Dioctanoylglycerol stimulates accumulation of [methyl-14C]choline and its incorporation into acetylcholine and phosphatidylcholine in a human cholinergic neuroblastoma cell line.
Dioctanoylglycerol, a synthetic diacylglycerol, stimulated [14C]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 microM dioctanoylglycerol the levels of [14C]choline attained during a 2 hour labeling period were elevated by 78 +/- 12%, while [14C]acetylcholine and long fatty acyl chain [14C]phosphatidylcholine levels increased by 26 +/- 2% and 19 +/- 5%, respectively (mean +/- S.E.M.). Total (long chain plus dioctanoyl-) [14C]phosphatidylcholine was increased by 198 +/- 33%. Kinetic analysis showed that dioctanoylglycerol reduced the apparent Km for choline uptake to 56 +/- 9% of control (n = 4). The Vmax was not significantly altered. The stimulation of [14C]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(o-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).(ABSTRACT TRUNCATED AT 250 WORDS)
Slack
B E
BE
Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge 02139.
Richardson
U I
UI
Nitsch
R M
RM
Wurtman
R J
RJ
D000109:Acetylcholine / Q000096:biosynthesis*; D002794:Choline / Q000378:metabolism*; D004075:Diglycerides / Q000494:pharmacology*; D006801:Humans; D009423:Nervous System Neoplasms / Q000378:metabolism* / Q000473:pathology; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D009994:Osmolar Concentration; D010275:Parasympathetic Nervous System; D010713:Phosphatidylcholines / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D004075:Diglycerides; D010713:Phosphatidylcholines; C044295:1,2-dioctanoylglycerol; D011493:Protein Kinase C; D002794:Choline; D000109:Acetylcholine; D013755:Tetradecanoylphorbol Acetate