n-caproylsphingosine and Necrosis

Ceramide is a lipid second messenger that was recently identified as mediator of pulmonary edema in vivo. Here, we investigated the effect of ceramide on the permeability of confluent endothelial cell monolayers. In monolayers of bovine pulmonary artery and human microvascular pulmonary endothelial cells, incubation with C6-ceramide for 3 h elevated permeability in a concentration-dependent manner, whereas dihydroceramide was without effect. After 3 h of incubation with ceramide, we found no signs of necrosis (release of lactate dehydrogenase, loss of thiazylyl blue reduction) or apoptosis (ssDNA, caspase-8 activity). The increased endothelial permeability in response to ceramide was attenuated by the Ser/Thr protein kinase inhibitors K252a, K252b and H-7, as well as by the phosphatidylinositol-specific phospholipase C inhibitor L108. Since in some systems sphingosine-1-phosphate (S1P) acts antagonistic to ceramide, the effect of S1P was studied. S1P transiently increased endothelial cell resistance, whether it was given together with ceramide or 90 min thereafter. These data provide a novel example of the antagonism between S1P and ceramide. Our findings further suggest that ceramide alters vascular permeability by activation of pathways dependent on unidentified phospholipase C and Ser/Thr kinase isoenzymes.

Topics: Animals; Apoptosis; Cattle; Cell Membrane Permeability; Cells, Cultured; Ceramides; Dose-Response Relationship, Drug; Endothelial Cells; Endothelium, Vascular; Enzyme Inhibitors; Lysophospholipids; Necrosis; Sphingosine