RESUMEN
The role of the phospholemman (PLM) on the efflux of taurine and chloride induced by swelling was studied in HEK293 cells overexpressing stable transfected PLM. PLM, a substrate for protein kinases C and A, is a protein that induces an anion current in Xenopus oocytes and forms taurine-selective channels in lipid bilayers. Taurine contributes as an osmolyte to regulatory volume decrease (RVD) and is highly permeable through PLM channels in bilayers. In PLM-overexpressing cells the process of RVD was more rapid and efficient (75%) than in control cells (44%). Also, [(3)H]taurine and (125)I efflux induced by hyposmolarity were markedly increased (30-100%) in two subclones of cells overexpressing PLM. This increased efflux was sensitive to the Cl channel blockers DDF, NPPB and DIDS. Acute treatment of control cells with isoproterenol and norepinephrine induced a significant potentiation (50-60%) of [(3)H]taurine release induced by hyposmolarity. In PLM-overexpressing cells the potentiation by these drugs was higher (100%). Insulin induced also an increase in [(3)H]taurine release, but only in PLM-overexpressing cells (50%). These results indicate that PLM may play a role in the RVD and that its phosphorylation may have a physiological significance during this process. The mechanisms involved in this process could include the activation of PLM itself as channel or the modulation of other preexisting channels.
Asunto(s)
Canales Iónicos/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Taurina/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Línea Celular , Tamaño de la Célula/efectos de los fármacos , Colforsina/análogos & derivados , Colforsina/farmacología , Regulación hacia Abajo , Humanos , Radioisótopos de Yodo , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Nitrobenzoatos/farmacología , Concentración Osmolar , Fosfoproteínas/biosíntesis , Fosfoproteínas/genética , Taurina/análisis , Transfección , TritioRESUMEN
Organic osmolyte and halide permeability pathways activated in epithelial HeLa cells by osmotically induced cell swelling were studied using electrophysiological and radiotracer efflux techniques. On hypotonic challenge, HeLa cells responded by activating an efflux pathway for [3H]taurine and a swelling-induced outwardly rectifying Cl- channel. Removal of extracellular Cl-, or its replacement by a less permeable anion, enhanced taurine efflux and decreased the inward current (Cl- efflux). The effect of Cl- removal on taurine efflux was not a consequence of changes in membrane potential. The degree of deactivation of the Cl- current at depolarized potentials was also Cl- dependent, suggesting that external Cl- is necessary for channel activity. The Cl- channel inhibitors 1,9-dideoxyforskolin, tamoxifen, and 4,4'- diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) inhibited swelling-activated taurine efflux, with DIDS being the most potent, at variance with the sensitivity of the Cl- channel. DIDS effect was dependent on external Cl-; concentrations of DIDS that inhibited 50% of taurine efflux were 0.2 and 4 microM at low and high Cl-, respectively. The results could be interpreted on the basis of separate pathways for swelling-activated taurine efflux and Cl- current differentially affected by Cl-. Alternatively, taurine and Cl- flux might occur through a common channel, with the two solutes interacting within the pore and being affected differentially by Cl- replacement.
Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Canales de Cloruro/fisiología , Cloruros/metabolismo , Cloruros/farmacología , Taurina/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Canales de Cloruro/antagonistas & inhibidores , Colforsina/análogos & derivados , Colforsina/farmacología , Células HeLa , Humanos , Cinética , Concentración Osmolar , Técnica de Dilución de Radioisótopos , Tamoxifeno/farmacología , Tritio , Equilibrio HidroelectrolíticoRESUMEN
Exposure of NIH/3T3 fibroblasts not expressing P-glycoprotein to 50, 30, 20, and 10% hyposmotic solutions led to cell volume increases of 70, 32, 21, and 12%, respectively. After swelling, NIH/3T3 cells exhibited regulatory volume decrease (RVD), attaining complete volume recovery after 30 min except in 50% hyposmotic solution, in which volume recovery was 76%. RVD was accelerated by gramicidin and inhibited by the Cl channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoic acid, 1,9-dideoxyforskolin, dipyridamole, and niflumic acid and by the K channel, blocker quinidine. RVD was reduced 15% by removal of extracellular Ca. The pathway opened by hypotonicity was highly permeable to K and Rb and only partly permeable to other cations. Most anions were able to permeate, with a permeability ranking of nitrate > benzoate = iodide > thiocyanate > chloride > > gluconate. The pathway was permeable to neutral amino acids, with a permeability ranking of glycine > alanine > glutamate > taurine > gamma-aminobutyric acid > glutamine. The pathway was not permeable to basic amino acids. These results show that, despite the absence of P-glycoprotein, NIH/3T3 cells exhibit RVD with properties similar to those expressed in most cell types.