Incorporation of 12(S)-hydroxyeicosatetraenoic acid into the phosphatidylcholine signaling pathway.
The incorporation of 12-lipoxygenase metabolites into phospholipids (PLs) could modify second messengers such as diacylglycerols (DAG) and phosphatidic acids. Incubation of [(14)C]12(S)-HETE (1 microM) with bovine pulmonary artery endothelial cells (BPAEC), resulted in its incorporation in PLs with concentration-dependent kinetics. After a 4 h incubation, the proportion of radioactive phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) + phosphatidylinositol (PI) isolated by TLC, was 77.9%, 16.4% and 5.7%, respectively. In PC, [(14)C]12(S)-HETE was incorporated at the position 2 of the glycerol. Three major peaks of radioactive PC were isolated on RP-HPLC which were hydrolysed by phospholipase C (PLC). The resulting diacylglycerols were derivatized and identified by GC/MS as 1-oleyl-, 1-stearoyl- and 1-palmitoyl-2-[12-HETE] PC. BPAEC were incubated with [(14)C]12(S)-HETE (1 microM) before stimulation with bradykinin (1 microM). (A) 1-acyl-2-[12-HETE] diacylglycerols were isolated, derivatized and analysed by MS. We identified 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 isolated and hydrolysed by alkaline phosphatase. The MS of resulting diacylglycerols 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 stimulation. Thus, the incorporation of 12(S)-HETE into PLs results in the production of altered phosphatidic acids and diacylglycerols. The time-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 decrease of both compounds. Propranolol, an inhibitor of PA phosphohydrolase, totally abolished the bradykinin-induced increase in 12-HETE DAG while 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 12-HETE DAG. This affords a novel method for confirming the major role of phospholipase D in PC metabolic pathways triggered during cell signaling.
Legrand
A B
AB
Laboratoire de Pharmacodynamie et de Pharmacologie Moléculaire, Université de Rennes I, France.
Wang
J
J
Sobo
G
G
Blair
I A
IA
Brash
A R
AR
Oates
J A
JA
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002417:Cattle; D004075:Diglycerides / Q000096:biosynthesis; D004730:Endothelium, Vascular / Q000378:metabolism; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000096:biosynthesis; D010713:Phosphatidylcholines / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013552:Swine
D004075:Diglycerides; D006893:Hydroxyeicosatetraenoic Acids; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D001920:Bradykinin