Metabolism and functional effects of sphingolipids in blood cells

Abstract

Summary. We examined the sphingolipid metabolism of peripheral blood cells, i.e. platelets, erythrocytes, neutrophils and mononuclear cells. A distinguishing characteristic of sphingolipid metabolism in these highly differentiated cells was their high sphingosine (Sph) kinase activity. The occurrence of [ 3 H]sphingosine 1-phosphate (Sph-1-P) from [ 3 H]Sph (actively incorporated from the outside) in the blood cells was strong, long-lasting, and independent of cell activation. Hence, the possibility of Sph-1-P playing a second messenger role is remote in these cells. About 40% of platelet Sph-1-P could be released extracellularly by 12-Otetradecanoylphorbol 13-acetate, possibly through mediation by protein kinase C. On the other hand, in erythrocytes, neutrophils and mononuclear cells a signi®cant percentage of Sph-1-P formed inside the cell was discharged without stimulation, whereas the stimulation-dependent release was marginal. In contrast to active [ ]sphingomyelin was barely detectable in the blood cells; this was especially true for anucleate platelets and erythrocytes. The Sph ! Sph-1-P pathway may become predominant over the Sph ! Cer ! sphingomyelin pathway during late-stage differentiation into platelets or erythrocytes. Sph and its methylated derivative, N,N-dimethylsphingosine, induced apoptosis not only in neutrophils but also in mononuclear cells, whereas Sph-1-P elicited Ca 2 mobilization in platelets. Our results suggest that all blood cells may remove plasma Sph, which is harmful or suppressive to cellular functions, and change it into Sph-1-P, acting as the source of plasma Sph-1-P, which may play a variety of important roles in blood vessels.

Determination of Sphingosine Kinase Activity for Cellular Signaling Studies

“…23 Consistent with our findings, it has been shown in other bulk cell studies of erythrocytes that the majority of intracellular sphingosine is in the phosphorylated form. 23,24,57 No other major fluorescent peaks except the internal standard were observed suggesting that under these conditions neither ceramide nor hexadecenal were formed in substantial quantities ( Figure 2 in the Supporting Information). This experiment demonstrated that SK activity could readily be detected in a cell lysate.…”

“…It is known that erythrocytes avidly take up extracellular sphingosine, while storing and actively releasing S1P. 23,57 This release of S1P appears to be in response to an as yet unidentified plasma factor. 23 Consistent with our findings, it has been shown in other bulk cell studies of erythrocytes that the majority of intracellular sphingosine is in the phosphorylated form.…”

Phosphorylation and Action of the Immunomodulator FTY720 Inhibits Vascular Endothelial Cell Growth Factor-induced Vascular Permeability

“…Platelets contain stored S1P (27) and release it upon activation, thus contributing to serum S1P levels. Moreover, neutrophils, erythrocytes, endothelial cells, and mononuclear cells also release S1P (28,29). However, the mechanisms whereby S1P is released are poorly understood.…”

The immunomodulator FTY720 is phosphorylated and released from platelets

“…Most, but not all, of the S1P release from platelets is dependent on stimuli such as thrombin and protein kinase C activation (Yatomi et al, 2000;Yatomi et al, 1997b), whereas erythrocytes release S1P constitutively (Ito et al, 2007;Yang et al, 1999). In contrast, it has been unclear where FTY720-P is produced.…”

“…Erythrocytes, platelets, and plasma were prepared as described elsewhere (Ito et al, 2007;Yang et al, 1999). Blood (~ 10 ml) was collected in 2 ml anticoagulant ACD (0.8% citric acid, 2.2% sodium citrate, and 2.45% glucose) then separated by centrifugation (120 x g for 15 min) into pellet and supernatant (platelet-rich plasma).…”