Stephan M. Huber

Liver cell death and anemia in Wilson disease involve acid sphingomyelinase and ceramide

Wilson disease is caused by accumulation of Cu2+ in cells, which results in liver cirrhosis and, occasionally, anemia. Here, we show that Cu2+ triggers hepatocyte apoptosis through activation of acid sphingomyelinase (Asm) and release of ceramide. Genetic deficiency or pharmacological inhibition of Asm prevented Cu2+-induced hepatocyte apoptosis and protected rats, genetically prone to develop Wilson disease, from acute hepatocyte death, liver failure and early death. Cu2+ 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.

Mechanisms of Suicidal Erythrocyte Death

Erythrocyte injury such as osmotic shock, oxidative stress or energy depletion stimulates the formation of prostaglandin E2 through activation of cyclooxygenase which in turn activates a Ca2+ permeable cation channel. Increasing cytosolic Ca2+ concentrations activate Ca2+ sensitive K+ channels leading to hyperpolarization, subsequent loss of KCl and (further) cell shrinkage. Ca2+ further stimulates a scramblase shifting phosphatidylserine from the inner to the outer cell membrane. The scramblase is sensitized for the effects of Ca2+ by ceramide which is formed by a sphingomyelinase following several stressors including osmotic shock. The sphingomyelinase is activated by platelet activating factor PAF which is released by activation of phospholipase A2. Phosphatidylserine at the erythrocyte surface is recognised by macrophages which engulf and degrade the affected cells. Moreover, phosphatidylserine exposing erythrocytes may adhere to the vascular wall and thus interfere with microcirculation. Erythrocyte shrinkage and phosphatidylserine exposure (‘eryptosis’) mimic features of apoptosis in nucleated cells which however, involves several mechanisms lacking in erythrocytes. In kidney medulla, exposure time is usually too short to induce eryptosis despite high osmolarity. Beyond that high Cl- concentrations inhibit the cation channel and high urea concentrations the sphingomyelinase. Eryptosis is inhibited by erythropoietin which thus extends the life span of circulating erythrocytes. Several conditions trigger premature eryptosis thus favouring the development of anemia. On the other hand, eryptosis may be a mechanism of defective erythrocytes to escape hemolysis. Beyond their significance for erythrocyte survival and death the mechanisms involved in ‘eryptosis’ may similarly contribute to apoptosis of nucleated cells.

Cation channels trigger apoptotic death of erythrocytes

AbstractErythrocytes are devoid of mitochondria and nuclei and were considered unable to undergo apoptosis. As shown recently, however, the Ca2+-ionophore ionomycin triggers breakdown of phosphatidylserine asymmetry (leading to annexin binding), membrane blebbing and shrinkage of erythrocytes, features typical for apoptosis in nucleated cells. In the present study, the effects of osmotic shrinkage and oxidative stress, well-known triggers of apoptosis in nucleated cells, were studied. Exposure to 850 mOsm for 24 h, to tert-butyl-hydroperoxide (1 mM) for 15 min, or to glucose-free medium for 48 h, all elicit erythrocyte shrinkage and annexin binding, both sequelae being blunted by removal of extracellular Ca2+ and mimicked by ionomycin (1 μM). Osmotic shrinkage and oxidative stress activate Ca2+-permeable cation channels and increase cytosolic Ca2+ concentration. The channels are inhibited by amiloride (1 mM), which further blunts annexin binding following osmotic shock, oxidative stress and glucose depletion. In conclusion, osmotic and oxidative stress open Ca2+-permeable cation channels in erythrocytes, thus increasing cytosolic Ca2+ activity and triggering erythrocyte apoptosis.

