Use of a fluorescent analog of CDP-DAG in human skin fibroblasts: characterization of metabolism, distribution, and application to studies of phosphatidylinositol turnover.
We studied the uptake metabolism, and distribution of a fluorescent analog of CDP-diacylglycerol [cytidine diphosphate-1, 2-oleoyl, (N-(4-nitrobenzo-2-oxa-1,3-diazole) aminocaproyl) diacylglycerol; CDP-NBD-DAG]. When cells were incubated with CDP-NBD-DAG for 60 min at 11 degrees C and washed, the fluorescent lipid was localized to the plasma membrane. However, upon warming to 37 degrees C, the fluorescent lipid redistributed into various intracellular membranes and was metabolized primarily to fluorescent analogs of DAG and phosphatidylcholine (PC), although small amounts of fluorescent phosphatidic acid and phosphatidylinositol (PI) were also formed. The incorporation of 32Pi into some of the fluorescent lipids was also determined in order to assess their turnover. Stimulation of cells with platelet-derived growth factor enhanced the synthesis of fluorescent PI relative to unstimulated cells by approximately 68%, while the synthesis of fluorescent PC was unaffected. In addition, the incorporation of 32Pi into fluorescent PI was enhanced. Stimulation of cells with interleukin-1 beta enhanced the synthesis of both fluorescent PI (approximately 88%) and PC (approximately 250%) compared to non-stimulated cells, but with less incorporation of 32Pi into fluorescent PI. Finally, incubation of CDP-NBD-DAG-treated cells with inhibitors of phosphatidic acid phosphohydrolase and DAG kinase resulted in a dramatic increase in the amount of fluorescent PI formed (approximately 64% of all the CDP-NBD-DAG metabolites). We conclude that CDP-NBD-DAG can be used for the de novo synthesis of fluorescent PI, and in combination with 32P labeling, provides a convenient method for studying PI turnover.
Salman
M
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Mayo Clinic and Foundation, Rochester, MN 55905, USA.
Pagano
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D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000378:metabolism; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D003512:Cyclohexanones / Q000494:pharmacology; D003567:Cytidine Diphosphate Diglycerides / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D008071:Lipoprotein Lipase / Q000037:antagonists & inhibitors; D008563:Membrane Lipids / Q000737:chemistry; D008856:Microscopy, Fluorescence; D019301:Oleic Acid / Q000378:metabolism; D019308:Palmitic Acid / Q000378:metabolism; D010712:Phosphatidic Acids / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011433:Propranolol / Q000494:pharmacology; D011744:Pyrimidinones / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology
D003512:Cyclohexanones; D003567:Cytidine Diphosphate Diglycerides; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007375:Interleukin-1; D008563:Membrane Lipids; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D010716:Phosphatidylinositols; D010982:Platelet-Derived Growth Factor; D011744:Pyrimidinones; D013844:Thiazoles; D019301:Oleic Acid; D019308:Palmitic Acid; C036535:1,6-bis(cyclohexyloximinocarbonyl)hexane; C047505:R 59022; D011433:Propranolol; D008071:Lipoprotein Lipase; D009327:4-Chloro-7-nitrobenzofurazan