sphingosine-1-phosphate and acetic-anhydride

Because sphingosine (Sph) is actively incorporated into platelets and rapidly converted to sphingosine 1-phosphate (Sph-1-P), which is then released extracellularly, it is important to study the effects of Sph and Sph-1-P on endothelial cells from the viewpoint of platelet-endothelial cell interaction. In this study, we found that Sph, as well as ceramide, induces apoptosis in human umbilical vein endothelial cells (HUVECs). In contrast, Sph-1-P acts as a HUVEC survival factor; this bioactive lipid was shown to protect HUVECs from apoptosis induced by the withdrawal of growth factors and to stimulate HUVEC DNA synthesis. In metabolic studies, [3H]Sph, incorporated into HUVECs, was converted to [3H]Cer and further to [3H]sphingomyelin in a time-dependent manner, whereas [3H]Sph-1-P formation from [3H]Sph was weak and transient. These findings in HUVECs are very different from those of platelets, which possess a highly active Sph kinase but lack Sph-1-P lyase. As a result, platelets abundantly store Sph-1-P, whereas HUVECs contain much less Sph-1-P. Finally, HUVECs, in contrast to platelets, failed to release Sph-1-P extracellularly, indicating that HUVECs themselves are not able to supply the survival factor Sph-1-P, but receive it from activated platelets. Our results suggest that platelets may maintain the integrity of endothelial cells by incorporating Sph and releasing Sph-1-P.

Topics: Acetic Anhydrides; Acylation; Apoptosis; Blood Platelets; Cell Communication; DNA; Endothelium, Vascular; Humans; Lysophospholipids; Microscopy, Fluorescence; Sphingolipids; Sphingosine; Umbilical Veins

We describe here in detail the development of a method to quantitatively measure sphingosine 1-phosphate (Sph-1-P), a bioactive sphingolipid. Sph-1-P was first extracted from cells into the upper aqueous phase under alkaline conditions by Folch's phase separation and then reextracted into the lower chloroform phase under acidic conditions. This phosphorylated sphingoid base extracted was quantitatively converted to N-[3H]-acetylated Sph-1-P, that is [3H]C2-ceramide 1-phosphate (C2-Cer-1-P), by N-acylation with [3H]acetic anhydride. The [3H]C2-Cer-1-P formed with the acylation was resolved by thin-layer chromatography, detected with autoradiography, and quantitated by scraping the corresponding band and counting its radioactivity with a scintillation counter. This assay allows quantification of Sph-1-P over a range from at least 100 pmol (often 30 pmol) to 10 nmol (the highest level tested). The utility and validity of our assay were demonstrated using human platelets. The amount of Sph-1-P in platelet extracts was proportional to the cell number and calculated as 141 +/- 4 pmol/10(8) cells (mean +/- SD, n = 3), which was about four times higher than that of sphingosine. The potent agonist thrombin did not affect the total Sph-1-P amounts in platelet suspensions but induced the release of Sph-1-P stored in the cells into the medium.

Topics: Acetic Anhydrides; Acylation; Adult; Blood Platelets; Humans; Lysophospholipids; Sphingosine