MOLECULAR-SPECIES OF PHOSPHOLIPIDS IN A MURINE STEM-CELL LINE RESPONSIVE TO ERYTHROPOIETIN

The generation of the lipid signalling molecules, diacylglycerol (DAG) and phosphatidic acid (PA), has been implicated in the transduction e vents essential for proliferation of murine B6SUt.EP stem cells respon sive to erythropoietin (EPO). Some of the responses were rapid and tra nsient while others were slower and sustained. In an attempt to better understand the biphasic nature of DAG and PA appearance in response t o EPO, an analysis of the molecular species of DAG, phosphatidylcholin e (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and PA in control and EPO-treated B6SUt.EP cells was made by HPLC and TLC. Fifteen to eighteen species were identified, which were increased non -uniformly by 0.2 unit/ml EPO. Greater increases (x 6) were observed i n 16:0,20:4 and 18:0,20:4 DAGs than in other species. The molecular sp ecies profiles of the stimulated DAGs were compared with the profiles of molecular species contained in the phospholipids. Comparison of the increase in DAG species caused by EPO with the molecular species pres ent in PC and PI showed both PI and PC as the source of the fast DAG a ccumulation and only PC as the source of the slow DAG accumulation. PE was involved in both phases. We found a consistent formation of ethan olamine over time, in larger amounts than choline, providing strong ev idence that, in addition to PC, PE is a major substrate. In addition, changes in molecular species of PA in response to EPO suggest that PI cannot account for the mass of PA formed during the first 30 s incubat ion with EPO, nor for PA formed during 30 min with EPO. It is conclude d that the majority of PA was formed by a direct action of phospholipa se D on PC.