Time-dependent inhibition of the cyclooxygenase pathway by 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid

We examined effects of small dose (1 microM or less) of exogenous 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) on the formation of cyclooxygenase products from exogenous arachidonic acid (AA) in washed human platelets. With a simultaneous addition of AA, 12-HPETE did not affect the formation of thromboxane (TX)B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT). However, by being preincubated with platelets before an addition of AA, 0.1 microM or greater of 12-HPETE inhibited the formation of TXB2 and HHT dose-dependently. In addition, the inhibitory effect of 12-HPETE increased as the preincubation time was prolonged. These results suggest that 12-HPETE is a strong inhibitor for the cyclooxygenase pathway.     

Synthesis of two hydroxy fatty acids from 7,10,13,16,19-docosapentaenoic acid by human platelets

Platelets metabolize 7,10,13,16,19-docosapentaenoic acid (22:5(n-3] into 11-hydroxy-7,9,13,16,19- and 14-hydroxy-7,10,12,16,19-docosapentaenoic acid via an indomethacin-insensitive pathway. Time-dependent studies with 20 microM substrate show a lag in the synthesis of both the 11- and 14-isomers which was not observed for the synthesis of thromboxane B2 (TXB2), 5,8,10-heptadecatrienoic acid, and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) from arachidonic acid. When platelets were incubated with increasing concentrations of 22:5(n-3), the 11- and 14-isomers were not produced until the substrate concentration exceeded 5 microM unless arachidonic acid was also added to the incubations. The stimulatory effect of arachidonic acid was not blocked by indomethacin thus suggesting that 12-hydroperoxyeicosatetraenoic acid or 12-HETE derived from arachidonic acid may activate the platelet lipoxygenase(s) which metabolize 22:5(n-3). Incubations containing 20 microM 22:5(n-3) and increasing levels of [1-14C]arachidonic acid show that the (n-3) acid inhibits the synthesis of both 5,8,10-heptadecatrienoic acid and TXB2 from arachidonic acid. At the same time, 12-HETE synthesis increased due to substrate shunting to the lipoxygenase pathway.     

The stimulation of arachidonic acid metabolism in human platelets by hydrodynamic stresses

Even though shear-induced platelet activation and aggregation have been studied for about 20 years, there remains some controversy concerning the arachidonic acid metabolites formed during stress activation and the role of thromboxane A2 in shear-induced platelet aggregation. In this study, platelets were labelled with [1-14C]arachidonic acid to follow the metabolism of arachidonic acid in stimulated platelets using HPLC and scintillation counting. Platelets activated by thrombin formed principally thromboxane A2, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE). In contrast, for platelets activated by shear--though arachidonic acid metabolism was stimulated--only 12-HETE was formed and essentially no cyclooxygenase metabolites were detected. This indicates that physical forces may initiate a different pathway for eicosanoid metabolism than most commonly used chemical stimuli and perhaps also implies that regulation of the cyclooxygenase activity may be a secondary level of regulation in eicosanoid metabolism.     

The effect of adrenalectomy on the aggregation of rat blood platelets and the endogenous formation of thromboxane B2 and 12-hydroxy-5,8,10,14-eicosatetraenoic acid

Rat platelets were isolated and labelled with [1-14C] arachidonic acid. After aggregation thromboxane B2, 12-hydroxy 5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-eicosatetraenoic acid (12-HETE) were the main metabolites formed. A comparison was made between several properties of the platelets of adrenalectomized and sham operated rats. There was no difference in collagen-induced aggregation. The amount of 12-HETE and the sum of TxB2 and 12-HETE formed from endogenous arachidonic acid after aggregation was higher in the first group.     

Selective inhibitors of platelet lipoxygenase: 4,7,10,13-eicosatetraynoic acid and 5,8,11,14-henicosatetraynoic acid

Nine acetylenic acids were evaluated to determine what structural features are required for selective inhibition of platelet lipoxygenase. Both 4,7,10,13-icosatetraynoic acid and 5,8,11,14-henicosatetraynoic acid inhibited the synthesis of 12-L-hydroxy-5,8,10,14-icosatetraenoic acid (HETE) more than 95 percent without significantly altering the production of either thromboxane B2 or 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT). The ID50 concentrations (microM) for inhibiting the synthesis of thromboxane B2 and HETE were respectively 51 and 0.46 with 4,7,10,13-icosatetraynoic acid while similar concentrations of 64 and 0.31 were found for 5,8,11,14-henicosatetraynoic acid.     

