EFFECTS OF PKC-ALPHA ACTIVATION ON CA2-CA CHANNEL IN DEOXYGENATED SICKLE CELLS( PUMP AND K)

We have previously shown that a pretreatment with phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), reduced deo xygenation-induced K+ loss and Ca2+ uptake and prevented cell dehydrat ion in sickle anemia red blood cells (SS cells) (H. Fathallah, E. Coez y, R.-S. De Neef, M.-D. Hardy-Dessources, and F. Giraud. Blood 86: 199 9-2007, 1995). The present study explores the detailed mechanism of th is PMA-induced inhibition. The main findings are, first; the detection of PKC alpha and PKC zeta in normal red blood cells and the demonstra tion that both isoforms are expressed at higher levels in SS cells. Th e 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, char ybdotoxin-sensitive K+ efflux in SS cells. This inhibition is partly i ndirect and explained by the reduced deoxygenation-induced rise in cyt osolic Ca2+ resulting from Ca2+ pump stimulation. However, a significa nt 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 da ta thus suggest a PKC alpha-mediated phosphorylation both of the Ca2pump and of the K-Ca channel or an auxiliary protein.