RESUMEN
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RESUMEN
The ectopic overexpression of the voltage-dependent Eag1 (Kv10.1) K+ channel is associated with the cancerous phenotype in about 70% of human cancers and tumor cell lines. Recent reports showed that, compared with the canonical Shaker-related Kv family, Kv10.1 presents unique structural and functional properties. Herein, we report the interaction of the class III anti-arrhythmic compound amiodarone with Kv10.1. Using whole-cell patch clamp, we found that amiodarone inhibits Kv10.1 channel conductance with nanomolar affinity. Additionally, and interestingly, we also report that amiodarone inhibits the characteristic Cole-Moore shift of Eag1 channels. Our observations are interpreted considering the structural-functional characteristics of these channels. We conclude that amiodarone possibly binds with high affinity to the voltage sensor module, altering the gating of Kv10.1.
Asunto(s)
Amiodarona/farmacología , Canales de Potasio Éter-A-Go-Go/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Potenciales de Acción/efectos de los fármacos , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Células HEK293 , Humanos , Activación del Canal Iónico , Unión ProteicaRESUMEN
La potencialidad terapéutica de fármacos se averigua mediante estudios bioquímicos y celulares que nos hablan de sus acciones sobre vías de señalización y receptores. Sin embargo, en algunas enfermedades -por ejemplo, enfermedades neurológicas conocidas como "desórdenes del movimiento"-, los bioensayos realizados miden las acciones farmacológicas mediante valoraciones conductuales en modelos animales de las mismas. No se han logrado bioensayos que correlacionen la acción terapéutica de fármacos sobre la actividad del tejido vivo. Se puede medir la actividad de decenas de neuronas mediante imagenología de calcio en tejido vivo. Ciertos parámetros de esta actividad neuronal registrada in vitro reflejan su estado patológico, así como la acción terapéutica de fármacos determinados. No hay un sistema integrado orientado a estos bioensayos, por lo que se combinan diferentes equipos comerciales de manera independiente con costo final de alrededor de 100,000 USD. Presentamos un prototipo de un sistema integral encaminado a realizar este tipo de bioensayos: microscopía de epifluorescencia con calidad suficiente para adquirir y medir cuantitativamente la actividad celular del tejido vivo registrada in vitro pero de costo 10 veces menor -alrededor de 10,000 USD-. Se pueden realizar satisfactoriamente bioensayos funcionales de uso potencial en la industria farmacéutica, investigación y docencia.
The therapeutic potential of drugs is determined by biochemical and cellular studies that inform us about their actions on signaling pathways and receptors. However, in some diseases -for example, neurological diseases such as "movement disorders"-, bioassays measure the pharmacological actions by evaluating behavior in animal models of the diseases. There are no bioassays that correlate drug therapeutic actions on living tissue. The neural activity of several neurons can be measured by using calcium imaging on living tissue. Certain parameters of the recorded neuronal activity in vitro reflect the pathological state and the therapeutic actions of specific drugs. There is no integrated system oriented to these bioassays, so different commercial equipment has to be integrated independently with costs about 100,000 USD. We present a prototype of an integral system aimed to perform bioassays in vitro: epifluorescence microscopy with enough quality for the acquisition and quantitative assessment of cell activity recorded in the living tissue with costs around 10 times less -about 10,000 USD-. It allows successfully functional bioassays of potential use in the pharmaceutical industry, research an education.
