Inhibition of erythrocyte phosphatidylserine exposure by urea and Cl⁻

Abstract

Osmotic shock by addition of sucrose to the medium stimulates erythrocyte sphingomyelinase with subsequent ceramide formation and triggers Ca(2+) entry through stimulation of cation channels. Both ceramide and Ca(2+) activate an erythrocyte scramblase, leading to breakdown of phosphatidylserine asymmetry, a typical feature of apoptosis. Because erythrocytes are regularly exposed to osmotic shock during passage of kidney medulla, the present study explored the influence of NaCl and urea on erythrocyte phosphatidylserine exposure as determined by annexin binding. The percentage of annexin-binding erythrocytes increased from <5 to 80 +/- 4% (n = 4) upon addition of 650 mM sucrose, an effect paralleled by activation of the cation channel and stimulation of ceramide formation. The number of annexin-binding erythrocytes increased only to 18% after addition of 325 mM NaCl and was not increased by addition of 650 mM urea. According to whole cell patch-clamp experiments, the cation conductance was activated by replacement of extracellular Cl(-) with gluconate at isotonic conditions or by addition of hypertonic sucrose or urea. Although stimulating the cation conductance, urea abrogated the annexin binding and concomitant increase of ceramide levels induced by osmotic cell shrinkage. In vitro sphingomyelinase assays demonstrated a direct inhibitory effect of urea on sphingomyelinase activity. Urea did not significantly interfere with annexin binding after addition of ceramide. In conclusion, both Cl(-) and urea blunt erythrocyte phosphatidylserine exposure after osmotic shock. Whereas Cl(-) is effective through inhibition of the cation conductance, urea exerts its effect through inhibition of sphingomyelinase, thus blunting formation of ceramide.

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

“…Moreover, those erythrocytes may bind to receptors in the vascular wall and thus impede microcirculation (4,5,24,47,50,51). Along those lines, we observed enhanced trapping of erythrocytes in renal medulla after ischemia of the mouse kidney (34). Phosphatidylserine-exposing cells may further participate in hemostasis (4,7,39,47,54).…”

Stimulation of erythrocyte ceramide formation by platelet-activating factor

“…On the contrary, urea protects erythrocytes against hyperosmotic shock, an effect proven to be due to direct inhibition of erythrocyte sphingomyelinase (Lang et al, 2004b). Accordingly, blocking the sphingomyelinase with 600 mM of urea blunted the effect of PAF on phosphatidylserine exposure.…”

“…We have recently shown that addition of 600 mM urea inhibits hyperosmotic-shock-induced ceramide formation by interfering with sphingomyelinase activity (Lang et al, 2004b). We thus used urea to demonstrate the link between PAF-induced ceramide formation and PAF-induced phosphatidylserine exposure.…”

PGE2 in the regulation of programmed erythrocyte death

“…Nevertheless, it is noteworthy that during acute renal failure erythrocytes may be trapped in renal medulla. 51,52 The subsequent erythrocyte 'apoptosis' may then contribute to the derangement of microcirculation. Beyond that any erythrocyte disorder facilitating erythrocyte shrinkage, such as sickle cell disease, 28,53 thalassemia 54 or iron deficiency 55 could, to the extent as it leads to activation of the cell volume regulatory cation channels, trigger premature 'apoptosis' and thus accelerate erythrocyte death.…”