RESUMEN
We have previously shown that despite the presence of mRNA encoding CFTR, renal proximal cells do not exhibit cAMP-sensitive Cl(-) conductance (Rubera I, Tauc M, Bidet M, Poujeol C, Cuiller B, Watrin A, Touret N, Poujeol P. Am J Physiol Renal Physiol 275: F651-F663, 1998). Nevertheless, in these cells, CFTR plays a crucial role in the control of the volume-sensitive outwardly rectifying (VSOR) activated Cl(-) currents during hypotonic shock. The aim of this study was to determine the role of CFTR in the regulation of apoptosis volume decrease (AVD) and the apoptosis phenomenon. For this purpose, renal cells were immortalized from primary cultures of proximal convoluted tubules from cftr(+/+) and cftr(-/-) mice. Apoptosis was induced by staurosporine (STS; 1 microM). Cell volume, Cl(-) conductance, caspase-3 activity, intracellular level of reactive oxygen species (ROS), and glutathione content (GSH/GSSG) were monitored during AVD. In cftr(+/+) cells, AVD and caspase-3 activation were strongly impaired by conventional Cl(-) channel blockers and by a specific CFTR inhibitor (CFTR(inh)-172; 5 microM). STS induced activation of CFTR conductance within 15 min, which was progressively replaced by VSOR Cl(-) currents after 60 min of exposure. In parallel, STS induced an increase in ROS content in the time course of VSOR Cl(-) current activation. This increase was impaired by CFTR(inh)-172 and was not observed in cftr(-/-) cells. Furthermore, the intracellular GSH/GSSG content decreased during STS exposure in cftr(+/+) cells only. In conclusion, CFTR could play a key role in the cascade of events leading to apoptosis. This role probably involves control of the intracellular ROS balance by some CFTR-dependent modulation of GSH concentration.
Asunto(s)
Apoptosis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Glutatión/metabolismo , Túbulos Renales Proximales/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Anticuerpos Monoclonales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Muerte Celular , Línea Celular Transformada , Canales de Cloruro/metabolismo , Canales de Cloruro/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/deficiencia , Regulador de Conductancia de Transmembrana de Fibrosis Quística/efectos de los fármacos , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , ADN Complementario , Regulación hacia Abajo , Conductividad Eléctrica , Activación Enzimática/efectos de los fármacos , Disulfuro de Glutatión/metabolismo , Humanos , Túbulos Renales Proximales/citología , Ratones , Ratones Noqueados , Estaurosporina/farmacología , Transfección , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The aim of this study was to characterize the role of CFTR during Cd(2+)-induced apoptosis. For this purpose primary cultures and cell lines originated from proximal tubules (PCT) of wild-type cftr(+/+) and cftr(-/-) mice were used. In cftr(+/+) cells, the application of Cd(2+) (5 microM) stimulated within 8 min an ERK1/2-activated CFTR-like Cl(-) conductance sensitive to CFTR(inh)-172. Thereafter Cd(2+) induced an apoptotic volume decrease (AVD) within 6 h followed by caspase-3 activation and apoptosis. The early increase in CFTR conductance was followed by the activation of volume-sensitive outwardly rectifying (VSOR) Cl(-) and TASK2 K(+) conductances. By contrast, cftr(-/-) cells exposed to Cd(2+) were unable to develop VSOR currents, caspase-3 activity, and AVD process and underwent necrosis. Moreover in cftr(+/+) cells, Cd(2+) enhanced reactive oxygen species (ROS) production and induced a 50% decrease in total glutathione content (major ROS scavenger in PCT). ROS generation and glutathione decrease depended on the presence of CFTR, since they did not occur in the presence of CFTR(inh)-172 or in cftr(-/-) cells. Additionally, Cd(2+) exposure accelerates effluxes of fluorescent glutathione S-conjugate in cftr(+/+) cells. Our data suggest that CFTR could modulate ROS levels to ensure apoptosis during Cd(2+) exposure by modulating the intracellular content of glutathione.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Fibrosis Quística/fisiopatología , Glutatión/metabolismo , Túbulos Renales Proximales/fisiología , Especies Reactivas de Oxígeno/metabolismo , Animales , Apoptosis/efectos de los fármacos , Benzoatos/farmacología , Cadmio/toxicidad , Caspasa 3/metabolismo , Técnicas de Cultivo de Célula , Línea Celular Transformada , Tamaño de la Célula , Fibrosis Quística/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Túbulos Renales Proximales/efectos de los fármacos , Túbulos Renales Proximales/patología , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos CFTR , Técnicas de Placa-Clamp , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Transducción de Señal/efectos de los fármacos , Tiazolidinas/farmacologíaRESUMEN
Apoptotic volume decrease (AVD) is prerequisite to apoptotic events that lead to cell death. In a previous study, we demonstrated in kidney proximal cells that the TASK2 channel was involved in the K+ efflux that occurred during regulatory volume decrease. The aim of the present study was to determine the role of the TASK2 channel in the regulation of AVD and apoptosis phenomenon. For this purpose renal cells were immortalized from primary cultures of proximal convoluted tubules (PCT) from wild type and TASK2 knock-out mice (task2-/-). Apoptosis was induced by staurosporine, cyclosporin A, or tumor necrosis factor alpha. Cell volume, K+ conductance, caspase-3, and intracellular reactive oxygen species (ROS) levels were monitored during AVD. In wild type PCT cells the K+ conductance activated during AVD exhibited characteristics of TASK2 currents. In task2-/- PCT cells, AVD and caspase activation were reduced by 59%. Whole cell recordings indicated that large conductance calcium-activated K+ currents inhibited by iberiotoxin (BK channels) partially compensated for the deletion of TASK2 K+ currents in the task2-/- PCT cells. This result explained the residual AVD measured in these cells. In both cell lines, apoptosis was mediated via intracellular ROS increase. Moreover AVD, K+ conductances, and caspase-3 were strongly impaired by ROS scavenger N-acetylcysteine. In conclusion, the main K+ channels involved in staurosporine, cyclosporin A, and tumor necrosis factor-alpha-induced AVD are TASK2 K+ channels in proximal wild type cells and iberiotoxin-sensitive BK channels in proximal task2-/- cells. Both K+ channels could be activated by ROS production.
