Molecular species analysis of 1,2-diglycerides on phorbol ester stimulation of LA-N-1 neuroblastoma cells during proliferation and differentiation.

1,2-Diacyl-sn-glycerol (DAG) is a product of cell activation that has emerged as an important intracellular messenger whose primary function appears to be the activation of protein kinase C. They originate by the activation of phospholipases, which hydrolyze different phospholipids depending on the external stimulus and the nature of the cells, leading to the production of different molecular species. In the present study the quantitative changes in the total mass and the molecular species of DAG formed on phorbol ester (12-O-tetradecanoyl-phorbol 13-acetate) stimulation were investigated in proliferating and retinoic acid (RA)-differentiated human LA-N-1 cells. The TPA treatment of both cell types elicited an increase in the total amount of DAG. The increase was biphasic; i.e., an initial peak at 2-5 min was followed by a sustained increase that persisted for > 30 min. The analysis of the molecular species of DAG and phospholipids showed that in proliferating LA-N-1 cells, the DAG increase corresponds to the production of mainly saturated/monounsaturated (16:0-18:1, 18:0-18:1) and saturated/saturated (16:0-16:0, 16:0-18:0) species, suggesting that they originate essentially from the hydrolysis of phosphatidylcholine. In contrast, RA-differentiated cells responded to TPA treatment by increasing the level of saturated/polyunsaturated (16:1-22:6, 18:0-22:6, 16:0-20:4, 18:0-20:4) and monounsaturated/monounsaturated (18:1-18:1) species, suggesting mainly a phosphatidylethanolamine origin. These findings indicate that the treatment of LA-N-1 cells with TPA generates different molecular species of DAG depending on their physiological state. These observations suggest in turn that different phospholipases are activated by TPA in proliferating and RA-differentiated cells.