Wilson disease is caused by accumulation of Cu(2+) in cells, which results in liver cirrhosis and, occasionally, anemia. Here, we show that Cu(2+) triggers hepatocyte apoptosis through activation of acid sphingomyelinase (Asm) and release of ceramide. Genetic deficiency or pharmacological inhibition of Asm prevented Cu(2+)-induced hepatocyte apoptosis and protected rats, genetically prone to develop Wilson disease, from acute hepatocyte death, liver failure and early death. Cu(2+) induced the secretion of activated Asm from leukocytes, leading to ceramide release in and phosphatidylserine exposure on erythrocytes, events also prevented by inhibition of Asm. Phosphatidylserine exposure resulted in immediate clearance of affected erythrocytes from the blood in mice. Accordingly, individuals with Wilson disease showed elevated plasma levels of Asm, and displayed a constitutive increase of ceramide- and phosphatidylserine-positive erythrocytes. Our data suggest a previously unidentified mechanism for liver cirrhosis and anemia in Wilson disease.

Similar to apoptosis of nucleated cells, suicidal erythrocyte death or eryptosis is characterized by cell shrinkage, membrane blebbing and membrane phospholipid scrambling with phosphatidylserine exposure at the cell surface. Signaling of eryptosis involves formation of prostaglandin E₂ with subsequent activation of cation channels and Ca²⁺-entry and/or release of platelet activating factor (PAF) with subsequent activation of sphingomyelinase and formation of ceramide. Ca²⁺ and ceramide stimulate cell membrane scrambling. Ca²⁺ further activates Ca²⁺-sensitive K⁺-channels leading to cellular KCl loss and cell shrinkage and stimulates the protease calpain resulting in degradation of the cytoskeleton. Injuries triggering eryptosis may similarly compromise survival of nucleated cells. The case is made that analysis of enhanced eryptosis may direct to the pathophysiology of systemic disease. Examples presented include drug side effects, sepsis, haemolytic uremic syndrome, Wilson´s disease, phosphate depletion and a rare condition caused by a mutation in GLUT1 turning the carrier into a cation channel.

Sequelae of sepsis include anemia which presumably results from accelerated clearance of erythrocytes from circulating blood. The underlying mechanisms, however, remained hitherto elusive. Most recent studies disclosed that increased cytosolic Ca2+ activity and ceramide both trigger suicidal erythrocyte death (i.e., eryptosis), which is characterized by lipid scrambling of the cell membrane leading to phosphatidylserine exposure at the erythrocyte surface. Phosphatidylserine exposing erythrocytes may adhere to vascular walls or may be engulfed by macrophages equipped with phosphatidylserine receptors. To explore whether sepsis leads to eryptosis, erythrocytes from healthy volunteers were exposed to plasma of patients suffering from sepsis, or to supernatants from sepsis producing pathogens. Then, phosphatidylserine exposure (annexin V binding), cell volume (forward scatter), cytosolic Ca2+ activity (Fluo3 fluorescence), and ceramide formation (anti-ceramide antibody) were determined by flow cytometry. Challenge of erythrocytes with plasma from the patients but not with plasma from healthy individuals triggered annexin V binding. The effect of patient plasma on erythrocyte annexin V binding was paralleled by formation of ceramide and a significant increase of cytosolic Ca2+ activity. Exposure of erythrocytes to supernatant of pathogens similarly induced eryptosis, an effect correlating with sphingomyelinase activity. The present observations disclose a novel pathophysiological mechanism leading to anemia and derangement of microcirculation during sepsis. Exposure to plasma from septic patients triggers phosphatidylserine exposure leading to adherence to the vascular wall and clearance from circulating blood.

Exposure of erythrocytes to osmotic shock, oxidative stress, and energy depletion activates Cl--sensitive and Ca2+-permeable cation channels. Subsequent Ca2+ entry triggers eryptosis, characterized by erythrocyte shrinkage, membrane blebbing, and phosphatidylserine exposure all features typical for apoptotic death of nucleated cells. Erythrocytes exposing phosphatidylserine are recognized, bound, engulfed, and degraded by macrophages. Eryptosis thus fosters clearance of affected erythrocytes from circulating blood. Iron deficiency leads to anemia, in part by decreasing erythrocyte life span. In this study, phosphatidylserine exposure, cell size, and cytosolic Ca2+ were measured by FACS analysis of annexin-V binding, forward scatter, and Fluo-3 fluorescence, respectively. Erythrocytes from mice on control diet were compared with erythrocytes from mice exposed 10 weeks to iron-deficient diet. Iron deficiency significantly (P<0.001) enhanced erythrocyte annexin-V binding (from 2.4 to 3.7%), decreased forward scatter (from 544 to 393), and increased cytosolic Ca2+ concentration. 45Ca2+ flux measurements and patch clamp experiments revealed enhanced Ca2+ uptake (by 2.3-fold) and cation channel activity. The half-life of fluorescence-labeled, iron-deficient, or Ca2+-loaded erythrocytes was significantly reduced compared with control erythrocytes. Thus, the experiments reveal a novel mechanism triggered by iron deficiency, which presumably contributes to accelerated clearance of erythrocytes in iron deficiency anemia.

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)