n-oleoylethanolamine and Hemolysis

Clostridium perfringens alpha-toxin induces hemolysis of rabbit erythrocytes through the activation of glycerophospholipid metabolism. Sheep erythrocytes contain large amounts of sphingomyelin (SM) but not phosphatidylcholine. We investigated the relationship between the toxin-induced hemolysis and SM metabolic system in sheep erythrocytes. Alpha-toxin simultaneously induced hemolysis and a reduction in the levels of SM and formation of ceramide and sphingosine 1-phosphate (S1P). N-Oleoylethanolamine, a ceramidase inhibitor, inhibited the toxin-induced hemolysis and caused ceramide to accumulate in the toxin-treated cells. Furthermore, dl-threo-dihydrosphingosine and B-5354c, isolated from a novel marine bacterium, both sphingosine kinase inhibitors, blocked the toxin-induced hemolysis and production of S1P and caused sphingosine to accumulate. These observations suggest that the toxin-induced activation of the SM metabolic system is closely related to hemolysis. S1P potentiated the toxin-induced hemolysis of saponin-permeabilized erythrocytes but had no effect on that of intact cells. Preincubation of lysated sheep erythrocytes with pertussis toxin blocked the alpha-toxin-induced formation of ceramide from SM. In addition, incubation of C. botulinum C3 exoenzyme-treated lysates of sheep erythrocytes with alpha-toxin caused an accumulation of sphingosine and inhibition of the formation of S1P. These observations suggest that the alpha-toxin-induced hemolysis of sheep erythrocytes is dependent on the activation of the SM metabolic system through GTP-binding proteins, especially the formation of S1P.

Topics: 4-Aminobenzoic Acid; ADP Ribose Transferases; Amidohydrolases; Animals; Bacterial Toxins; Botulinum Toxins; Calcium-Binding Proteins; Ceramidases; Chromatography, Thin Layer; Diglycerides; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Erythrocytes; Ethanolamines; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Hemolysis; Inositol 1,4,5-Trisphosphate; Lysophospholipids; Oleic Acids; para-Aminobenzoates; Pertussis Toxin; Phosphatidylcholines; Phosphorylcholine; Phosphotransferases (Alcohol Group Acceptor); Rabbits; Sheep; Sphingomyelins; Sphingosine; Time Factors; Toxins, Biological; Type C Phospholipases