Ethanolamine as well as choline is released to the external medium on phorbol ester and foetal calf serum stimulation of glial cells in primary culture.

Stimulation of protein kinase C (PKC) in a variety of cells causes enhanced catabolism of phosphatidylcholine [ 1,2]. Diacylglycerol (DAG) released from phosphatidylcholine turnover functions as a second messenger in plasma membranes activating PKC [3]. In the CNS at least, choline released extracellularly as a result of enhanced phosphatidylcholine (PC) turnover may provide free choline for acetylcholine synthesis [4]. Basal turnover of phosphatidylcholine in primary glial cells may occur through 1 so-PC suggesting a phospholipase Amediated mechanism r5]. However, phorbol ester treatment of glial cultures activates a phospholipase D [6]. Phosphatidylethanolamine (PE) in several cell lines also shows increased catabolism on stimulation with phorbol esters [7]. Here we show that phorbol ester and foetal calf serum stimulate the turnover of PE and PC in glial cells in primary culture. As a result of this enhanced catabolism ethanolamine, as well as choline, is released to the external medium. Glial cell cultures were prepared from cerebra of 1 -2 da (Nuncr. Preliminary experiments (data not shown) revealed that incorporation of 3H-choline or 3H-ethanolamine into lipids reached equilibrium after about 12 hours. For routine labelling of lipids,cultures at seven days in DMEM containing 0.1% FCS and 2% glutamine were incubated with 5pCi per well of either 3H-choline or 3Hethanolamine (Amersham plc) for 24 hours at 37O. After incubation cultures were rinsed with DMEM and fresh DMEM (-FCS) added. After agonist stimulation ethanolamine and choline metabolites in the extracellular and intracellular phases were recovered by solvent extracrat pups [8] and cells grown in 6-well plates