Abstract
The life span of erythrocytes is tightly regulated. Therefore, a mechanism is required to remove senescent or damaged erythrocytes without rupture of the cell membrane resulting in the release of hemoglobin which may impair kidney function. The mechanism of suicidal erythrocyte death is called eryptosis and shares similarities with apoptosis of nucleated cells such as exposure of phosphatidylserine at the cell surface, increase in cytosolic Ca2+ concentration, blebbing of the membrane, cell shrinkage and enzymatic degradation of the cytoskeletton. The cell shrinkage of eryptotic cells is mediated by a Ca2+-dependent K+ channel, the Gardos channel. Its activation by an increase in the intracellular Ca2+ concentration results in the efflux of K+, Cl- and osmotically obliged water. Phosphatidylserine-exposing erythrocytes are rapidly engulfed by macrophages equipped with phosphatidylserine receptors and degraded. Excessive eryptosis may lead to anemia, the pathological lack of erythrocytes. The present study was performed to elucidate mechanisms regulating erythrocyte survival and suicidal cell death. First, the functional significance of the Gardos channel for suicidal erythrocyte death and erythrocyte clearance was studied. Furthermore, the protective role of Gardos channels during exposure to hemolytic toxins was elucidated. Both issues were addressed by experiments performed in mice lacking the Ca2+-dependent K+ channel KCa3.1, the Gardos channel, and their wildtype littermates. Using patch-clamp recording, flow cytometry, in vitro hemolysis and a mouse sepsis model, it is shown that Gardos channel activity and Gardos effect delay hemolysis of injured erythrocytes and, thus, prevent the disastrous filtration of released hemoglobin into the renal tubular system. In a further series of experiments, the role of the NO/cGMP pathway, a powerful regulator of the life span of a variety of cells, for erythrocyte survival is investigated. Flow cytometry, Western Blotting, hematological counts, and MRI imaging were used to illustrate by means of a cGKI-deficient mouse model that cGKI is a mediator of erythrocyte survival in vitro and in vivo. Moreover, the participation of the phosphoinositide-dependent kinase PDK1, a key element in the phosphoinositol-3-kinase signalling pathway, which is involved in the regulation of ion channels, transporters, cell volume and cell survival, in the regulation of suicdal erythrocyte death was studied. Experiments performed in hypomorphic mice with some 20% of normal PDK1 acitivity and their wildtype littermates revealed that PDK1 deficiency is associated with decreased Ca2+ entry into erythrocytes and thus with blunted eryptotic effects of oxidative stress, osmotic shock and chloride removal. Finally, the functional significance of host pathogen interactions for suicidal erythrocyte death was investigated. Using flow cytometry, it could be shown that peptidoglycan, a main component of the bacterial cell wall, is a potent stimulus of eryptosis and thereby impairs erythrocyte survival. Peptidoglycan-induced eryptosis may therefore, at least in part, account for anemia observed in patients with bacterial infections.
Author and committee
dc:creator, dc:contributor.*- Author
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- Föller, Michael
Identifiers
dc:identifier.*- Identifier
- hdl:10900/49186