INCORPORATION OF 12(S)-HYDROXYEICOSATETRAENOIC ACID INTO THE PHOSPHATIDYLCHOLINE SIGNALING PATHWAY

The incorporation of 12-lipoxygenase metabolites into phospholipids (P Ls) could modify second messengers such as diacylglycerols (DAG) and p hosphatidic acids. Incubation of [C-14]12(S)-HETE (1 mu M) with bovine pulmonary artery endothelial cells (BPAEC), resulted in its incorpora tion in PLs with concentration-dependent kinetics. After a 4 h incubat ion, the proportion of radioactive phosphatidylcholine (PC), phosphati dylethanolamine (PE), phosphatidylserine (PS) + phosphatidylinositol ( PI) isolated by TLC, was 77.9%, 16.4% and 5.7%, respectively. In PC, [ C-14]12(S)-HETE was incorporated at the position 2 of the glycerol. Th ree major peaks of radioactive PC were isolated on RP-HPLC which were hydrolysed by phospholipase C (PLC). The resulting diacylglycerols wer e derivatized and identified by GC/MS as 1-oleyl-, 1-stearoyl- and 1-p almitoyl-2-[12-HETE] PC, BPAEC were incubated with [C-14]12(S)-HETE(1 mu M) before stimulation by bradykinin (1 mu M). (A) 1-acyl-2-[12-KETE ] diacylglycerols were isolated, derivatized and analysed by MS. We id entified a major ion with m/z = 926 that corresponds to the molecular ion of authentic 1-stearoyl-2-12(S)-HETE DAG, and 2 other ions with m/ z = 924 and 898 that correspond to the molecular ions of 1-oleyl- and 1-palmitoyl-2-12(S)-HETE DAG, respectively. (B) Radioactive PA was iso lated and hydrolysed by alkaline phosphatase, The MS of resulting diac ylglycerols identified 1-stearoyl-, 1-oleyl-, and 1-palmitoyl-2-12(S)- HETE phosphatidic acids. The quantities of 12-HETE PA and the 3 major 12-HETE diacylglycerols were shown to increase following bradykinin st imulation. Thus, the incorporation of 12(S)-HETE into PLs results in t he production of altered phosphatidic acids and diacylglycerols. The t ime-course of increases in 1-acyl-2-(12-HETE) phosphatidic acids and 1 -acyl-2-(12-HETE) diacylglycerols showed maximal concentrations 1 and 2 min after bradykinin stimulation, respectively, followed by the decr ease of both compounds. Propranolol, an inhibitor of PA phosphohydrola se, totally abolished the bradykinin-induced increase in 12-HETE DAG w hile increasing the magnitude and duration of 12-HETE PA release. The inhibiting effect of propranolol on bradykinin-induced increase of 12- HETE DAG demonstrates that 12-HETE PA is the principal precursor for 1 2-HETE DAG. This affords a novel method for confirming the major role of phospholipase D in PC metabolic pathways triggered during cell sign aling.