Protein kinase C mediates erythrocyte “programmed cell death” following glucose depletion

Abstract

depletion of erythrocytes leads to activation of Ca 2ϩ -permeable cation channels, Ca 2ϩ entry, activation of a Ca 2ϩ -sensitive erythrocyte scramblase, and subsequent exposure of phosphatidylserine at the erythrocyte surface. Ca 2ϩ entry into erythrocytes was previously shown to be stimulated by phorbol esters and to be inhibited by staurosporine and chelerythrine and is thus thought to be regulated by protein phosphorylation/dephosphorylation, presumably via protein kinase C (PKC) and the corresponding phosphoserine/threonine phosphatases. The present experiments explored whether PKC could contribute to effects of energy depletion on erythrocyte phosphatidylserine exposure and cell volume. Phosphatidylserine exposure was estimated from annexin binding and cell volume from forward scatter in fluorescence-activated cell sorter analysis. Removal of extracellular glucose led to depletion of cellular ATP, stimulated PKC activity, led to translocation of PKC␣, enhanced serine phosphorylation of membrane proteins, decreased cell volume, and increased annexin binding, the latter effect being blunted but not abolished in the presence of 1 M staurosporine or 50 nM calphostin C. The PKC stimulator phorbol-12-myristate-13-acetate (3 M) and the phosphatase inhibitor okadaic acid (1-10 M) mimicked the effect of glucose depletion and similarly led to translocation of PKC␣ and enhanced serine phosphorylation, increased annexin binding, and decreased forward scatter, the latter effects being abrogated by PKC inhibitor staurosporine (1 M). Fluo-3 fluorescence measurements revealed that okadaic acid also enhanced erythrocyte Ca 2ϩ activity. The present observations suggest that protein phosphorylation and dephosphorylation via PKC and the corresponding protein phosphatases contribute to phosphatidylserine exposure and cell shrinkage after energy depletion. cell volume; eryptosis; calcium; okadaic acid; staurosporine AS SHOWN RECENTLY, erythrocytes exposed to oxidative stress, osmotic shock, or glucose depletion activate a Ca 2ϩ -permeable cation channel (22,29,30,32)

Procoagulant properties of microparticles released from red blood cells in paroxysmal nocturnal haemoglobinuria

“…It is known that human RBC contain PKC, which mediates the phosphorylation of cytoskeletal proteins (Danilov & Cohen, 1989) and the human Na + /H + antiport (Bourikas et al, 2003). PKCa, which translocates to the RBC membrane upon stimulation with PMA, mediates PS exposure following glucose depletion of RBC (Klarl et al, 2006). In the case of platelet activation accompanied by MP release in response to calcium ionophore, the involvement of calpain-mediated proteolysis of cytoskeletal proteins was also suggested (Fox et al, 1990), but not in the response to C5b-9 (Wiedmer et al, 1990).…”

Erythrocyte programmed cell death

“…The stimulation of eryptosis by energy depletion involves activation of PKC and PKC-dependent phosphorylation of membrane proteins with subsequent phosphatidylserine exposure and cell shrinkage (82). The effects of energy depletion are mimicked by stimulation of PKC with phorbolesters or inhibition of protein phosphatases such as okadaic acid (82).…”

“…The effects of energy depletion are mimicked by stimulation of PKC with phorbolesters or inhibition of protein phosphatases such as okadaic acid (82). Protein kinase C (PKC) activation results in stimulation of erythrocyte Ca 21 entry (83) and phosphatidylserine exposure (84).…”

Stimulation of human red blood cells leads to Ca2+-mediated intercellular adhesion

“…The dependence on high Ca 2+ concentrations and evidence from further studies reporting enhanced aggregation of PS liposomes in the presence of polymers [42] suggest that additional membrane constituents in the RBC contribute to the aggregation process. This leads to other Ca 2+ -dependent proteins in RBCs, such as PKC α [43,44] or the nitric oxide synthase [45,46]. Further research is required to address the question of the molecular identity of the additional components in the adhesion process.…”