DIOCTANOYLGLYCEROL STIMULATES ACCUMULATION OF [METHYL-C-14]CHOLINE AND ITS INCORPORATION INTO ACETYLCHOLINE AND PHOSPHATIDYLCHOLINE IN A HUMAN CHOLINERGIC NEUROBLASTOMA CELL-LINE

Dioctanoylglycerol, a synthetic diacylglycerol, stimulated [C-14]choline uptake in cultured human neuroblastoma (LA-N-2) cells. As this effect has not, to our knowledge, been reported before, it was of interest to characterize it in more detail. In the presence of 500-mu-M dioctanoylglycerol the levels of [C-14]choline attained during a 2 hour labeling period were elevated by 78 +/- 12%, while [C-14]acetylcholine and long fatty acyl chain [C-14]phosphatidylcholine levels increased by 26 +/- 2% and 19 +/- 5%, respectively (mean +/- S.E.M.). Total (long chain plus dioctanoyl-) [C-14]phosphatidylcholine was increased by 198 +/- 33%. Kinetic analysis showed that dioctanoylglycerol reduced the apparent K(m) for choline uptake to 56 +/- 9% of control (n = 4). The V(max) was not significantly altered. The stimulation of [C-14]choline accumulation by dioctanoylglycerol was not dependent on protein kinase C activation; the effect was not mimicked by phorbol ester or by 1-oleoyl-2-acetylglycerol, and was not inhibited by the protein kinase C inhibitors H-7 or staurosporine, or by prolonged pretreatment with phorbol 12-myristate 13-acetate. The effect of dioctanoylglycerol was slightly (but not significantly) reduced by EGTA and strongly inhibited by the cell-permeant calcium chelator bis(omicron-aminophenoxy)-ethane-N,N,N'N'-tetraacetic acid, tetra(acetoxymethyl)ester. Although these results implicate elevated intracellular calcium in the response, dioctanoylglycerol did not increase phosphatidylinositol hydrolysis in LA-N-2 cells, and its effect was not inhibited by the diacylglycerol kinase inhibitor R 59 022 (which blocks the conversion of diacylglycerol to phosphatidic acid, a known stimulator of phosphatidylinositol hydrolysis). The enhancement of choline uptake by diacylglycerol, a phospholipid metabolite, may represent a mechanism by which precursor levels can be replenished more rapidly when membrane turnover is increased.