GLUT1 mutations are a cause of paroxysmal exertion-induced dyskinesias and induce hemolytic anemia by a cation leak

Paroxysmal dyskinesias are episodic movement disorders that can be inherited or are sporadic in nature. The pathophysiology underlying these disorders remains largely unknown but may involve disrupted ion homeostasis due to defects in cell-surface channels or nutrient transporters. In this study, we describe a family with paroxysmal exertion-induced dyskinesia (PED) over 3 generations. Their PED was accompanied by epilepsy, mild developmental delay, reduced CSF glucose levels, hemolytic anemia with echinocytosis, and altered erythrocyte ion concentrations. Using a candidate gene approach, we identified a causative deletion of 4 highly conserved amino acids (Q282_S285del) in the pore region of the glucose transporter 1 (GLUT1). Functional studies in Xenopus oocytes and human erythrocytes revealed that this mutation decreased glucose transport and caused a cation leak that alters intracellular concentrations of sodium, potassium, and calcium. We screened 4 additional families, in which PED is combined with epilepsy, developmental delay, or migraine, but not with hemolysis or echinocytosis, and identified 2 additional GLUT1 mutations (A275T, G314S) that decreased glucose transport but did not affect cation permeability. Combining these data with brain imaging studies, we propose that the dyskinesias result from an exertion-induced energy deficit that may cause episodic dysfunction of the basal ganglia, and that the hemolysis with echinocytosis may result from alterations in intracellular electrolytes caused by a cation leak through mutant GLUT1.

Ion channels in cell proliferation and apoptotic cell death.

Cell proliferation and apoptosis are paralleled by altered regulation of ion channels that play an active part in the signaling of those fundamental cellular mechanisms. Cell proliferation must - at some time point - increase cell volume and apoptosis is typically paralleled by cell shrinkage. Cell volume changes require the participation of ion transport across the cell membrane, including appropriate activity of Cl− and K+ channels. Besides regulating cytosolic Cl− activity, osmolyte flux and, thus, cell volume, most Cl− channels allow HCO3− exit and cytosolic acidification, which inhibits cell proliferation and favors apoptosis. K+ exit through K+ channels may decrease intracellular K+ concentration, which in turn favors apoptotic cell death. K+ channel activity further maintains the cell membrane potential, a critical determinant of Ca2+ entry through Ca2+ channels. Cytosolic Ca2+ may trigger mechanisms required for cell proliferation and stimulate enzymes executing apoptosis. The switch between cell proliferation and apoptosis apparently depends on the magnitude and temporal organization of Ca2+ entry and on the functional state of the cell. Due to complex interaction with other signaling pathways, a given ion channel may play a dual role in both cell proliferation and apoptosis. Thus, specific ion channel blockers may abrogate both fundamental cellular mechanisms, depending on cell type, regulatory environment and condition of the cell. Clearly, considerable further experimental effort is required to fully understand the complex interplay between ion channels, cell proliferation and apoptosis.

Cell volume in the regulation of cell proliferation and apoptotic cell death.

Cell proliferation must – at some time point – lead to increase of cell volume and one of the hallmarks of apoptosis is cell shrinkage. At constant extracellular osmolarity those alterations of cell volume must reflect respective changes of cellular osmolarity which are hardly possible without the participation of cell volume regulatory mechanisms. Indeed, as shown for ras oncogene expressing 3T3 fibroblasts, cell proliferation is paralleled by activation of Na+/H+ exchange and Na+,K+,2Cl- cotransport, the major transport systems accomplishing regulatory cell volume increase. Conversely, as evident from CD95-induced apoptotic cell death, apoptosis is paralleled by inhibition of Na+/H+ exchanger and by activation of Cl- channels and release of the organic osmolyte taurine, major components of regulatory cell volume decrease. However, ras oncogene activation leads to activation and CD95 receptor triggering to inhibition of K+ channels. The effects counteract the respective cell volume changes. Presumably, they serve to regulate cell membrane potential, which is decisive for Ca++ entry through ICRAC and the generation of cytosolic Ca++ oscillations in proliferating cells. As a matter of fact ICRAC is activated in ras oncogene expressing cells and inhibited in CD95-triggered cells. Activation of K+ channels and Na+/H+ exchanger as well as Ca++ oscillations have been observed in a wide variety of cells upon exposure to diverse mitogenic factors. Conversely, diverse apoptotic factors have been shown to activate Cl- channels and organic osmolyte release. Inhibition of K+ channels is apparently, however, not a constant phenomenon paralleling apoptosis which in some cells may even require the operation of K+ channels. Moreover, cell proliferation may at some point require activation of Cl- channels. In any case, the alterations of cell volume are obviously important for the outcome, as cell shrinkage impedes cell proliferation and apoptosis can be elicited by increase of extracellular osmolarity. At this stage little is known about the interplay of cell volume regulatory mechanisms and the cellular machinery leading to mitosis or death of the cell. Thus, considerable further experimental effort is required in this exciting area of cell physiology.

