Hepoxilin analogs, potential new therapeutics in disease.

We have chemically synthesized several stable analogs of the naturally occurring hepoxilins, 12-LO products derived from arachidonic acid, which we found to have promising actions in a variety of test systems of disease. The analogs, PBTs, afford chemical and biological stability to the hepoxilin molecule. This article reviews some of our latest observations with the PBTs in the areas of inflammation (inhibition of the bleomycin-evoked lung fibrosis in mice in vivo), platelet aggregation (antagonism of the thromboxane receptor in human platelets in vitro) and thrombosis (inhibitors in vivo), and cancer (apoptosis of the human leukemia cell line, K562 in vitro and in vivo). The demonstration that the PBTs are active in vivo suggests that they can serve as a platform for their further development as novel therapeutics in disease.

Hepoxilins, potential endogenous mediators of insulin release.

Evidence is presented to show that pancreatic islets of Langerhans are capable of producing hepoxilins A3 and B3 from endogenous substrates as well as 14C-labeled 12-HPETE. Both hepoxilins are active in stimulating the release of insulin from these cells in the presence of 10 mM glucose. These experiments suggest that the hepoxilins may participate as potential endogenous mediators of insulin release in islets of Langerhans.

Hepoxilins: a review on their cellular actions.

This review is intended to summarize the biological actions of the hepoxilins reported to date. These actions appear to have, as their basis, changes in intracellular concentrations of ions including calcium and potassium ions as well as changes in second messenger systems. Recent evidence suggests that the biological actions of the hepoxilins may be receptor-mediated as indicated from data showing the existence of hepoxilin-specific binding proteins in the human neutrophil. Such evidence also implicates the association of G-proteins both in hepoxilin-binding as well as in hepoxilin action. The potential use of stable analogs of the hepoxilins is discussed as well as the directions in which this area is heading.

Hemoglobin- and hemin-catalyzed transformation of 12L-hydroperoxy-5,8,10,14-eicosatetraenoic acid.

12L-Hydroperoxy-5,8,10,14-eicosatetraenoic acid (12L-HPETE) containing 2H at positions 5,6,8,9,11,12,14,15 was prepared during brief incubations of rat platelets with a mixture of [1-14C]arachidonic acid and 5,6,8,9,11,12,14,15-[ 2H8 ]arachidonic acid. The labelled 12L-HPETE was converted into a mixture of products, including 8- and 10-hydroxy-11,12- epoxyeicosatrienoic acids, during incubations in phosphate buffer containing bovine hemin. Hemoglobin could replace hemin in this reaction, which did not proceed in buffer alone. Mass spectral analysis showed these hydroxy epoxides to be identical to those previously identified from rat lung. These epoxides were hydrolyzed by a rat lung enzyme previously shown to contain epoxide hydratase activity. These results show that the hydroxy epoxides could be formed from 12L-HPETE nonenzymatically.

Docosahexaenoic acid causes accumulation of free arachidonic acid in rat pineal gland and hippocampus to form hepoxilins from both substrates.

Hepoxilins (Hx) are biologically active metabolites of arachidonic acid (AA) formed regioselectively from 12(S)-HPETE by 'hepoxilin synthase'. Hx modulate synaptic neurotransmission in hippocampal CA1 neurons, and inhibit norepinephrine release in hippocampal slices. During the course of our studies we investigated whether docosahexaenoic acid (DHA) was a substrate for hepoxilin formation. We used two tissues, the pineal gland and hippocampal slices. Tissues were incubated alone or with AA (20 microg/ml) or DHA (20 microg/ml). After 60 min at 37 degrees C, samples were acid-extracted to convert Hx into their stable trioxilin (TrX) form and analyzed as the Me-TMSi derivatives by EI-GC/MS to determine the structures of the DHA metabolites, and as PFB-TMSi derivatives by GC/MS in the NICI mode using SIM to simultaneously quantify TrX products of the 3-series (derived from AA) monitored at m/z 569, while those of the 5-series (derived from DHA) were monitored at m/z 593. Results show good conversion of both substrate fatty acids by the rat pineal gland and hippocampal slices, into the 3-series (21.3 +/- 5.8 and 12.5 +/- 2.2 ng/microg protein, respectively) and 5-series TrX (12.3 +/- 2.7 and 2.9 +/- 0.4 ng/microg protein, respectively). Surprisingly though, experiments with DHA, in both tissues, also showed formation of TrX derived from endogenous AA (3-series) (10.4 +/- 8.3 and 3.1 +/- 2.1 ng/microg protein, respectively). These experiments demonstrate previously unreported actions of DHA causing the accumulation of AA, which is converted into hepoxilins. In order to prove that AA is accumulated during DHA stimulation of the tissue, we carried out separate experiments with hippocampal slices in which the neutral lipids and phospholipids were labeled with [14C]AA. DHA caused a time-dependent appearance of free [14C]AA which was released mostly from the TG pool. Measurement of the AA/DHA ratio in the TG pool by GC/MS further indicated that DHA is incorporated into the TG at the expense of AA. These results demonstrate that DHA competes with AA for acylation into the metabolically active TG fraction, and both fatty acids are converted into hepoxilins of the corresponding series.

