A murine haematopoietic stem-cell line, B6SUt.EP, responsive to erythropoietin (EPO), has been found to exhibit both early and late changes in diacylglycerol (DAG) and phosphatidic acid (PA) as measured by HPLC and TLC. DAG levels peaked at 5 s with a 28.1% increase compared with control levels (from 17.3 to 22.2 pmol/106 cells) with a later peak at 30 min (84.2% increase from 17.3 to 31.9 pmol). These changes were concentration-dependent from 0.025 to 10 units/ml EPO (5 s, EC50 = 0.82 unit/ml; 30 min, EC50 = 0.10 unit/ml). In addition, PA levels increased 752.3% compared with control levels (from 8.6 to 64.7 μg/106 cells) with an early peak at 20 s, as measured by both HPLC and TLC (5 s, EC50 = 0.07 unit/ml). G-protein regulation was investigated by studying the effects of the non-hydrolysable GTP analogue guanosine 5´-[γ-thio]triphosphate (GTP[S]) on PA synthesis. The addition of GTP[S] (10 μM) in permeabilized cells increased PA content from 6.3 μg to 48.6 μg per 106 cells. In the presence of EPO and GTP[S], PA levels increased to 64.8 μg. An antagonist of G-proteins, guanosine 5´-[β-thio]diphosphate (GDP[S]), had no effect on control levels of PA (5.9 μg/106 cells) but blocked the effect of EPO on PA (30.6 μg/106 cells). Thus, EPO stimulated both lipid second messengers, DAG and PA. Our results demonstrate DAG kinetics to be biphasic, as observed with a high concentration of EPO, or monophasic, as observed with low concentrations of EPO. The PA accumulation preceding that of DAG in the slower and sustaining phase suggests that PA was not derived from DAG. This was confirmed by the stimulation of PA (without ATP) by GTP[S], effectively excluding phosphorylation of DAG by DAG kinase in the formation of PA. In addition, phospholipase D (PLD) activation was demonstrated with a maximal increase in phosphatidylethanol at 5 min, suggesting that EPO increases PA via a guanine nucleotide-binding protein coupled to PLD. The temporal relationship of the evolution of PA and DAG is further strengthened by experiments with ethanol and propranolol as inhibitors of the DAG/PA phosphohydrolase reaction and R59022 as an inhibitor of the DAG kinase reaction.

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