The ω‐hydroxylation of arachidonic acid by human polymorphonuclear leukocytes

Abstract

Incubations of [1-'4C]arachidonic acid with unstimulated human polymorphonuclear leukocytes resulted in the formation of four new metabolites in a previously described reverse-phase HPLC system. Three of these metabolites were largely suppressed in a CO/02 (80/20, by vol.) atmosphere indicating a cytochrome-P450-dependent monooxygenase reaction. In agreement with this assumption is their NADPH/02-dependent formation in the microsomal fraction. One metabolite was identified by gas chiomatography/mass spectrometry analysis as o-hydroxy-arachidonic acid and the two others were secondary products identified as w-carboxy-arachidonic acid and 5,2O-dihydroxy-E,Z,Z,Z-6,8,11 ,14-eicosatetraenoic acid. Since the affinity for arachidonate of the w-monooxygenase was quite low and the presence of LTB4 suppressed the w-hydroxylation of arachidonate, we conclude that the known LTB4 w-monooxygenase is responsible for the formation of w-hydroxy-arachidonate. It is unlikely, however, that significant concentrations of these metabolites are formed by activated polymorphonuclear leukocytes in vivo. The fourth metabolite remains tightly associated with the leukocytes but has not been further characterized.The release of arachidonic acid from phospholipids catalyzed by the action of phospholipase A2 is a frequent event subsequent to increases in intracellular cytosolic calcium levels. Depending on the various cell types in which this is observed, the further metabolism of arachidonic acid leads to highly active autacoids derived from either the cyclooxygenase or lipoxygenase pathways. In human polymorphonuclear leukocytes (PMN), two lipoxygenases are present and convert arachidonate to 15-hydroperoxyeicosatetraenoic acid (1 5-HPETE) or 15-hydroxyeicosatetraenoic acid (1 5-HETE) and to a major part to leukotriene B4 (LTB4), a highly chemotactic agent, which is formed from the primary 5-lipoxygenase product 5-HPETE via LTA4 [l]. The activity of 5-lipoxygenase was reported to depend strongly on elevated Ca2+ levels [2,3], but we recently showed that glutathione depletion also has an activating effect, probably by increasing organic peroxide concentrations [4]. Without Ca2+ or peroxides, the addition of arachidonic acid to PMN results in only minor amounts of 5-lipoxygenase-derived metabolites as detected in HPLC chromatograms by monitoring their triene absorption at 280 nm and diene absorption at 237 nm, respectively.In the course of these experiments, we also employed [l-14C]arachidonate and monitored the metabolites by means of a radioactivity detector. The corresponding chromatograms showed four additional metabolites. They all escaped ultraviolet detection because of a lack of an absorbance at the respective wavelength. Since this was an indication against lipoxygenase metabolites, we investigated other routes of Correspondence to V. Ullrich,

Cyclical binding, processing, and functional interactions of neutrophils with leukotriene B4

“…The receptors thereby couple to intracellular signals that promote function. However, the PMN soon counteract these signals by: a) degrading phospholipid products (tl12 5 1 min), pumping Ca2' out of cytosol (t, 5 1 min), and terminating PKC mobilization (t1,2 5 0.25 min) (see previous references); b) metabolizing LTB, to less potent C-20 oxygenated derivatives (tl,2 -10 min) (Kreisle and Parker, 1983;Shak and Goldstein, 1985;O'Flaherty et al, 1986b,c;Soberman et al, 1987;Hatzelmann and Ullrich, 1988); c) decreasing LTB, receptor availability (observed at 10 min) (Goldman and Goetzl, 1984); and d) becoming insensitive to ambient LTB, (t 2 2.5 min) (O'Flaherty et al, 1981;Goldman and Goetzl, 1984;O'Flaherty, 1985). Like many other agonists, then, LTB, causes a burst of cellular excitation followed by a period of quiesence and desensitization.…”

Quantitation of 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) produced by human polymorphonuclear leukocytes using electron capture ionization gas chromatography/mass spectrometry

“…Synthetic 20-HETE and 19(S)-HETE were a gift from Dr J. R. Falck (University of Texas, Southwest Medical Center, Dallas, Texas). 5,6,8,9,11,12,14,1 5-(3H)LTB, (220 Ci mmol-') was obtained from Dupont-New England Nuclear, and ("O)H,O was obtained from Isotec Inc. (Miamisburg, Ohio). Formylmethionylleucylphenylalanine (FMLP) was obtained from Sigma Chemical Co. (St Louis, Missouri); l-O-hexadecyl-2acetylglycerophosphocholine (PAF) was obtained from Bachem (Bubendorf, Switzerland).…”

Biological activity and metabolism of 20‐hydroxyeicosatetraenoic acid in the human platelet

“…Hepatocytes as well as unstimulated neutrophils have also been found to transform exogenous free arachiconic acid into this metabolite (Hatzelmann & Ullrich, 1988;. Recently, 20-HETE biosynthesis was found to be elevated in the kidneys of young spontaneous hypertensive rats (Sacerdoti et al, 1988).…”

“…Hepatocytes , renal cortical, and medulla preparations (Morrison & Pascoe, 1981;Oliw et al, 1981;Schwartzman et al, 1986;Takahashi et al, 1990) contain an active cytochrome P-450 which readily catalyzes the formation of 20-hydroxyeicosatetraenoic acid (20-HETE) as well as . In addition, 20-HETE is a major metabolite of arachidonic acid formed within the resting polymorphonuclear leukocyte (PMN) following the addition of exogenous arachidonic acid (Hatzelmann & Ullrich, 1988).…”