Mechanistic studies on the biosynthesis of 6-ketoprostaglandin F1alpha.

The isolation, structure and biosynthesis of a novel metabolite of arachidonic acid formed during incubations of rat stomach homogenates is reported. This product was formed from unlabeled, tritium-labeled and deuterium-labeled arachidonic acid and the prostaglandin endoperoxides, G2 and H2, demonstrating its formation via the "prostaglandin endoperoxide synthetase" pathway. Its structure, 6-ketoprostaglandin F1alpha, was based on mass spectral interpretation of several derivatives (undeuterated and deuterated) and confirmed through chemical synthesis. 6-Ketoprostaglandin F1alpha is unique in being formed from a substrate of the "2" series of prostaglandins, i.e. arachidonic acid, yet belonging to the "1" series. A mechanism for its biosynthesis is proposed involving a specific cyclising enzyme which we term, 6(9)-oxycyclase.

Distribution of prostaglandin biosynthetic pathways in several rat tissues. Formation of 6-ketoprostaglandin F1alpha.

The capacity of homogenates of rat lung, liver, spleen, whole stomach, stomach fundus, kidney and heart to form prostaglandin E2, F2alpha, D2, thromboxane B2 and 6-ketoprostaglandin F1alpha was investigated. All products were quantitated by mass spectrometry. The results demonstrated interesting differences between tissues in the direction of the 'prostaglandin synthetase' to specific products (e.g. as in spleen and stomach).

The 6-ketoprostaglandin F1alpha pathway in the lamb ductus arteriousus.

Homogenates of the lamb ductus arteriosus at term have the capacity to convert exogenous arachidonic acid into prostaglandins E2, F2alpha and 6-ketoprostaglandin F1alpha. No evidence was obtained for the formation of prostaglandin D2 and thromboxane B2. All products were quantitated by mass spectrometry. 6-Ketoprostaglandin F1alpha was the major compound, formed in amounts in excess of 10 fold over prostalgandins E2 and F2alpha.

Catabolism of an isolated, purified intermediate of prostaglandin biosynthesis by regions of the adult rat kidney.

1. A heat labile, cold-stable, stannous chloride-reducible intermediate of prostaglandin biosynthesis was formed in good yield (greater than 60%) from 3H-labeled arachidonic acid during brief incubations (30--90 s, 37 degrees C) with sheep seminal vesicle microsomes in the presence of p-hydroxymercuribenzoate (4 mM). This intermediate appears to have properties similar to one of the endoperoxides (15-hydroxyprostaglandin-9,11-endoperoxide) recently isolated by Hamberg and Samuelsson (Proc. Natl. Acad. Sci. U.S. (1973) 70, 889-903) AND Nugteren and Hazelhof (Biochem. Biophys. Acta. (1973) 326, 448-461). 2. Treatment of the purified intermediate with homogenates of rat kidney cortex, medulla and papilla resulted within 2 min (37 degrees C) in complete conversion into several compounds including prostaglandins E2 and F2alpha. The main product (40-50% yield formed by papilla homogenates was prostaglandin E2. The conversion into prostaglandin E2 was largely abolished by previous bo9ling of the homogenate whereas the conversion into prostaglandin F2alpha was not. The intermediate was stable in buffer for the same period of incubation. 3. The ratio of tritiated prostaglandins E2: F2alpha obtained were: papilla, 1.90; medulla, 0.76; cortex, 0.48. 4. These observations indicate that both types of prostaglandins can be formed by all three regions of the rat kidney and that regional differences exist in the proportion of E2 : F2alpha that is formed. Whereas prostaglandin E2 is mostly formed by an enzymatic process, prostaglandin F2alpha is not.

9-Hydroxyprostaglandin dehydrogenase in rat kidney cortex converts prostaglandin I2 into 15-keto-13,14-dihydro 6-ketoprostaglandin E1.