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In this study, we investigated in groups of female BALB/c mice injected with Crotalus durissus terrificus venom (Cdt) the renal function based on creatinine clearance, percentage of fractional excretion cytokines and histological examination of renal tissue. Cdt caused renal alterations that induced proteinuria during the initial hours post-venom and reduced creatinine clearance 15 min. up to 2 hours post-venom administration. In urine from mice injected with Cdt induced a decrease in IL-4 levels. More pronounced increments of IL-5, IL-6 and IFN-γ were observed after 15 and 30 min, respectively. The highest levels of TNF and IL-10 were observed at 1 and 4 hs, respectively. The ratios of pro- and anti-inflammatory cytokines in animals injected with Cdt, which may be manifested in the inflammatory status during the envenoming. In groups of animals treated with Cdt were observed a decreasing in creatinine clearance and its effect on glomerular filtration rate was accompanied by decreased fractional excretion of cytokines and morphologic disturbances. This loss of change selectively in envenomation could thus explain why the relatively excretion of cytokines is reduced while of total proteins increases. In conclusion the fractional excretion of cytokines is significantly reduced in mice injected with Cdt, despite proteinuria.
Asunto(s)
Venenos de Crotálidos/farmacología , Crotalus , Mediadores de Inflamación/orina , Riñón/efectos de los fármacos , Animales , Creatinina/orina , Femenino , Humanos , Interferón gamma/orina , Interleucina-10/orina , Interleucina-4/orina , Interleucina-5/orina , Interleucina-6/orina , Riñón/fisiología , Pruebas de Función Renal , Ratones , Ratones Endogámicos BALB CRESUMEN
The effects of Crotalus durissus terrificus venom (Cdt) were analyzed with respect to the susceptibility and the inflammatory mediators in an experimental model of severe envenomation. BALB/c female mice injected intraperitoneally presented sensibility to Cdt, with changes in specific signs, blood biochemical and inflammatory mediators. The venom induced reduction of glucose and urea levels and an increment of creatinine levels in serum from mice. Significant differences were observed in the time-course of mediator levels in sera from mice injected with Cdt. The maximum levels of IL-6, NO, IL-5, TNF, IL-4 and IL-10 were observed 15 min, 30 min, 1, 2 and 4 hours post-injection, respectively. No difference was observed for levels of IFN-gamma. Taken together, these data indicate that the envenomation by Cdt is regulated both pro- and anti-inflammatory cytokine responses at time-dependent manner. In serum from mice injected with Cdt at the two first hours revealed of pro-inflammatory dominance. However, with an increment of time an increase of anti-inflammatory cytokines was observed and the balance toward to anti-inflammatory dominance. In conclusion, the observation that Cdt affects the production of pro- and anti-inflammatory cytokines provides further evidence for the role played by Cdt in modulating pro/anti-inflammatory cytokine balance.
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Venenos de Crotálidos/toxicidad , Crotalus/metabolismo , Citocinas/sangre , Animales , Creatina/sangre , Venenos de Crotálidos/administración & dosificación , Modelos Animales de Enfermedad , Femenino , Interleucina-10/sangre , Interleucina-4/sangre , Interleucina-6/sangre , Cinética , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico/sangre , Mordeduras de Serpientes/sangre , Mordeduras de Serpientes/inducido químicamente , Factor de Necrosis Tumoral alfa/sangreRESUMEN
We recently reported that rodent anterior pituitary (AP) cells (with the exception of corticotrophs and melanotrophs) express neuronal markers, including 68-kDa neurofilaments (NF68) in an oestrogen-dependent manner. The functional significance of neurofilament (NF) expression in the AP is unknown, but recent data in myelinated nerve fibres from NF-null mice suggest that NFs can regulate ion channel function. Because Ca(2+) influx through voltage-gated Ca(2+) channels is required for hormone secretion in AP cells, and oestrogen regulates the expression of Ca(2+) channels in AP cells, the present study examined the expression of alpha1 subunits of voltage gated Ca(2+) channels in relation to that of NF68. Using quantitative immunofluorescence, we demonstrate that alpha 1C and alpha 1D subunits are abundantly expressed in female AP cells, alpha 1A subunits are moderately expressed, and alpha 1G and alpha 1B subunits are expressed at the lowest levels. Double-immunostaining showed that NF68 expression is not correlated with that of alpha 1C, alpha 1D or alpha 1B. Expression of alpha 1G and NF68 appear to be mutually exclusive from each other. Moreover, alpha 1A subunit and NF68 expression are significantly correlated and alpha 1A immunoreactivity is sexually dimorphic (i.e. low in males and high in females) and its levels of expression vary during the oestrous cycle, similar to NF68. Finally, omega-agatoxin IVA, a specific blocker of P/Q type Ca(2+) currents that are a result of the activity of alpha 1A subunits, inhibited to a greater extent spontaneous [Ca(2+)](i) fluctuations in AP cells from females in oestrous and dioestrous, whereas cells from females in pro-oestrous and males were less affected by this toxin. These results suggest a preferential participation of P/Q-type Ca(2+) channels and hence alpha 1A subunits, in regulating spontaneous Ca(2+) transients in AP cells under conditions where the proportion of NF68-expressing cells is high. It remains to be determined whether the expression of NF68 affects that of alpha 1A Ca(2+) channel subunits or vice versa.
