RESUMEN
The signaling cascade linking insulin receptor stimulation to the activation of Na/H exchanger (NHE) was investigated in human erythrocytes, a simple cell model expressing the NHE1 isoform and protein kinase C (PKC) alpha and zeta isoforms only. Our results demonstrate the presence of phosphatidylinositol (PtdIns) 3-kinase in these cells and its activation by insulin. With a similar time-course, insulin also promoted both the translocation and activation of PKC zeta, but had no effect on PKC alpha. Inhibition of PtdIns 3-kinase with wortmannin prevented the activation of PKC zeta by insulin. Stimulation of NHE1 was observed after 10 min of insulin treatment and persisted for at least 60 min. This effect was totally abolished by wortmannin or GF 109203X, an inhibitor of all PKC isoforms, but not by Gö 6976, a specific inhibitor of conventional and novel PKCs (e.g. PKC alpha). These data indicate that PKC zeta activation is mediated by a PtdIns 3-kinase-dependent mechanism and that NHE1 stimulation involves the sequential activation of PtdIns 3-kinase and PKC zeta. In addition, insulin stimulation of NHE1 occurred without altering the phosphorylation state of the exchanger, suggesting that the phosphorylation of an ancillary protein by PKC zeta would be responsible for activation of the transporter.
Asunto(s)
Eritrocitos/enzimología , Insulina/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Androstadienos/farmacología , Transporte Biológico/efectos de los fármacos , Carbazoles/farmacología , Activación Enzimática/efectos de los fármacos , Eritrocitos/citología , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Humanos , Indoles/farmacología , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Maleimidas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Fosfotirosina/metabolismo , Pruebas de Precipitina , Unión Proteica/efectos de los fármacos , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C-alfa , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , WortmaninaRESUMEN
KCl cotransport activated by swelling of sickle red blood cells (SS RBC)is inhibited by deoxygenation. Yet recent studies found a Cl--dependent increase in sickle reticulocyte density with cyclic deoxygenation. This study sought to demonstrate cotransporter stimulation by deoxygenation of SS RBC in isotonic media with normal pH. Low-density SS RBC exhibited a Cl--dependent component of the deoxygenation-induced net K+ efflux, which was blocked by two inhibitors of KCl cotransport, [(dihydroindenyl)oxy]alkanoic acid and okadaic acid. Cl--dependent K+ efflux stimulated by deoxygenation was enhanced 2.5-fold by clamping of cellular Mg2+ at the level in oxygenated cells using ionophore A-23187. Incubating cells in high external K+ or Rb+ minimized inhibition of KCl cotransport by internal Mg2+, and under these conditions deoxygenation markedly stimulated KCl cotransport in the absence of ionophore. Activation of KCl cotransport by deoxygenation of SS RBC in isotonic media at normal pH is consistent with the generalized dephosphorylation of membrane proteins induced by deoxygenation and activation of the cotransporter by a dephosphorylation mechanism. Na+/H+ exchange activity, known to be modulated by cytosolic Ca2+ elevation and cell shrinkage, remained silent under deoxygenation conditions.
Asunto(s)
Eritrocitos/metabolismo , Hemoglobina Falciforme , Cloruro de Potasio/metabolismo , Intercambiadores de Sodio-Hidrógeno/fisiología , Calcimicina/farmacología , Ácidos Carboxílicos/farmacología , Hipoxia de la Célula , Cloruros/fisiología , Eritrocitos/efectos de los fármacos , Humanos , Indenos/farmacología , Magnesio/metabolismo , Magnesio/fisiología , Ácido Ocadaico/farmacología , Ouabaína/farmacología , Potasio/metabolismo , Rubidio/metabolismo , Rubidio/fisiología , Sodio/metabolismo , Intercambiadores de Sodio-Hidrógeno/efectos de los fármacosRESUMEN
We have previously shown that a pretreatment with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), reduced deoxygenation-induced K+ loss and Ca2+ uptake and prevented cell dehydration in sickle anemia red blood cells (SS cells) (H. Fathallah, E. Coezy, R.-S. De Neef, M.-D. Hardy-Dessources, and F. Giraud. Blood 86: 1999-2007, 1995). The present study explores the detailed mechanism of this PMA-induced inhibition. The main findings are, first, the detection of PKC alpha and PKC zeta in normal red blood cells and the demonstration that both isoforms are expressed at higher levels in SS cells. The alpha-isoform only is translocated to the membrane and activated by PMA and by elevation of cytosolic Ca2+. Second, PMA is demonstrated to activate Ca2+ efflux in deoxygenated SS cells by a direct stimulation of the Ca2+ pump. PMA, moreover, inhibits deoxygenation-induced, charybdotoxin-sensitive K+ efflux in SS cells. This inhibition is partly indirect and explained by the reduced deoxygenation-induced rise in cytosolic Ca2+ resulting from Ca2+ pump stimulation. However, a significant inhibition of the Ca2+-activated K+ channels (K(Ca) channels) by PMA can also be demonstrated when the channels are activated by Ca2+ plus ionophore, under conditions in which the Ca2+ pump is operating near its maximal extrusion rate, but swamped by Ca2+ plus ionophore. The data thus suggest a PKC alpha-mediated phosphorylation both of the Ca2+ pump and of the K(Ca) channel or an auxiliary protein.
Asunto(s)
Anemia de Células Falciformes/enzimología , ATPasas Transportadoras de Calcio/sangre , Eritrocitos/enzimología , Isoenzimas/sangre , Proteína Quinasa C/sangre , Anemia de Células Falciformes/sangre , Ácido Araquidónico/farmacología , Calcimicina/farmacología , Calcio/sangre , Calcio/farmacología , Cromatografía DEAE-Celulosa , Citosol/metabolismo , Diglicéridos/farmacología , Ácido Egtácico/farmacología , Activación Enzimática , Membrana Eritrocítica/metabolismo , Humanos , Técnicas In Vitro , Isoenzimas/aislamiento & purificación , Cinética , Peso Molecular , Proteína Quinasa C/aislamiento & purificación , Proteína Quinasa C-alfa , Valores de Referencia , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Deoxygenation (DO) of sickle cell anemia red blood cells (SS cells) induces membrane permeabilization to Ca2+, Na+, and K+ and cell dehydration mostly through the activation of the Ca(2+)-dependent K+ channels. We show that DO of both SS cells and normal red blood cells was accompanied by a nonspecific dephosphorylation of membrane proteins. After treatment with a protein kinase C activator (phorbol myristate acetate) or a phosphoprotein phosphatase inhibitor (okadaic acid), the level of membrane protein phosphorylation in deoxygenated cells was maintained higher or equal, respectively, to that of the oxygenated controls. We found that these drugs in SS cells (1) inhibited by 40% the DO-stimulated net Ca2+ uptake, without affecting the DO-stimulated Ca2+ influx, suggesting that they activated the Ca2+ efflux; (2) slightly increased the DO-induced Na+ uptake and decreased the DO-induced K+ loss; and (3) prevented the DO-induced cell dehydration. Both drugs are known to stimulate both phosphorylation and activity of the Ca pump and of the Na/H antiport. Inhibition of SS cell dehydration might be due to an activation of the Ca pump preventing [Ca2+]i elevation responsible for the stimulation of the K+ channels and/or to an activation of the Na/H exchange resulting in cell water gain.