15-Keto-13,14-dihydro 6-ketoprostaglandin E1 was positively identified by gas chromatography-mass spectrometry with negative-ion chemical ionisation detection from samples of rat kidney high-speed supernatant incubated with prostaglandin I2 in the presence of NAD+. A decreased formation of this product was observed when NAD+ was substituted with NADP+ and none was observed in the absence of nucleotide or substrate prostaglandin I2. Experiments with [9 beta-3H]prostaglandin I2 showed a time- and concentration-dependent loss of tritium which appeared as tritiated water, typical of reaction of [9 beta-3H]prostaglandin substrates with the enzyme, 9-hydroxyprostaglandin dehydrogenase. Time-course measurements of the appearance of tritiated water showed similar rates with 6-keto[9 beta-3H]prostaglandin F1 alpha and 15-keto-13,14-dihydro 6-keto[9 beta-3H]prostaglandin F1 alpha as substrates. These experiments suggest that the transformation of prostaglandin I2 and 6-ketoprostaglandin F1 alpha into the 15-keto-13,14-dihydro 6-ketoprostaglandin E1 catabolite occurs in this in vitro preparation via the corresponding 15-keto-13,14-dihydro catabolite of 6-ketoprostaglandin F1 alpha.

Application of prostaglandin-profiling techniques to study the release of prostaglandins from the fetal lamb ductus arteriosus.

Through gas chromatographic techniques with capillary columns and electron-capture detection which allow the resolution of prostaglandins F2 alpha, D2, E2, thromboxane B2 and 6-ketoprostaglandin F1 alpha and their 15-keto-and 15-keto-13,14-dihydro metabolites, we have studied the release of these products from the ductus arteriosus in vitro. 6-Ketoprostaglandin F1 alpha was the major product in the incubation fluid while 15-keto-13,14-dihydro prostaglandin F2 alpha was the major product in the tissue. Prostaglandin E2 was almost undetectable in the fluid and tissue. Prostaglandin I2 formed in major proportions by the tissue is released mostly as 6-ketoprostaglandin F1 alpha, although minor amounts of its 15-keto-13,14-dihydro metabolites were detected. These results show differential release of prostaglandin types by this tissue, demonstrating formation and metabolism of endogenous prostaglandins at the same time.

Distribution of prostaglandin biosynthetic pathways in organs and tissues of the fetal lamb.

Five prostaglandins, i.e. prostaglandins E2, F2alpha and D2, 6-keto-prostaglandin F1alpha and thromboxane B2, were measured by mass spectrometry. Homogenates of fetal lamb brain, lung, liver, spleen and kidney and the ductus arteriosus, aorta and pulmonary artery formed different amounts of each product. Although the main prostaglandin in the fetal organs was prostaglandin E2, arterial tissue formed mostly 6-keto-prostaglandin F1alpha. These results demonstrate significant differences between organs and tissues in the relative direction of the 'prostaglandin synthetase' enzyme complex.

Prostaglandin biosynthesis and catabolism in the lamb ductus arteriosus, aorta and pulmonary artery.

Homogenates of tissues from fetal and neonatal lamb ductus arteriosus, aorta and pulmonary artery have the capacity to convert arachidonic acid as well as the intermediate prostaglandin endoperoxide, prostaglandin H2, into three products: prostaglandins E2, F2alpha and a major product 6-ketoprostaglandin F1alpha. The three tissues also displayed prostaglandin 15-hydroxydehydrogenase and 13-reductase catabolic activities. The catabolishing system showed considerable substrate specificity: prostaglandin E1 was a good substrate whereas prostaglandins F1alpha and F2alpha were completely devoid of catabolism. The complete system was observed in immature as well as mature arterial vessels, in the fetus as well as the neonate (up to 7 days old). These experiments demonstrate the presence of several components of the prostaglandin system in these tissues and offer biochemical evidence for the implication of prostaglandins E2 and I2 in the maintenance of the ductus and neighboring vessels in a relaxed state in the fetus.

Age-dependent increase in the formation of prostaglandin I2 by intact and homogenised aortae from the developing spontaneously hypertensive rat.

The prostaglandin I2 biosynthetic capacity of aortae from spontaneously hypertensive rats of various ages (1, 2, 3, 4, and 5 months) was investigated. An age-dependent increase in enzyme activity was observed reaching maximum by three months of age which correlated well with the age- related increase in the systolic blood pressure. These results support our notion that the enhanced aortic synthesis of the potent vasodepressor prostaglandin I2 by the spontaneously hypertensive rat likely represents an adaptive mechanism for the attenuation of the sustained elevation in blood pressure in this animal model.

Comparison between the in vivo rate of metabolism of prostaglandin I2 and its blood-pressure-lowering response after intravenous administration in the rat.

Intravenous bolus injection of prostaglandin I2 in the Inactin-anaesthetised rat produces a slow dose-dependant vasodepression which reaches maximum approximately 15 s. after injection. Administration of 9 beta-[3H1]-prostaglandin I2 by the same route followed by serial arterial sampling and TLC analysis revealed a slow conversion into one less polar metabolite starting after 20 s and reaching 40% by two minutes in the circulation. These experiments indicate that prostaglandin I2 survives pulmonary transit for a sufficiently long time to elicit a biological action. Thus its continuous systemic vascular synthesis could play an important role in the control of hypertension.

Catabolism of 6-ketoprostaglandin F1alpha by the rat kidney cortex.

