Inhibition of deoxygenation-induced membrane protein dephosphorylation and cell dehydration by phorbol esters and okadaic acid in sickle cells

Abstract

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.