Generation of thromboxane A2-like activity from prostaglandin endoperoxides by polymorphonuclear leukocyte homogenates [proceedings].

Rabbit aorta contracting substance (RCS) released from isolated perfused lungs of guinea pigs during anaphylaxis (Piper & Vane, 1969) is now thought to be a mixture of thromboxane A2 (TXA2) and prostaglandin endoperoxides (PGG2, PGH2; Hamberg, Svensson & Samuelsson, 1975; Bunting, Moncada & Vane, 1976). TXA2 is generated enzymically from PGG2 or PGH2 by horse or human platelet microsomes (Bunting, Moncada, Needleman & Vane, 1975). TXA2 is distinguished from endoperoxides by its shorter half life and its increased potency in contracting the rabbit aorta; moreover, while prostaglandin endoperoxides relax the mesenteric and coeliac arteries of the rabbit, TXA2 is a powerful constrictor of both (Bunting, Moncada & Vane, 1976). Rabbit peritoneal polymorphs (PMNs) release prostaglanin during phagocytosis (Higgs, McCall & Youlten, 1975). We have now shown that homogenates of these cells contain an enzyme which converts PG endoperoxides into a substance with increased RCS activity which contracts the rabbit coeliac artery and has a similar half life to TXA2. PMN leukocyte suspensions obtained by the method of Hirsch & Church (1960) were centrifuged (50 g for O min), washed once with 0.9% saline and resuspended in 5 ml Krebs bicarbonate solution at a concentration of 4-18 x 107 cells/ml. The cells were disrupted by freezing and thawing five times and homogenized in a glass homogenizer. The homogenates were then centrifuged at 100,000g for 1 h and resuspended in 2 ml 100 mM Tris buffer (pH 7.5) prior to incubation with PGG2 or PGH2 (1 jg/ml). The biological activity of the incubation mixtures was assayed on spirally cut strips of rabbit aorta and rabbit coeliac artery, superfused at 10 ml/min with Krebs bicarbonate containing a mixture of antagonists (Gilmore, Vane & Wyllie, 1968) plus indomethacin (2 gLg/ml). Changes in length of the assay tissues were detected by Harvard smooth muscle transducers and displayed on a Watanabe pen recorder. Homogenates of PMNs were incubated with PGG2 or PGH2 for 2 min at 0°C to test for thromboxane synthetase activity. None was detected in resting PMNs but when they were incubated with killed bacteria (Pertussis vaccine, Burroughs Wellcome) for 1 h at 370C prior to homogenization, increased RCS activity was generated and the coeliac artery contracted. This conversion was prevented by boiling the homogenate prior to incubation, or by addition of benzydamine (200 gg/ml) a drug which inhibits the formation of TXA2 by horse platelet microsomes (Moncada, Needleman, Bunting & Vane, unpublished) but not indomethacin (10 gg/ml). Incubation mixtures at neutral pH and 0°C were rapidly extracted with dry ether; the ether was evaporated under nitrogen and the residue taken up in buffer. Extracts contained RCS activity with a half life of 10-11 min at 0°C, 1-2 min at room temperature and less than 1 min at 37°C; values similar to those described for TXA2. These results show that homogenates of phagocytosing PMNs generate thromboxane A2-like activity. Thromboxane B2, the metabolite of TXA2 is chemotactic (Boot, Dawson & Kitchen, 1976); thus the production of thromboxanes by PMNs during phagocytosis could contribute to the inflammatory process.