Homogenates of the rat kidney cortex converted 5,8,9,11,12,14,15-hepta-tritiated 6-ketoprostaglandin F 1alpha into one major product identified by gas chromatography-mass spectrometry of the methoxime-methyl ester trimethylsilyl ether derivative as 6,15-diketo-9,11-dihydroxyprost-13-enoic acid. The sequence of derivatisation i.e. methoximation prior to methylation, was crucial as methylation of 15-keto catabolites of the E, F and 6-keto-F series affords degradation products. The corresponding 15-keto-13,14-dihydro catabolite was formed in much smaller quantities. Time course studies indicated that 6-keto-prostaglandin F1alpha was catabolised at a slower rate (about 2-5 fold) than prostaglandin F1alpha. The catabolic activity was blocked by NADH.

The enzymatic conversion of arachidonic acid into 8,11,12-trihydroxyeicosatrienoic acid. Resolution of rat lung enzyme into two active fractions.

The conversion of arachidonic acid into 8,11,12-trihydroxyeicosatrienoic acid by rat lung high-speed supernatant has been resolved into two separate stages through ammonium sulfate precipitation. The first stage is catalysed by 0-30% ammonium sulfate fraction and converts arachidonic acid and 12-hydroperoxyeicosatetraenoic acid into an intermediate, X. X is subsequently utilized in the second stage by the fraction sedimented at 30-50% saturation in ammonium sulfate to form two isomeric 8,11,12-trihydroxyeicosatrienoic acids.

Identification of a 'urinary-type' metabolite of prostaglandin F2 alpha in the rat circulation.

During constant slow intravenous infusion of tritiated prostaglandin F2 alpha into male adult rats, a major portion of radioactivity in blood appeared as a new metabolite, identified as tetranor-15-keto-13,14-dihydroprostaglandin F2 alpha (18.8 +/- 4.2%, n = 7). The previously recognised blood metabolite, 15-keto-13,14-dihydroprostaglandin F2 alpha, was also observed (15.1 +/- 4.1%, n = 7). 15-keto-13,14-Dihydroprostaglandin F2 alpha disappeared quickly from the circulation while tetranor-15-keto-13,14-dihydroprostaglandin F2 alpha was still detected (8.6 +/- 2.8%, n = 3) 2 h after infusion was stopped. These results indicate that tetranor-15-keto-13,14-dihydroprostaglandin F2 alpha, because of its slow disappearance from the circulation, may provide a better indicator than 15-keto-13,14-dihydroprostaglandin F2 alpha of prostaglandin F2 alpha synthesis in vivo.

Formation of 8,11,12-trihydroxyeicosatrienoic acid by a rat lung high-speed supernatant fraction.

An enzyme was found in the high-speed (100 000 x g) supernatant fraction of a rat lung homogenate which catalysed the conversion of arachidonic acid into 8,11,12-trihydroxyeicosatrienoic acid. The isomeric 8,9,12-triol was not detected. The structure of the isolated product was confirmed by mass spectrometric analysis of the methyl ester t-butyldimethylsilyl ether derivative. These results indicate that formation of both positional isomers is carried out by separate enzymes, the distributions of which are not restricted to platelets.

Antibodies to 5,6-dihydroprostaglandin I2 trap endogenously produced prostaglandin I2 in the rat circulation.

Rabbit immunoglobulins raised against 5,6-dihydroprostaglandin I2 which crossreact with prostaglandin I2 were infused intravenously into Inactin-anaesthetised male adult rats. Clearance of intravenously administered [3H]prostaglandin I2 from the blood, which is normally rapid (t 1/2 approx. 45 s), was delayed strikingly in the presence of antibody (t 1/2 approx. 60 min). The antibodies also sequestered the endogeneously synthesized prostaglandin I2 and inhibited its metabolism. The rate of appearance of endogenous prostaglandin I2 in the circulation of the rat was measured in the following way: arterial blood samples (0.5 ml) were withdrawn before, during and at various time intervals (up to 180 min) after infusion of antibodies had terminated; the prostaglandins were extracted from the blood with ethanol, and the extracts were assayed by radioimmunoassay (before and after separation by high-pressure liquid chromatography) for the following prostaglandins: 6-keto-F1 alpha, E2, F2 alpha and 13,14-dihydro-15-keto-metabolites of E2 and F2 alpha. Rapid and specific time-related increments of prostaglandin I2 (detected serologically as 6-keto-F1 alpha) were observed. At 180 min these increases ranged from 1500- to 2500-fold over preinfusion levels. No significant increases were observed in the other prostaglandins measured; nor were there increases in 6-keto-F1 alpha when saline or immunoglobulins from non-immune plasma were infused into rats. When measured by these procedures, no appreciable differences in 6-keto-F1 alpha production were found between Japanese normotensive and spontaneously hypertensive rats.