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Previous studies of endothelin-1 (ET) synthesis have shown that some cultured endothelial cells secrete an intermediate product, big-endothelin-1 (bigET), suggesting that the processing of secreted bigET to ET may be physiologically significant. In this study, two pertinent ET converting enzyme activities, mast cell chymase I (EC 3.4.21.39) and a phosphoramidon-sensitive, neutral metalloprotease, were identified in a rat lung particulate fraction. We perfused rat lungs with bigET and chymostatin or phosphoramidon to study the relevance of these two proteases to the processing of extracellular bigET in vivo. Addition of compound 48/80 (a compound which activates mast cells, causing degranulation and release of chymase) to the perfusion buffer greatly increased hydrolysis of exogenously added bigET to ET. ET formation was inhibited completely by 32 microM chymostatin, whereas inhibition by 50 microM phosphoramidon was incomplete and variable. Perfusate histamine levels were used to monitor the extent of mast cell degranulation, and inhibition of ET production by phosphoramidon was attributed to inhibition of degranulation, per se. There was a direct correlation between perfusate ET and histamine levels in both control and phosphoramidon-treated (but not chymostatin-treated) lungs. Our results suggest that chymase from lung mast cells is capable of physiologically relevant extracellular processing by bigET to ET in the perfused rat lung.

The products of arachidonic acid metabolism in the 15,000xg supernatant of sonicated rat PMN are described. Only products derived from 5-lipoxygenase are observed. These products are 5-HETE and products derived from hydrolysis of LTA4, particularly LTB4. Some minor products derived from decomposition of 5-HPETE are also observed. The dependence of the activity of 5-lipoxygenase on enzyme and on substrate concentrations is presented and discussed in terms of a kinetic model that includes enzyme inactivation during turnover and substrate inhibition. The 5-lipoxygenase activity is stimulated by Ca++ and ATP.

The 15,000xg supernatant of sonicated rat PMN contains 5-lipoxygenase that converts arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene A4 and an HPETE peroxidase that catalyzes reduction of the 5-HPETE. The specificity of this HPETE peroxidase for peroxides, reducing agents, and inhibitors has been characterized to distinguish this enzyme from other peroxidase activities. In addition to 5-HPETE, the HPETE peroxidase will catalyze reduction of 15-hydroperoxyeicosatetraenoic acid, 13-hydroperoxyoctadecadienoic acid, and 15-hydroperoxy-8,11,13-eicosatrienoic acid, but not cumene or t-butylhydroperoxides. The HPETE peroxidase accepted 5 of 11 thiols tested as reducing agents. However, glutathione is greater than 15 times more effective than any other thiol tested. Other reducing agents, ascorbate, NADH, NADPH, phenol, p-cresol, and homovanillic acid, were not accepted by HPETE peroxidase. This enzyme is not inhibited by 10 mM KCN, 2 mM aspirin, 2 mM salicylic acid, or 0.5 mM indomethacin. When 5-[14C]HPETE is generated from [14C]arachidonic acid in the presence of unlabeled 5-HPETE and the HPETE peroxidase, the 5-[14C]HETE produced is of much lower specific activity than the [14C]arachidonic acid. This indicates that the 5-[14C]HPETE leaves the active site of 5-lipoxygenase and mixes with the unlabeled 5-HPETE in solution prior to reduction and is a kinetic demonstration that 5-lipoxygenase has no peroxidase activity. Specificity for peroxides, reducing agents, and inhibitors differentiates HPETE peroxidase from glutathione peroxidase, phospholipid-hydroperoxide glutathione peroxidase, a 12-HPETE peroxidase, and heme peroxidases. The HPETE peroxidase could be a glutathione S-transferase selective for fatty acid hydroperoxides.

In the 5-lipoxygenase pathway for arachidonic acid metabolism, reduction of 5-hydroperoxyeicosatetraenoic acid (5-HPETE) to 5-hydroxyeicosatetraenoic acid (5-HETE) is catalyzed by an activity different from glutathione peroxidase. Glutathione peroxidase here refers to the nonspecific peroxidase that catalyzes the reduction by glutathione of cumene hydroperoxide and a variety of other peroxides including 5-HPETE. This enzyme is inhibited by mercaptosuccinic acid. Preparations of the 15,000xg supernatant from lysed rat peritoneal polymorphonuclear leukocytes were the source of these activities. Thus, when glutathione peroxidase is inhibited to less than 0.5% of its normal activity by mercaptosuccinic acid, 5-HPETE is reduced as efficiently as in the absence of mercaptosuccinate. In lysate preparations from which endogenous glutathione has been removed, reduction of 5-HPETE is still observed but only in the presence of added reducing agents, e.g., 0.2 mM glutathione. When endogenous glutathione peroxidase is not inhibited, reduction of 5-HPETE occurs at a rate greater than 15-fold faster than can be accounted for by this activity. We conclude, therefore, that the glutathione peroxidase in rat PMNs is not kinetically competent to account for reduction of 5-HPETE. There is a distinct peroxidase that catalyzes this reaction. The 5-HPETE peroxidase can utilize glutathione as reducing agent but is not inhibited by mercaptosuccinate, and additional results indicate that it is inactivated during turnover.