Oxidation induces a Cl−-dependent cation conductance in human red blood cells

Oxidative stress induces complex alterations of membrane proteins in red blood cells (RBCs) eventually leading to haemolysis. To study changes of membrane ion permeability induced by oxidative stress, whole‐cell patch‐clamp recordings and haemolysis experiments were performed in control and oxidised human RBCs. Control RBCs exhibited a small cation‐selective whole‐cell conductance (236 ± 38 pS; n= 8) which was highly sensitive to the external Cl− concentration: replacement of NaCl in the bath by sodium gluconate induced an increase of this cation conductance by about 85 %. Exposing RBCs to t‐butylhydroxyperoxide (1 mm for 10 min) induced a twofold increase in this cation conductance which was further stimulated after replacement of extracellular Cl− by gluconate, Br−, I− or SCN−. In addition, lowering the ionic strength of the bath solution by isosmotic substitution of NaCl by sorbitol activated the cation conductance. The Cl−‐sensitive and oxidation‐induced cation conductance was Ca2+ permeable, exhibited a permselectivity of Cs+ > K+ > Na+= Li+ >> NMDG+, and was partially inhibited by amiloride (1 mm) and almost completely inhibited by GdCl3 (150 μm), but was insensitive to TEA, BaCl2, NPPB, flufenamic acid or quinidine. DIDS (100 μm) reversibly inhibited the activation of the cation conductance by removal of external Cl−. Oxidation induced haemolysis in NaCl‐bathed human RBCs. This haemolysis was attenuated by amiloride (1 mm) and inhibited by replacement of bath Na+ by the impermeant cation NMDG+. The Na+‐ and Ca2+‐permeable conductance might be involved in haemolytic diseases induced by elevated oxidative stress, such as glucose‐6‐phosphate dehydrogenase deficiency.

Enhanced programmed cell death of iron-deficient erythrocytes

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.

Involvement of ceramide in hyperosmotic shock- induced death of erythrocytes

AbstractErythrocytes lack nuclei and mitochondria, the organelles important for apoptosis of nucleated cells. However, following increase of cytosolic Ca2+ activity, erythrocytes undergo cell shrinkage, cell membrane blebbing and breakdown of phosphatidylserine asymmetry, all features typical for apoptosis in nucleated cells. The same events are observed following osmotic shock, an effect mediated in part by activation of Ca2+-permeable cation channels. However, erythrocyte death following osmotic shock is blunted but not prevented in the absence of extracellular Ca2+ pointing to additional mechanisms. As shown in this study, osmotic shock (950 mOsm) triggers sphingomyelin breakdown and formation of ceramide. The stimulation of annexin binding following osmotic shock is mimicked by addition of ceramide or purified sphingomyelinase and significantly blunted by genetic (aSM-deficient mice) or pharmacologic (50 μM 3,4-dichloroisocoumarin) knockout of sphingomyelinase. The effect of ceramide is blunted but not abolished in the absence of Ca2+. Conversely, osmotic shock-induced annexin binding is potentiated in the presence of sublethal concentrations of ceramide. In conclusion, ceramide and Ca2+ entry through cation channels concert to trigger erythrocyte death during osmotic shock.

Eryptosis, a Window to Systemic 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.