Microsoft Word - IAA115BF

Introduction The outstanding role of polymorphonuclear granulocytes (PMN) to produce mediators of inflammation has been clearly established [2, 6–9]. After activation by extracellular stimuli the cells convert arachi-donic acid to three major groups of derivatives: the prostaglandins, the thromboxanes and the recently discovered leukotrienes are formed by oxygenation and further transformation. Among the lipoxygenase transformation products are factors with pronounced effect on leukocyte migration (leukotriene B4 and iso-mers) and vascular permeability [11]. The biological activity referred to for 40 years as slow-reacting substance of anaphylaxis (SRS-A) consists of three related metabolites LTC4, D4 and E4 [5, 10, 12]. The lipoxygenase pathway within human granulocytes is initiated by a 5-lipoxygenase which transforms arachi-donic acid to 5-HPETE. LTA4 is converted via gluta-thione-S-transferase to LTC4; LTC4 is metabolized by a y-glutamyltranspeptidase (y-GT) to LTD4 and LTD4 by a dipeptidase to LTE4. It was the purpose of the present investigation to determine the release of leukotriene-inducing (5lipoxygenase, glutathione-S-transferase) as well as metabolizing (y-GT, dipeptidase) enzymes [3, 10, 11]. Material and Methods Human PMNs were obtained from heparinized blood of healthy donors and separated on a Ficoll Metrizoate gradient followed by dextran sedimentation [3, 4]. Homogenization was performed in a Potter-Elvehjem Teflon glass homogenizer; differential centrifu-gation was carried out at 400, 3,000 and 20,000 gforl5 min and at 200,000 g for 60 min leading to four pellets and a final supernatant fraction. Equilibrium gradient centrifugation was performed with a postnuclear supernatant on a 19–54% (w/w) continuous sucrose gradient and centrifuged for 11 h at 100,000 g. Leukotriene-inducing and -metabolizing enzymes were determined as follows: PMNs were stimulated with the calcium ionophore in the presence of 14C-arachidonic acid and indomethacin; the release of 5-HETE, 5-HPETE, LTB4, 20-OH LTB4 and phospholipids was detected by thin-layer chromatography of the labeled