RESUMEN
A role for acid-sensing ion channels (ASICs) to serve as epithelial channels for Na(+) uptake by the gill of freshwater rainbow trout was investigated. We found that the ASIC inhibitors 4',6-diamidino-2-phenylindole and diminazene decreased Na(+) uptake in adult rainbow trout in a dose-dependent manner, with IC50 values of 0.12 and 0.96 µM, respectively. Furthermore, we cloned the trout ASIC1 and ASIC4 homologs and demonstrated that they are expressed differentially in the tissues of the rainbow trout, including gills and isolated mitochondrion-rich cells. Immunohistochemical analysis using custom-made anti-zASIC4.2 antibody and the Na(+)-K(+)-ATPase (α5-subunit) antibody demonstrated that the trout ASIC localizes to Na(+)/K(+)-ATPase-rich cells in the gill. Moreover, three-dimensional rendering of confocal micrographs demonstrated that ASIC is found in the apical region of mitochondrion-rich cells. We present a revised model whereby ASIC4 is proposed as one mechanism for Na(+) uptake from dilute freshwater in the gill of rainbow trout.
Asunto(s)
Canales Iónicos Sensibles al Ácido/metabolismo , Branquias/metabolismo , Oncorhynchus mykiss/metabolismo , Sodio/metabolismo , Bloqueadores del Canal Iónico Sensible al Ácido/farmacología , Canales Iónicos Sensibles al Ácido/biosíntesis , Canales Iónicos Sensibles al Ácido/farmacocinética , Amilorida/análogos & derivados , Amilorida/farmacología , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Transporte Biológico Activo , Clonación Molecular , Diminazeno/farmacología , Indoles/farmacología , Alineación de Secuencia , ATPasa Intercambiadora de Sodio-Potasio/inmunología , Tripanocidas/farmacologíaRESUMEN
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Acid-sensing ion channel 1a (ASIC1a) and 2a (ASIC2a) subunits are widely expressed throughout mammalian central nervous system. Activation of Ca2+-permeable ASIC1a homomultimers is largely responsible for acidosis-mediated, glutamate receptor-independent, ischemic neuronal injury. The function of ASIC2a in brain ischemia is less known except that transient global ischemia induces ASIC2a protein expression up-regulation in neurons that survived ischemia. Acidosis is assumed to play a critical role in brain ischemia injury. In the present experiment, rat C6 neuroglioma cells were used to explore the function of ASIC2a. MTT and relative LDH release assay revealed that knockdown of ASIC2a could aggravate the acidosis-induced injury of C6 cells. Through changing extracellular Ca2+ concentration and measuring intracellular calcium fluorescence intensity, it was found that aggravated damage was due to toxic Ca2+ overload via ASICs mechanisms. The current results indicated that, different from ASIC1a, ASIC2a probably played a protective role against the injury induced by extracellular acidosis in C6 cells (AU)