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Aniones/metabolismo , Canales de Cloruro/fisiología , Cloruros/metabolismo , Equilibrio Hidroelectrolítico/fisiología , Células 3T3 , 4-Aminopiridina/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/análisis , Animales , Bario/farmacología , Permeabilidad de la Membrana Celular , Canales de Cloruro/antagonistas & inhibidores , Cloruros/farmacología , Colchicina/toxicidad , Colforsina/análogos & derivados , Colforsina/farmacología , Dipiridamol/farmacología , Gramicidina/farmacología , Cinética , Ratones , Ácido Niflúmico/farmacología , Nitrobenzoatos/farmacología , Bloqueadores de los Canales de Potasio , Canales de Potasio/fisiología , Quinidina/farmacología , Tetraetilamonio , Compuestos de Tetraetilamonio/farmacología , Factores de Tiempo , Equilibrio Hidroelectrolítico/efectos de los fármacosRESUMEN
The osmolyte function of amino acids and Cl in native NIH/3T3 cells not expressing the P-glycoprotein was examined by investigating the free amino acid concentration and the swelling-activated efflux of [3H]taurine, as representative of amino acids, and of 125I, as a tracer for Cl. Taurine and 125I efflux was activated by 20 and 30% hyposmotic solutions. At 50% hyposmotic solutions, the osmolyte pool was essentially depleted. The Cl channel blockers 5-nitro-2-(3-phenylpropyl-amino)benzoic acid, 1,9-dideoxyforskolin, dipyridamole, and niflumic acid inhibited the release of the two osmolytes by 80-95%. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (400 microM) decreased the efflux of taurine 80% without affecting that of 125I. Linolenic and arachidonic acids (5-20 microM) showed a concentration-dependent inhibitory effect on taurine and 125I fluxes. Omission of Ca decreased osmolyte fluxes by 16%. Verapamil inhibited the osmolyte release only at 500 microM. Nimodipine at 25 and 50 microM decreased the release of [3H]taurine and 125I by approximately 60 and 80%, respectively, but this effect was independent of the presence of extracellular Ca. These results indicate that amino acids and Cl function as osmolytes during regulatory volume decrease in native NIH/ 3T3 cells.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Aminoácidos/metabolismo , Canales de Cloruro/fisiología , Cloruros/metabolismo , Taurina/metabolismo , Equilibrio Hidroelectrolítico/fisiología , Células 3T3 , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/fisiología , Canales de Cloruro/antagonistas & inhibidores , Colforsina/análogos & derivados , Colforsina/farmacología , Dipiridamol/farmacología , Ácidos Grasos no Esterificados/farmacología , Líquido Intracelular/fisiología , Radioisótopos de Yodo , Cinética , Ratones , Ácido Niflúmico/farmacología , Nitrobenzoatos/farmacología , Concentración OsmolarRESUMEN
Forskolin, a naturally occurring activator of adenylate cyclase, inhibits total and high-affinity cyclic AMP phosphodiesterase activity in soluble and particulate fractions of cultured LLC-PK1 renal epithelial cells. The naturally occurring forskolin analogue 1,9-dideoxyforskolin, which does not stimulate adenylate cyclase activity, is a more potent inhibitor of cyclic AMP phosphodiesterase activity than forskolin. To clarify the structural feature of the forskolin molecule responsible for inhibition of cyclic AMP phosphodiesterase activity, the effects of two agents which share structural identity with portions of the forskolin ring were tested. The steroid 5-pregnenolone, but not the hexose alpha-D-galactose, inhibited cyclic AMP phosphodiesterase activity in LLC-PK1 cells. Forskolin and 1,9-dideoxyforskolin both stimulate protein kinase C activity in LLC-PK1 cells. The effect of 1,9-dideoxyforskolin in stimulating LLC-PK1 protein kinase C activity can be attenuated by staurosporine. Both 5-pregnenolone and alpha-D-galactose also stimulate protein kinase C activity in LLC-PK1 cells. 5-Pregnenolone and the phorbol ester phorbol 12-myristate 13-acetate cause translocation of protein kinase C from a soluble to a particulate fraction, while both 1,9-dideoxyforskolin and alpha-D-galactose increase protein kinase C activity in both soluble and particulate fractions. Our results demonstrate that forskolin exerts diverse enzymic effects in cultured LLC-PK1 cells.