| Abstract: |
Prior studies demonstrated that conversion of sphingomyelin to ceramide via sphingomyelinase action resulted in the generation of free sphingoid bases and inactivation of protein kinase C in human leukemia (HL-60) cells (Kolesnick, R.N. (1989) J. Biol. Chem. 264, 7617-7623). The present studies define the novel phospholipid ceramide 1-phosphate in these cells and present evidence for formation of this compound by preferential utilization of ceramide derived from sphingomyelin. A ceramide 1-phosphate standard, prepared enzymatically via diacylglycerol kinase, was utilized for localization. In cells labeled to equilibrium with 32P(i) to label the head group of the molecule, the basal ceramide 1-phosphate level was 30 ± 2 pmol/106 cells. Generation of ceramide via the use of exogenous sphingomyelinase resulted in time- and concentration-dependent formation of ceramide 1-phosphate. As little as 3.8 x 10-5 units/ml was effective and a 3-fold increase was observed with a maximal concentration of 3.8 x 10-a2 units/ml; ED50 ~ 2 x 10-4 units/ml. This effect was observed by 5 min and maximal at 30 min. Similarly, in cells labeled with [3H]serine to probe the sphingoid base backbone, the basal level of ceramide 1-phosphate was 39 ± 5 pmol/106 and increased 2.5-fold with sphingomyelinase; ED50 ~ 5 x 10-5 units/ml. To determine the source of the phosphate moiety, studies were performed with cells short term labeled with 32P(i) and resuspended in medium without radiolabel. Under these conditions, sphingomyelin was virtually unlabeled. Nevertheless, sphingomyelinase (3.8 x 10-2 units/ml) induced a 12-fold increase in radiolabel incorporation, suggesting ceramide 1-phosphate formation occurred via ceramide phosphorylation. This event appeared specific for ceramide derived from sphingomyelin since ceramide from glycosphingolipids was not converted to ceramide 1-phosphate. In sum, these studies demonstrate the novel phospholipid ceramide 1-phosphate in HL-60 cells and suggest the possibility that a path exists from sphingomyelin to ceramide 1-phosphate via the phosphorylation of ceramide. |
| Keywords: |
human cell; cell line; tumor cells, cultured; leukemia, promyelocytic, acute; kinetics; cell culture; leukemia cell; phosphates; autoradiography; ceramide; ceramides; sphingomyelin phosphodiesterase; sphingomyelin; glycoside hydrolases; phospholipid metabolism; cell strain hl 60; sphingomyelins; phosphorus radioisotopes; human; priority journal; article; support, non-u.s. gov't; support, u.s. gov't, p.h.s.; sphingiodea
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