James C. McGuire

The chemical structure of prostaglandin X (prostacyclin)

The chemical structure of prostaglandin X, the anti-aggregatory substance derived from prostaglandin endoperoxides, is 9-deoxy-6, 6alpha-epoxy-delta5-PGF1alpha. The stable compound formed when prostaglandin X undergoes a chemical transformation in biological systems in 6-keto-PGF1alpha. Prostaglandin X is stabilized in aqueous preparations by raising the pH to 8.5 or higher. The trivial name prostacyclin is proposed for 9-deoxy-6, 9alpha-epoxy-delta5-PGF1alpha.

Metabolism of arachidonic acid by human neutrophils: Characterization of the enzymatic reactions that lead to the synthesis of leukotriene B4

Human neutrophils stimulated with calcium ionophore A23187 synthesized 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4. Time-course studies showed that the concentrations of both products reached a maximum after 2 min after which the products were rapidly removed. In longer incubations, 5-HETE was esterified into membrane lipids, and leukotriene B4 was converted to 20- hydroxyleukotriene B4 and/or 20- carboxyleukotriene B4. The reaction is apparently self-limiting. After the maximum was reached, addition of fresh ionophore, Ca2+ or oxygen had little effect. Fresh arachidonic acid increases the yields of 5-HETE and delta 6-trans-leukotriene B4 but not additional leukotriene B4. Only the addition of fresh neutrophils gave additional leukotriene B4. This finding suggests that leukotriene B4 synthesis is limited by both substrate availability and enzyme inhibition by hydroperoxide intermediate. Exogenous arachidonic acid added with ionophore had different effects on the syntheses of leukotriene B4, delta 6-trans-leukotriene B4, and 5-HETE. As the arachidonic acid concentration increases, product formation increases in the following order: 5-HETE greater than delta 6-trans-leukotriene B4 greater than leukotriene B4. At a high concentration (more than 10 microM) of arachidonic acid, the synthesis of delta 6-trans-leukotriene B4 was greater than leukotriene B4 itself. Since delta 6-trans-leukotriene B4 represents the nonenzymatic decomposition of leukotriene A4, we suggest that one of the rate-limiting steps in the synthesis of leukotriene B4 is the leukotriene A4 hydrolase. Our data suggest the synthesis of leukotriene B4 is under the control of three factors: (1) substrate availability; (2) limited capacity of the leukotriene A4 hydrolase, and (3) enzyme inactivators generated during the reaction, such as hydroperoxide intermediate. The tightly controlled system assures only a finite amount of this powerful bioactive substance will be produced under most conditions.

Inhibition of human neutrophil arachidonate 5-lipoxygenase by 6, 9-deepoxy-6, 9-(phenylimino)- Δ 6, 8-prostaglandin I1 (U-60257)

6,9-Deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin I1 (U-60257) and its methyl ester (U-56467) are selective inhibitors of leukotriene C and D biosynthesis both in vitro and in vivo. In this study, we demonstrated that the principal site of inhibition may be arachidonate 5-lipoxygenase, the initial enzyme of leukotriene biosynthesis. U-60257 and its methyl ester block LTB4 synthesis in human peripheral neutrophils with an ID50 of 1.8 and 0.42 microM respectively. This inhibitory action of U-60257 on neutrophil 5-lipoxygenase can be reduced or reversed by a high concentration of exogenous arachidonic acid. U-60257 at 100 microM has no apparent effect on the following enzymes. 1) cyclooxygenase of sheep vesicular gland or human platelets; 2) 12-lipoxygenase of human platelets and 3) soybean 15-lipoxygenase. Thus, we conclude that U-60257 and its methyl ester are potent and selective inhibitors of arachidonate 5-lipoxygenase.

Effect of 6,9-deepoxy-6,9-(phenylimino)-Δ 6,8-prostaglandin 11 (U-60,257), an inhibitor of leukotriene synthesis, on human neutrophil function

Abstract A23187, a calcium ionophore, stimulated a time-dependent generation of 5(S), 12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid (leukotriene B4), production of superoxide anion (O2−) and release of granule-associated β-glucuronidase and lysozyme by human neutrophils. Leukotriene B4 also elicited the selective release of granule enzymes from cytochalasin B-treated neutrophils. U-60,257, a recently identified inhibitor of leukotriene (LT) C4 and D4 synthesis, caused a dose-related (1–10 μM) suppression of LTB4 production by A23187-activated neutrophils. Degranulation and O2− generation by neutrophils exposed to A23187 and the chemotactic oligopeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), were also inhibited with U-60,257.

