Effects of PKC alpha activation on Ca2+ pump and K(Ca) channel in deoxygenated sickle cells.
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.
Fathallah
H
H
Unité de Recherches Associée 1116, Centre National de la Recherche Scientifique, Université Paris XI, Orsay, France.
Sauvage
M
M
Romero
J R
JR
Canessa
M
M
Giraud
F
F
D000755:Anemia, Sickle Cell / Q000097:blood / Q000201:enzymology*; D016718:Arachidonic Acid / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000097:blood / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000097:blood*; D002848:Chromatography, DEAE-Cellulose; D003600:Cytosol / Q000378:metabolism; D004075:Diglycerides / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000097:blood* / Q000302:isolation & purification; D007700:Kinetics; D008970:Molecular Weight; D011493:Protein Kinase C / Q000097:blood* / Q000302:isolation & purification; D051571:Protein Kinase C-alpha; D012016:Reference Values; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D004075:Diglycerides; D007527:Isoenzymes; D016718:Arachidonic Acid; D000001:Calcimycin; D004533:Egtazic Acid; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D000252:Calcium-Transporting ATPases; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium; C013965:diolein