Immunoaffinity purification and characterization of thromboxane synthase from porcine lung

Thromboxane synthase has been purified 620-fold from porcine lung microsomes by a three-step purification procedure including Lubrol-PX solubilization, reactive blue-agarose chromatography, and immunoaffinity chromatography. The purified enzyme exhibited a single protein band (53,000 daltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Rabbit antiserum raised against the purified enzyme immunoprecipitated thromboxane synthase activity from crude enzyme preparations of porcine lung, cow lung, and human platelets, indicating the existence of structural homology of the enzyme in these species. Immunoblotting experiment identified the same polypeptide (53,000 daltons) in porcine lung and a polypeptide of 50,000 daltons in human platelets, confirming the identity of the enzyme and the specificity of the antiserum. Purified thromboxane synthase is a hemoprotein with a Soret-like absorption peak at 418 nm. The enzyme reaction has a Km for 15-hydroxy-9 alpha, 11 alpha-peroxidoprosta-5, 13-dienoic acid of 12 microM, an optimal pH of 7.5, and an optimal temperature of reaction at 30 degrees C. Purified thromboxane synthase catalyzed the formation of both thromboxane B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT). The ratios of HHT to thromboxane B2 varied from 1.6 to 2.1 dependent on the reaction conditions. Except that HHT was formed at a greater rate, the formation of HHT and that of thromboxane responded identically to pH, temperature, substrate concentration, kinetics of formation, metal ions, and inhibitors suggesting that the two products are probably formed at the same active site via a common intermediate. Thromboxane synthase was irreversibly inactivated by 15-hydroxy-9 alpha, 11 alpha-peroxidoprosta-5,13-dienoic acid during catalysis and by treatment of 15-hydroperoxyeicosatetraenoic acid. The irreversible inactivation, however, could be protected by reversible inhibitors such as sodium (E)-3-[4-(1-imidazolylmethyl)phenyl]-2-propenoate and 15-hydroxy-11 alpha,9 alpha-(epoxymethano)-prosta-5,13-dienoic acid, suggesting that the inactivation occurred at the active site of the enzyme. The catalytic inactivation of thromboxane synthase and the greater rate of formation of HHT in thromboxane-synthesizing system may probably play important regulatory roles in the control of thromboxane synthesis.     

Metabolism of 7,10,13,16-docosatetraenoic acid to dihomo-thromboxane, 14-hydroxy-7,10,12-nonadecatrienoic acid and hydroxy fatty acids by human platelets

Human platelets metabolize 7,10,13,16-docosatetraenoic acid (22:4(n - 6)) into dihomo-thromboxane B2 and 14-hydroxy-7,10,12-nonadecatrienoic acid at about twenty percent of the rate they convert arachidonic acid to thromboxane B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid. 14-Hydroxy-7,10,12,16-docosatetraenoic was the major metabolite produce via the lipoxygenase pathway. Several other hydroxy acids were also produced in small amounts via an indomethacin-insensitive pathway. Incubation of 20 microM arachidonic acid with various levels of 22:4(n - 6) resulted in a dose-dependent inhibition of both thromboxane B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid production. Conversely, 12-hydroxy-5,8,10,14-eicosatetraenoic acid synthesis was stimulated because of substrate shunting to the lipoxygenase pathway. These results show that 22:4(n - 6) may modify platelet function both by serving as a precursor for a 22-carbon thromboxane and by suppressing the synthesis of thromboxane A2 from arachidonic acid. In addition, our results suggest that simultaneous release of 22:4(n - 6) and arachidonic acid from platelet phospholipids will result in an elevation of both 12-hydroxy-5,8,10,14-eicosatetraenoic acid levels as well as simultaneous synthesis of 14-hydroxy-7,10,12,16-docosatetraenoic acid.     

Monohydroxylated fatty acid substrate specificity of human leukocyte 5-lipoxygenase and omega-hydroxylase.

Various monohydroxylated fatty acids were synthesized from eicosapolyenoic acids, namely arachidonic (20:4 omega-6), timnodonic (20:5 omega-3), dihomogammalinolenic (20:3 omega-6) and mead (20:3 omega-9) acids. 12-Hydroxy derivatives, as well as 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), were produced with platelets as the enzyme source, and 15-hydroxy derivatives were produced by soya bean lipoxygenase treatment. Each monohydroxylated fatty acid was incubated with human leukocytes in the presence or absence of the calcium ionophore A23187, and dihydroxylated products were analysed by h.p.l.c. 12-Hydroxy derivatives of 20:4 omega-6, 20:5 omega-3 and 20:3 omega-9 were similarly oxygenated by both the 5-lipoxygenase and the omega-hydroxylase. As expected, the 12-hydroxy derivative of 20:3 omega-6 was not a substrate for 5-lipoxygenase, but surprisingly, omega-6 oxygenated products, like 15-OH-20:4 or HHT, were not converted by the enzyme, although being potential substrates because of the presence of two double bonds at C-5 and C-8. omega-6 oxygenated derivatives were also poorly converted by leukotriene B4 omega-hydroxylase, a cytochrome P-450-dependent enzyme. It is concluded that both leukocyte 5-lipoxygenase and omega-hydroxylase exhibit a substrate specificity towards monohydroxylated fatty acids with respect to their double bonds and/or the carbon position of the alcohol function.     

