Molecular species analysis of a product of phospholipase D activation. Phosphatidylethanol is formed from phosphatidylcholine in phorbol ester- and bradykinin-stimulated PC12 cells.
Tumor-promoting phorbol esters or calcium-mobilizing receptor ligands stimulate phosphatidylcholine breakdown and in many cells this is accompanied by phospholipase D (PLD) activation. We tested whether or not a direct relationship exists between these two phenomena. Pheochromocytoma (PC12) cells were stimulated with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate or with the calcium-mobilizing receptor ligand bradykinin in media containing 1% ethanol. The fatty acid composition of the molecular species of phosphatidylethanol (PEt), a product of PLD activation, formed in stimulated cells was compared with the molecular species of endogenous phospholipids isolated from unstimulated PC12 cells. PEt was isolated and analyzed by fast atom bombardment-mass spectrometry (FAB-MS) in the negative ion mode. Fatty acid composition and headgroup structure of the major PEt molecular ions were confirmed by linked scan analysis. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol were isolated from unstimulated cells and converted into phosphatidic acids using PLD. Mass spectra of the respective phosphatidic acids were obtained by fast atom bombardment-mass spectrometry as described above. The molecular species of PEt formed in 12-O-tetradecanoylphorbol-13-acetate- and bradykinin-stimulated PC12 cell were identical to those of phosphatidylcholine isolated from untreated cells.
Holbrook
P G
PG
Laboratory of Bioorganic Chemistry, National Institute of Diabetes Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Pannell
L K
LK
Murata
Y
Y
Daly
J W
JW
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology*; D020404:Glycerophospholipids; D007700:Kinetics; D013058:Mass Spectrometry; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism; D016716:PC12 Cells; D010712:Phosphatidic Acids / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism*; D010743:Phospholipids / Q000302:isolation & purification / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D020404:Glycerophospholipids; D009829:Oleic Acids; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D010743:Phospholipids; C051521:phosphatidylethanol; D019301:Oleic Acid; D010739:Phospholipase D; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin