Asunto(s)
Enfermedades Renales Quísticas/diagnóstico , Amaurosis Congénita de Leber/diagnóstico , Atrofias Ópticas Hereditarias/diagnóstico , Ciliopatías , Diagnóstico Precoz , Exoma , Enfermedades Hereditarias del Ojo/diagnóstico , Enfermedades Hereditarias del Ojo/genética , Femenino , Estudios de Seguimiento , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Lactante , Enfermedades Renales Quísticas/genética , Amaurosis Congénita de Leber/genética , Masculino , Atrofias Ópticas Hereditarias/genética , Distrofias Retinianas/diagnóstico , Distrofias Retinianas/genética , Análisis de Secuencia de ADN/métodosRESUMEN
We have generated and characterised a clone of chicken DT40 lymphocytes stably transfected with the rat P2X(7) receptor (rP2X(7)). Successful transfection was confirmed by Western blotting. Under voltage clamp, P2X(7)-expressing cells responded to ATP and dibenzoyl-ATP (Bz-ATP) (a more potent P2X(7) receptor agonist) with a rapidly activating and sustained inward current. The EC(50) values for these agonists were 305 and 15 microM, respectively. Bz-ATP evoked Ca(2+) and Mn(2+) influx into transfected cells as determined by Fura-2 spectrofluorimetry. Responses to Bz-ATP were inhibited by pre-treatment of cells with oxidised ATP. Treatment of cells with Bz-ATP for up to 24hr produced time- and concentration-dependent cell death. This was associated with an increase in caspase-3-like activity, exposure of phosphatidylserine on the outside of cell membrane and DNA cleavage, indicating death by apoptosis. Pre-treatment with Z-VAD-fmk, a pan-caspase inhibitor, reduced the DNA fragmentation and phosphatidylserine externalisation, but did not affect overall rates of cell death at 24hr, implicating caspase-independent mechanisms. The properties of rP2X(7) receptors expressed in DT40 cells are similar to those described for other expression systems. Because DT40 cells lack functionally detectable endogenous P2 receptors and are highly amenable to genetic manipulation, stably transfected DT40 cells provide a novel and potentially useful model system in which to investigate the intracellular signal transduction pathways associated with P2X(7) receptor stimulation, in particular those involved in induction of cell death.
Asunto(s)
Apoptosis/fisiología , Receptores Purinérgicos P2/fisiología , Animales , Calcio/metabolismo , Caspasa 3 , Caspasas/metabolismo , Muerte Celular/fisiología , Células Cultivadas , Pollos , Electrofisiología , Activación Enzimática , Permeabilidad , Agonistas del Receptor Purinérgico P2 , Ratas , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X7 , TransfecciónRESUMEN
P2X(7), a purinergic receptor, is expressed in renal collecting ducts as they undergo fulminant cystogenesis in the cpk/cpk mouse model of autosomal recessive polycystic kidney disease (ARPKD). Dissociated cpk/cpk kidneys generate cysts from cell aggregates within 24h of suspension culture and we demonstrate that BzATP, a P2X(7) agonist, reduces cystogenesis. This effect is P2X(7)-specific, because: (i) equimolar concentrations of other purinergic agonists, ATP and UTP, had lesser effects and (ii) the P2X(7) inhibitor, oxidized ATP, abrogated the BzATP-mediated reduction in cystogenesis. BzATP did not significantly affect total cell number, proliferation, LDH release or caspase 3 activity, and zVAD-fmk, a caspase blocker, failed to modulate BzATP effects. In addition, this P2X(7) agonist did not significantly alter cyst size, probably excluding altered vectorial transport. In vivo, ATP was detected in cyst fluid from cpk/cpk kidneys; moreover, P2X(7) protein was also upregulated in human fetal ARPKD epithelia versus normal fetal collecting ducts. Thus, ATP may inhibit pathological renal cyst growth through P2X(7) signaling.