Substitution of 15-hydroxyeicosatetraenoic acid in the phosphoinositide signaling pathway.
Consideration of how 15-hydroxyeicosatetraenoic acid (15-HETE) might exert its biological actions led us to investigate the consequences of its incorporation into bovine pulmonary arterial endothelial cell (BPAEC) phospholipids [3H]15(S)-HETE was incorporated mainly (89%) into phosphatidylinositols, predominantly as 1-stearoyl-2-(15-HETE) phosphatidylinositol. By contrast 5(S)- and 12(S)-HETE are incorporated largely into phosphatidylcholine. 15-HETE had a long persistence in the phosphatidylinositols of BPAEC with a half-life of 12 h; its uptake was concentration-dependent, and it accumulated so that 2-(15-HETE) phosphatidylinositol accounted for 10.9% of total phosphatidylinositol after four sequential 1-h incubations of cells with 1 microM 15(S)-HETE. After incubating BPAEC with 15(S)-HETE, stimulation of the cells with bradykinin led to an increase in the levels of 15-HETE. Following addition of bradykinin to cells exposed to [3H]15(S)-HETE, a radiolabeled diacylglycerol was isolated. A mass spectrum of its pentafluorobenzoyl (PFBO) trimethylsilyl (Me3Si) derivative obtained with direct electron capture negative ion chemical ionization mass spectrometry (DNICI/MS) revealed a molecular anion and fragment ions that were identical with those observed with the PFBO/Me3Si derivative of authentic 1-stearoyl-2-(15-HETE) diacylglycerol. There was a lesser quantity of 1-oleoyl-2-(15-HETE) diacylglycerol. An increase in the quantity of 1-stearoyl-2-(15-HETE) diacylglycerol from 6 +/- 1.4 pmol/10(7) cells in the basal state to 12.7 +/- 3.5 after bradykinin was measured by DNICI/MS utilizing a deuterium-labeled analog as an internal standard. Thus, incorporation of 15(S)-HETE into the phosphatidylinositol of these cells led to the release of altered second messengers.
Legrand
A B
AB
Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Lawson
J A
JA
Meyrick
B O
BO
Blair
I A
IA
Oates
J A
JA
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D019852:Diacylglycerol Kinase; D004075:Diglycerides / Q000378:metabolism; D004730:Endothelium, Vascular / Q000201:enzymology / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism*; D007700:Kinetics; D010716:Phosphatidylinositols / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D010770:Phosphotransferases / Q000378:metabolism; D015398:Signal Transduction
D004075:Diglycerides; D006893:Hydroxyeicosatetraenoic Acids; D010716:Phosphatidylinositols; D010743:Phospholipids; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; D010770:Phosphotransferases; D019852:Diacylglycerol Kinase; D001920:Bradykinin