSION: The identities of K+ channels in the human umbilical artery were shown using the patch-clamp technique, and the physiologic effect of K+ channels on resting tone was documented.
Asunto(s)
Canales de Potasio/análisis , Arterias Umbilicales/química , 4-Aminopiridina/farmacología , Calcio/farmacología , Conductividad Eléctrica , Femenino , Humanos , Potenciales de la Membrana , Músculo Liso Vascular/química , Técnicas de Placa-Clamp , Péptidos/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/fisiología , Embarazo , Tetraetilamonio/farmacologíaRESUMEN
Potassium (K+) channels are believed to regulate mammalian sperm acquisition of fertilising capacity. However, the molecular identity of these proteins in sperm has not been elucidated. In this report, using immunoconfocal and electron microscopy we show that a minimum of four different classes of K+ channels (Kv1.1, Kv1.2, Kv3.1 and GIRK1) are present and regionally distributed over the surface of mouse epididymal sperm. In addition, the use of reverse transcription polymerase chain reaction on RNA from mouse spermatogenic cells allowed the amplification of multiple transcripts corresponding to the channels identified by immunocytochemistry. Consistent with this, whole-cell patch-clamp recordings showed the expression of at least two different outwardly rectifying K+ currents in spermatogenic cells.
Asunto(s)
Canales de Potasio/análisis , Espermatozoides/metabolismo , Animales , Electrofisiología , Epidídimo/citología , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Masculino , Ratones , Microscopía Confocal , Microscopía Electrónica , Técnicas de Placa-Clamp , Canales de Potasio/clasificación , Espermatogénesis , Espermatozoides/ultraestructuraRESUMEN
Whole cell patch-clamp recordings were used to study the electrical properties of the macrophage-like cell line J774.1, after infection with Leishmania amazonensis. Infection induced a significant increase in cell size and membrane capacitance, suggesting that parasite invasion leads to the addition of plasma membrane to the host cell. By 24 hr after infection, the host cell membrane potential was significantly more hyperpolarized than control cells, and this difference remained for the subsequent 72 hr post-infection. The hyperpolarization was paralleled by an increase in the density of inward rectifying K(+) currents. The shape of the conductance vs. voltage curve, the kinetic properties and the pharmacological profile of these currents were not significantly altered by infection. These results suggest that infection by L. amazonensis causes an increase in the number of functional inward rectifying K(+) channels, leading to hyperpolarization of the host cell membrane.
Asunto(s)
Leishmaniasis Cutánea/fisiopatología , Macrófagos/fisiología , Macrófagos/parasitología , Animales , Línea Celular/parasitología , Conductividad Eléctrica , Electrofisiología , Leishmania mexicana , Potenciales de la Membrana/fisiología , Ratones , Microscopía Electrónica , Técnicas de Placa-Clamp , Canales de Potasio/análisis , Canales de Potasio/fisiologíaRESUMEN
A 107-pS (symmetrical 150 mM KCl), nonselective cation channel was reconstituted from a microsomal membrane fraction of the larval stage of the tapeworm Echinococcus granulosus. Most of the time, it displayed a high open probability (>>0.95) irrespective of either the applied voltage, Ca2+, Ba2+, or tetraethylammonium concentration. Nevertheless, in contrast with this "leaklike" behavior, less frequently this "all-the-time-open" channel reversibly entered two different kinetic modes. One of them was characterized by lower Po values and some voltage sensitivity (V(1/2) congruent with 129 mV, and an equilibrium constant for channel closing changing e-fold per 63-mV change) the kinetic analysis revealing that it resulted from the appearance of voltage-sensitivity in the mean closed times and a sixfold increase in the equilibrium constant for channel closing at 0 mV. The other mode was characterized by a very fast open-close activity leading to poorly resolved current levels and a Po around 0.6-0.7 which, occasionally and in a voltage-sensitive manner, entered a long-lived nonconducting state. However, the rare nature of these mode-shifting transitions precluded a more detailed analysis of their kinetics. The conductive properties of the channel were not affected by these switches. Model gating alone does not seem to ensure any physiological role of this channel and, instead, some other channel changes must occur if this phenomenon were to be of regulatory importance in vivo. Thus, mode-shifting might constitute an alternative target for channel activity modulation also in tapeworms.
Asunto(s)
Activación del Canal Iónico/fisiología , Animales , Echinococcus/química , Echinococcus/fisiología , Cinética , Membrana Dobles de Lípidos/química , Canales de Potasio/análisis , Canales de Potasio/fisiologíaRESUMEN
A peptide toxin, ShK, that blocks voltage-dependent potassium channels was isolated from the whole body extract of the Caribbean sea anemone Stichodactyla helianthus. It competes with dendrotoxin I and alpha-dendrotoxin for binding to synaptosomal membranes of rat brain, facilities acetylcholine release at an avian neuromuscular junction and suppresses K+ currents in rat dorsal root ganglion neurones in culture. Its amino acid sequence is R1SCIDTIPKS10RCTAFQCKHS20MKYRLSFCRK30TCGTC35. There is no homology with other K+ channel-blocking peptides, except for BgK from the sea anemone Bunodosoma granulifera. ShK and BgK appear to be in a different structural class from other toxins affecting K+ channels.