USE OF A FLUORESCENT ANALOG OF CDP-DAG IN HUMAN SKIN FIBROBLASTS - CHARACTERIZATION OF METABOLISM, DISTRIBUTION, AND APPLICATION TO STUDIESOF PHOSPHATIDYLINOSITOL TURNOVER

We studied the uptake, metabolism, and distribution of a fluorescent a nalog of CDP-diacylglycerol [cytidine diphosphate-1, 2-oleoyl, {N-(4-n itrobenzo-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 tile plasma memb rane. However, upon warming to 37 degrees C, the fluorescent lipid red istributed into various intracellular membranes and was metabolized pr imarily to fluorescent analogs of DAG; and phosphatidylcholine (PC), a lthough small amounts of fluorescent phosphatidic acid and phosphatidy linositol (PI) were also formed. The incorporation of P-32(i) into som e of the fluorescent lipids was also determined in order to assess the ir turnover. Stimulation of cells with platelet-derived growth factor enhanced the synthesis of fluorescent PI relative to unstimulated cell s by similar to 68%, while the synthesis of fluorescent PC was unaffec ted. In addition, the incorporation of P-32(i) into fluorescent PI was enhanced. Stimulation of cells with interleukin-1 beta enhanced the s ynthesis of both fluorescent PI (similar to 88%) and PC (similar to 25 0%) compared to non-stimulated cells, but with less incorporation of P -32(i) into fluorescent PI. Finally, incubation of CDP-NBD-DAG-treated cells with inhibitors of phosphatidic acid phosphohydrolase and DAG k inase resulted in a dramatic increase in the amount of fluorescent PI formed (similar to 64% of all the CDP-NBD-DAG metabolites). We conclud e that CDP-NBD-DAG can be used for the de novo synthesis of fluorescen t PI, and in combination with P-32 labeling, provides a convenient met hod for studying PI turnover.