Asunto(s)
Apoptosis/fisiología , Tamaño de la Célula , Túbulos Renales Proximales/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Acetilcisteína/farmacología , Animales , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Caspasa 3/metabolismo , Línea Celular Transformada , Tamaño de la Célula/efectos de los fármacos , Ciclosporina/farmacología , Inhibidores Enzimáticos/farmacología , Depuradores de Radicales Libres/farmacología , Túbulos Renales Proximales/citología , Ratones , Ratones Noqueados , Péptidos/farmacología , Potasio/metabolismo , Estaurosporina/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Several papers reported the role of TASK2 channels in cell volume regulation and regulatory volume decrease (RVD). To check the possibility that the TASK2 channel modulates the RVD process in kidney, we performed primary cultures of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) from wild-type and TASK2 knockout (KO) mice. In KO mice, the TASK2 coding sequence was in part replaced by the lac-Z gene. This allows for the precise localization of TASK2 in kidney sections using beta-galactosidase staining. TASK2 was only localized in PCT cells. K+ currents were analyzed by the whole-cell clamp technique with 125 mM K-gluconate in the pipette and 140 mM Na-gluconate in the bath. In PCT cells from wild-type mice, hypotonicity induced swelling-activated K+ currents insensitive to 1 mM tetraethylammonium, 10 nM charybdotoxin, and 10 microM 293B, but blocked by 500 microM quinidine and 10 microM clofilium. These currents were increased in alkaline pH and decreased in acidic pH. In PCT cells from TASK2 KO, swelling-activated K+ currents were completely impaired. In conclusion, the TASK2 channel is expressed in kidney proximal cells and could be the swelling-activated K+ channel responsible for the cell volume regulation process during osmolyte absorptions in the proximal tubules.
Asunto(s)
Túbulos Renales Proximales/citología , Túbulos Renales Proximales/metabolismo , Potenciales de la Membrana/fisiología , Ósmosis/fisiología , Canales de Potasio de Dominio Poro en Tándem , Canales de Potasio/metabolismo , Animales , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Soluciones Hipotónicas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Presión Osmótica , Canales de Potasio/deficienciaRESUMEN
The role of cystic fibrosis transmembrane conductance regulator (CFTR) in the control of Cl(-) currents was studied in mouse kidney. Whole cell clamp was used to analyze Cl(-) currents in primary cultures of proximal and distal convoluted and cortical collecting tubules from wild-type (WT) and cftr knockout (KO) mice. In WT mice, forskolin activated a linear Cl(-) current only in distal convoluted and cortical collecting tubule cells. This current was not recorded in KO mice. In both mice, Ca(2+)-dependent Cl(-) currents were recorded in all segments. In WT mice, volume-sensitive Cl(-) currents were implicated in regulatory volume decrease during hypotonicity. In KO mice, regulatory volume decrease and swelling-activated Cl(-) current were impaired but were restored by adenosine perfusion. Extracellular ATP also restored swelling-activated Cl(-) currents. The effect of ATP or adenosine was blocked by 8-cyclopentyl-1,3-diproxylxanthine. The ecto-ATPase inhibitor ARL-67156 inhibited the effect of hypotonicity and ATP. Finally, in KO mice, volume-sensitive Cl(-) currents are potentially functional, but the absence of CFTR precludes their activation by extracellular nucleosides. This observation strengthens the hypothesis that CFTR is a modulator of ATP release in epithelia.
Asunto(s)
Canales de Cloruro/metabolismo , Cloruros/metabolismo , AMP Cíclico/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/deficiencia , Nefronas/metabolismo , Adenosina/farmacología , Animales , Calcio/farmacología , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas , Colforsina/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Inhibidores Enzimáticos/farmacología , Soluciones Hipotónicas/farmacología , Túbulos Renales/citología , Túbulos Renales/efectos de los fármacos , Túbulos Renales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CFTR , Ratones Noqueados , Mutación , Nefronas/citología , Nefronas/efectos de los fármacos , Técnicas de Placa-Clamp , TransfecciónRESUMEN
The role of CFTR in the control of K(+) currents was studied in mouse kidney. Whole cell clamp was used to identify K(+) currents on the basis of pharmacological sensitivities in primary cultures of proximal (PCT) and distal convoluted tubule (DCT) and cortical collecting tubule (CCT) from wild-type (WT) and CFTR knockout (KO) mice. In DCT and CCT cells, forskolin activated a 293B-sensitive K(+) current in WT, but not in KO, mice. In these cells, a hypotonic shock induced K(+) currents blocked by charybdotoxin in WT, but not in KO, mice. In PCT cells from WT and KO mice, the hypotonicity-induced K(+) currents were insensitive to these toxins and were activated at extracellular pH 8.0 and inhibited at pH 6.0, suggesting that the corresponding channel was TASK2. In conclusion, CFTR is implicated in the control of KCNQ1 and Ca(2+)-sensitive swelling-activated K(+) conductances in DCT and CCT, but not in proximal convoluted tubule, cells. In KO mice, impairment of the regulatory volume decrease process in DCT and CCT could be due to the loss of an autocrine mechanism, implicating ATP and adenosine, which controls swelling-activated Cl(-) and K(+) channels.