An evaluation of the relationship between arachidonic acid lipoxygenation and human neutrophil degranulation

Abstract Exposure of neutrophils to the calcium ionophore A23187 (5 μ M ) resulted in a time-dependent generation of 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and the selective release of the granule-associated enzymes, β-glucuronidase and lysozyme. The acetylenic fatty acids, 5,8,11,14-eicosatetraynoic acid, 4,7,10,13-eicosatetraynoic acid, 6,9,12-octadecatriynoic acid, and 10,13-eicosadiynoic acid, which inhibit the lipoxygenase and/or cyclooxygenase pathways of arachidonic acid metabolism, caused a dose-related inhibition of 5-HETE production (50–100 μ M ) by and enzyme release (0.01–100 μ M ) from A23187-stimulated neutrophils. However, these compounds also enhanced 5-HETE synthesis at concentrations (1–10 μ M ) which suppressed enzyme release. Certain phenylhydrazone derivatives, while having no effect on β-glucuronidase or lysozyme release, caused a significant inhibition of A23187-induced 5-HETE generation. Further, the aforementioned acetylenic acids as well as 5,8-tetradecadiynoic acid, which is inactive against 5-lipoxygenase, inhibited N -formyl-methionyl-leucyl-phenylalanine-induced granule exocytosis even though this secretagogue failed to stimulate 5-HETE synthesis. These data suggest that the 5-lipoxygenation of arachidonic acid may not be required for granule enzyme release from human neutrophils.

9,11-Iminoepoxyprosta-5,13-dienoic acid is a selective thromboxane A2 synthetase inhibitor.

9,11-Iminoepoxyprosta-5,13-dienoic acid inhibits the thromboxane A2 synthetase in platelet and lung microsomal enzyme preparations and in intact platelets. It does not inhibit the protaglandin I2 synthetase in aorta or lung microsomes and intact Balb 3T3 fibroblasts. In lung microsomes, which contain both enzymes, 9,11-iminoepoxyprosta-5,13-dienoic acid inhibits only thromboxane A2 formation and augments prostaglandin I2 formation. This inhibitor is more selective than other reported prostaglandin endoperoxide analogs which inhibit the platelet thromboxane synthetase.

Production of prostaglandin D2 by murine macrophage cell lines

Several tumor-derived murine macrophage cell lines were evaluated in vitro as cloned prototypes of tissue macrophages for their ability to metabolize arachidonic acid. Unexpectedly, two cell lines, J774A.1 and WR19M.1, rapidly converted exogenous 14C-arachidonic acid (AA) to a single major prostaglandin metabolite. The compound, PGD2, was positively identified by TLC, HPLC, and GC-MS. The enzymatic formation of the PGD2 was shown by inhibition of its formation by indomethacin and reduced formation of 14C-PGD2 from 14C-PGH2 in boiled cells. When J774A.1 cells were prelabeled with 3H-AA, cultured for 24 hours, and stimulated with lipopolysaccharide (LPS), PGD2 was again the predominant product. No other tumor derived cell lines, including several other murine macrophage lines, produced significant amounts of PGD2. Elicited and activated murine peritoneal macrophages produced only small amounts of PGD2, but resident peritoneal macrophages produced modest amounts of PGD2. Exaggerated formation of PGD2 by J774A.1 and WR19M.1 cells may be a consequence of neoplastic transformation or the clonal expansion of a minor subpopulation of normal tissue macrophages.

Metabolism of (5E)-6a-carboprostaglandin I2 by rhesus monkey lung 15-OH prostaglandin dehydrogenase

(5E)-6a-Carbaprostaglandin I2 (carbacyclin) was oxidized to (5E)-15-dehydro-6a-carbaprostaglandin I2 (15-dehydrocarbacyclin) by partially purified rhesus monkey lung prostaglandin dehydrogenase (PGDH). The (5E-15-dehydro-6a-carbaprostaglandin I2 was isolated by preparative thin-layer chromatography and identified by gas chromatography-mass spectrometry. A Lineweaver-Burke plot gave an apparent Km value of 2.9 microM and a Vmax of 35.7 nmoles carbacyclin oxidized/mg protein/min. These values are similar to previously reported Km and Vmax values for PGI2 and PGE1.

THE STRUCTURE OF PROSTAGLANDIN I 2

The chemical structure of prostaglandin I, the anti-aggretory substance derived from prostaglandin endoperoxides , is 9-deoxy-6, 9α-epoxy-Δ 5 -PGF 1 α .The stable compound formed when prostaglandin X undergoes a chemical transformation in biological systems is 6-keto-PGF 1 α. Prostaglandin I is stabilized in aqueous preparations by raising the pH to 8.5 or higher. The trivial name prostacyclin is proposed for 9-deoxy-6, 9α-epoxy-Δ 5 -PGF 1 α