Douglas R. Morton

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.

Acetyl glyceryl ether phosphorylcholine stimulates leukotriene B4 synthesis in human polymorphonuclear leukocytes.

Acetyl glyceryl ether phosphorylcholine (AGEPC) and leukotriene B4 (LTB4) induce concentration-dependent neutrophil aggregation. On a molar basis, LTB4 is approximately 10 to 100 times more potent than AGEPC. AGEPC-induced aggregation is attenuated by two inhibitors of arachidonate lipoxygenation, eicosatetraynoic acid and nordihydroguaiaretic acid, and to a lesser extent by the cyclooxygenase inhibitor, indomethacin. LTB4-induced aggregation is not readily reduced by the above inhibitors of arachidonic acid metabolism. Reverse phase high performance liquid chromatography, coupled with selective ion gas chromatography/mass spectrometry, shows that AGEPC stimulates neutrophils to synthesize sufficient LTB4 to account for the AGEPC response. In addition, the rate of LTB4 biosynthesis in response to AGEPC correlates well with the rate of AGEPC- and/or LTB4-induced neutrophils aggregation, and desensitization experiments indicate that AGEPC and LTB4 cross-desensitize. These data suggest that AGEPC-induced neutrophil aggregation may be mediated by LTB4.

Leukotriene C synthetase, a special glutathione S-transferase: Properties of the enzyme and inhibitor studies with special reference to the mode of action of U-60,257, a selective inhibitor of leukotriene synthesis

The cytosolic glutathione S-transferases of rat liver have been fractionated by chromatofocusing into 10 distinct fractions based on their reactivity with 2,4-dinitrochlorobenzene. All these fractions were capable of generating leukotriene C4 (LTC4) from leukotriene A4 (LTA4) to some extent. An inhibitor of leukotriene synthesis, U-60,257, inhibited the activity of these enzymes. The cytosolic glutathione S-transferases of rat basophil leukemia (RBL) cells have been similarly fractionated. U-60,257 inhibited the activity of some of these fractions but not that of others. None of the fractions of the enzyme from RBL cells formed LTC4 from LTA4. The microsomal glutathione S-transferase from rat liver also produced LTC4 from LTA4. It differs from the microsomal LTC synthetase of RBL cells in at least two respects: (1) The enzyme from RBL cells did not react with chromophoric substrates like dinitrochlorobenzene while the enzyme from liver did react. (2) Triton X-100 potentiated the activity of the enzyme from basophil leukemia cells and solubilized it, while it inhibited the activity of the leukotriene-synthesizing enzyme in the rat liver preparation. These results, along with a distinctly different inhibitor profile, indicate that LTC synthetase is a new and distinct glutathione S-transferase.

Feline polymorphonuclear leukocytes respond chemotactically to leukotriene B4 and activated serum but not to F-Met-Leu-Phe.

The chemotactic response of feline polymorphonuclear leukocytes (PMNs) to three types of chemoattractants was studied. Feline PMNs responded to leukotriene B4 as well as to agarose-activated autologous and homologous serum. However, no response was obtained to N-formylmethionylleucylphenylalanine (FMLP), and four similar peptides that activate the FMLP receptor (N-formylnorleucylleucylphenylalanine, N-formylmethionylphenylalanine, methionylleucylphenylalanine, and pepstatin.) Thus, feline PMNs are similar to equine, porcine, bovine and canine PMNs which also do not respond chemotactically to these peptides.

Diphenhydramine blocks the leukotriene-C4 enhanced mucus secretion in canine tracheain vivo

Leukotrienes (C4, D4) have been shown to enhance mucus seeretion in both isolated human airway tissue and intact canine tracheain vivo. They also have been implicated as putative mediators in several airways diseases. In previous canine studies the mucus enhancing effect of leukotriene-C4 was blocked by atropine, FLP 55,712, and hexamethonium but not by cutting the superior laryngeal and vagus nerves. We anesthetized mongrel dogs with chloralose (100 mg/kg) and urethane (500 mg/kg) and ventilated them on a pump. To visualize the secretions from submucosal glands, we exposed the mucosa of the upper trachea and coated its surface with powdered tantalum. Seeretions from the glands formed elevation in the tantalum layer (hillocks) with time: the number of tracheal hillocks (an index of mucus secretion) was measured at one or more of the four time points on six dogs after each treatment of the treatment sequence: no LTC4, LTC4, no LTC4+ blocker, and LTC4+ blocker. The potential blocker was diphenhydramine, an H1 antagonist for histamine. LTC4 was injected into the cranial thyroid artery which directly feeds the tracheal segment. We observed hillocks through a dissecting microscope, and the number of hillocks per 1.2 cm2 were counted for a 1–4 min interval. In 6 dogs with 12 responses, LTC4 (10 μg) gave a positive response that was significantly different from control (p<0.01–0.05) at 2–4 min.Diphenhydramine (n=6), 0.5 mg/kg, a dose which blocked a histamine challenge without blocking an acetylcholine challenge of secretion, gave a statistically significant (p<0.01–0.05) reduction in mucus secretion at 1–4 min. These results support the conclusion that leukotriene C4 induces mucus secretion in dogs that is blocked by prior diphenhydramine administration. This would indicate histamine has a role, but as yet an unknown mechanism in the action of leukotriene-C4 in enhancing mucus.

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.

Hydrolysis of an orally active platelet inhibitory prostanoid amide in the plasma of several species.

The prostanoid 3-oxa-4,5,6-trinor-3,7-inter-m-phenylene-PGE1-amide (OI-PGE1-amide) has a prolonged duration of oral platelet aggregation inhibitory activity when compared to the parent free acid (OI-PGE1) in the rat. When incubated in rat plasma at 1 microgram/ml for 30 seconds prior to addition of ADP, OI-PGE1-amide inhibits in vitro rat platelet aggregation approximately 50%. OI-PGE1 inhibits at 1 ng/ml. Inhibition of platelet aggregation by plasma incubated with OI-PGE1-amide (1 microgram/ml) increases with time and the rate of this increase differs with species. Incubation of OI-PGE1 in plasma does not result in an increase of platelet inhibitory activity with time. The increase of platelet inhibitory activity was assumed to indicate hydrolysis of OI-PGE1-amide to the more active OI-PGE1. A compound, different from OI-PGE1-amide, was isolated by an ion exchange/silica gel separation sequence from an incubation of OI-PGE1-amide in rat plasma. It had potent platelet aggregation inhibitory activity. This material was shown to be OI-PGE1 by thin-layer chromatography, gas chromatography and mass spectral analysis. Studies with [3H]-OI-PGE1-amide confirmed the formation of OI-PGE1 in plasma incubations. Amide hydrolytic activity was significantly different between species, the rank order being: rat greater than guine pig greater than monkey = human greater than dog. This relationship corresponded with that determined by measuring the increase in platelet inhibitory activity with time in plasma incubations of OI-PGE1-amide reported above. Present data indicate that (a) OI-PGE1-amide is hydrolyzed to the parent acid by plasma enzymes of several species and (b) hydrolytic activity of plasma varies widely between species.

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