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Canales de Calcio Tipo P/metabolismo , Canales de Calcio Tipo Q/metabolismo , Inmunohistoquímica , Proteínas del Tejido Nervioso/metabolismo , Proteínas de Neurofilamentos/metabolismo , Subunidades de Proteína/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio Tipo N , Canales de Calcio Tipo P/genética , Canales de Calcio Tipo Q/genética , Ciclo Estral/fisiología , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Subunidades de Proteína/genéticaRESUMEN
The plant alkaloid ryanodine (Ry) is a high-affinity modulator of ryanodine receptor (RyR) Ca(2+) release channels. Although these channels are present in a variety of cell types, their functional role in nerve cells is still puzzling. Here, a monosubstituted fluorescent Ry analogue, B-FL-X Ry, was used to reveal the distribution of RyRs in cultured rat sympathetic neurons. B-FL-X Ry competitively inhibited the binding of [3H]Ry to rabbit skeletal muscle SR membranes, with an IC(50) of 150 nM, compared to 7 nM of unlabeled Ry. Binding of B-FL-X Ry to the cytoplasm of sympathetic neurons is saturable, reversible and of high affinity. The pharmacology of B-FL-X Ry showed marked differences with unlabeled Ry, which are partially explained by its lower affinity: (1) use-dependent reversible inhibition of caffeine-induced intracellular Ca(2+) release; (2) diminished voltage-gated Ca(2+) influx, due to a positive shift in the activation of voltage gated Ca(2+) currents. B-FL-X Ry-stained sympathetic neurons, viewed under confocal microscopy, showed conspicuous labeling of crescent-shaped structures pertaining to the Golgi complex, a conclusion supported by experiments showing co-localization with Golgi-specific fluorescent probes and the breaking up of crescent-shaped staining after treatment with drugs that disassemble Golgi complex. The presence of RyRs to the Golgi could be confirmed with specific anti-RyR(2) antibodies, but evidence of caffeine-induced Ca(2+) release from this organelle could not be obtained using fast confocal microscopy. Rather, an apparent decrease of the cytosolic Ca(2+) signal was detected close to this organelle. In spite of that, short-term incubation with brefeldin A (BFA) suppressed the fast component of caffeine-induced Ca(2+) release, and the Ca(2+) release process lasted longer and appeared less organized. These observations, which suggest a possible role of the Golgi complex in Ca(2+) homeostasis and signaling in nerve cells, could be relevant to reports involving derangement of the Golgi complex as a probable cause of some forms of progressive neuronal degeneration, such as Alzheimer's disease and amyotrophic lateral sclerosis.
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Señalización del Calcio/fisiología , Aparato de Golgi/metabolismo , Neuronas/fisiología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Rianodina/análogos & derivados , Rianodina/metabolismo , Animales , Antibacterianos/farmacología , Especificidad de Anticuerpos , Compuestos de Boro/metabolismo , Brefeldino A/farmacología , Cafeína/farmacología , Calcio/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Colorantes Fluorescentes , Aparato de Golgi/química , Macrólidos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas/química , Neuronas/ultraestructura , Inhibidores de Fosfodiesterasa/farmacología , Ratas , Canal Liberador de Calcio Receptor de Rianodina/análisis , Canal Liberador de Calcio Receptor de Rianodina/inmunología , Ganglio Cervical Superior/citologíaRESUMEN
During spermatogenesis the activity of intracellular Ca(2+)-release channels is likely to play an important role in different specific cellular functions. Accordingly, messenger RNAs for the three inositol 1,4,5-trisphosphate receptor (IP3R) subtypes were found to be present throughout spermatogenesis. Immunocytochemical analysis revealed distinct distribution patterns of the mature IP3Rs during sperm differentiation. At early stages, IP3Rs are distributed throughout the cytoplasm, and as differentiation proceeds they become selectively localised to the Golgi complex. Consistently, spermatogonia underwent large intracellular Ca2+ release in response to thapsigargin (TG), while smaller responses were detected in late spermatocytes and spermatids. The distribution of IP3Rs and the larger Ca(2+)-release responses found in spermatogonia, suggest that IP3Rs may be involved in cell proliferation at this stage. This notion is supported by our observations in a spermatogenic cell line that depletion of intracellular Ca2+ pools using TG inhibits cell division, and that incubation with an IP3R-I antisense oligonucleotide completely inhibited proliferation. Furthermore, the three genes encoding ryanodine receptor proteins (RyRs) are expressed at all stages of spermatogenesis. However, immunocytochemical studies with specific antibodies against each of the RyR subtypes detected types 1 and 3 in spermatogenic cells and only type 3 in mature sperm. In contrast to IP3Rs, RyRs remain scattered in the cytoplasm throughout differentiation. Functional responses to caffeine and ryanodine were absent in spermatogenic cells and in mature sperm. These findings suggest that IP3Rs have significantly more important roles in spermatogenesis than RyRs, and that one of these roles is crucial for cell proliferation.
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Canales de Calcio/aislamiento & purificación , Señalización del Calcio , Receptores Citoplasmáticos y Nucleares/aislamiento & purificación , Canal Liberador de Calcio Receptor de Rianodina/aislamiento & purificación , Espermatogénesis , Animales , Canales de Calcio/genética , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Compartimento Celular , Diferenciación Celular , División Celular/efectos de los fármacos , Epidídimo/citología , Inmunohistoquímica , Indoles/farmacología , Receptores de Inositol 1,4,5-Trifosfato , Masculino , Ratones , Oligonucleótidos Antisentido/farmacología , Reacción en Cadena de la Polimerasa , ARN Mensajero/aislamiento & purificación , Receptores Citoplasmáticos y Nucleares/genética , Canal Liberador de Calcio Receptor de Rianodina/genética , Espermátides/fisiología , Espermatogonias/fisiología , Tapsigargina/farmacologíaRESUMEN
There is pharmacological evidence that Ca2+ channels play an essential role in triggering the mammalian sperm acrosome reaction, an exocytotic process required for sperm to fertilize the egg. Spermatozoa are small terminally differentiated cells that are difficult to study by conventional electrophysiological techniques. To identify the members of the voltage-dependent Ca2+ channel family possibly present in sperm, we have looked for the expression of the alpha 1A, alpha 1B, alpha 1C, alpha 1D and alpha 1E genes in mouse testis and in purified spermatogenic cell populations with RT-PCR. Our results indicate that all 5 genes are expressed in mouse testis, and in contrast only alpha 1E, and to a minor extent alpha 1A, are expressed in spermatogenic cells. In agreement with these findings, only T-type Ca2+ channels sensitive to the dihydropyridine nifedipine were observed in patch-clamp recordings of pachytene spermatocytes. These results suggest that low-threshold Ca2+ channels are the dihydropyridine-sensitive channels involved in the sperm acrosome reaction.