Metabolism of 4,7,10,13,16-docosapentaenoic acid by human platelet cyclooxygenase and lipoxygenase

Washed human platelets are shown to metabolize 4,7,10,13,16-docosapentaenoic acid into three major metabolites which were purified by reverse-phase HPLC. The mass spectra of the methyl ester-trimethylsilyl ether and ethyl ester-trimethylsilyl ether of compound A established it as delta 4-dihomo-thromboxane B2. Compound B was shown to be 14-hydroxy-4,7,10,12-nonadecatetraenoic acid, which is analogous to 12-hydroxy-5,8,10-heptadecatrienoic acid from arachidonic acid. Compound C was produced via an indomethacin-insensitive pathway and was identified as 14-hydroxy-4,7,10,12,16-docosapentaenoic acid. Time- and substrate-dependent studies showed that compounds A,B and C were produced approximately 10,15 and 65% of the extent to which thromboxane B2, 12-hydroxy-5,8,10-heptadecatrienoic acid and 12-hydroxy-5,8,10,14-eicosatetraenoic acid were produced, respectively, from arachidonic acid.     

Regeneration of vitamin E in human platelets

Human platelets possess active lipoxygenase and cyclooxygenase which convert arachidonic acid to (12S)-12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) plus (12S)-12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and thromboxane B2 plus 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), respectively. When platelet homogenates were incubated with arachidonate, there was a rapid consumption of platelet tocopherol. Time course analysis revealed that within 0.5 min, over half of arachidonate and tocopherol were metabolized. Mass formation of 12-HPETE and 12-HETE or thromboxane B2 and HHT exceeded that of the mass of tocopherol oxidized. Preincubation with the lipoxygenase inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA) completely abolished this arachidonate-induced tocopherol oxidation whereas cyclooxygenase inhibitors (indomethacin and aspirin) further potentiated tocopherol oxidation, indicating that this oxidation is closely linked with platelet 12-lipoxygenase activity. Incubation with lipoxygenase metabolites of arachidonic acid showed that only 12-HPETE caused a rapid tocopherol oxidation which was followed by a gradual tocopherol regeneration. By using nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor which is also a strong reductant, over 60% of the arachidonate-induced oxidized tocopherol was regenerated. Tocopherol regeneration declined with increasing oxidation time induced by arachidonate, and after 30-60 min virtually no regeneration could be observed, suggesting that the precursor molecule was unstable. We postulate that the precursor molecule is the tocopheroxyl radical. In the presence of ETYA, a lipoxygenase inhibitor without antioxidant properties, either ascorbate or GSH provided significant tocopherol regeneration. Kinetic studies showed that tocopherol regeneration after the addition of ascorbate was essentially completed by 1 min. By contrast, GSH addition caused a steady increase in tocopherol which peaked after 10 min of its addition. To determine whether this rapid regeneration is chemical or enzymic, regeneration was studied in the presence of chloroform and methanol. Comparison of various reductants in this denaturing condition for enzymes showed that ascorbate and NDGA afforded significant regeneration whereas GSH was ineffective, indicating that there are distinct enzymic and non-enzymic mechanisms for tocopherol regeneration. This study provides direct evidence from mass analysis that tocopherol can be regenerated in human cell homogenates. This finding implies that maintenance of membrane tocopherol status may be an essential function of ascorbate and GSH which operate in concert to ensure maximum membrane protection against oxidative damage.     

Products, kinetics, and substrate specificity of homogeneous thromboxane synthase from human platelets: development of a novel enzyme assay

Homogeneous thromboxane synthase from human platelets converted prostaglandin H2 (PGH2) to thromboxane A2 (measured as thromboxane B2, TxB2), 12(L)-hydroxy-5,8,10-heptadecatrienoic acid (HHT), and malondialdehyde (MDA) in equimolar amounts under a variety of experimental conditions. PGG2 was transformed to MDA and corresponding 15- and 12-hydroperoxy products. PGH1 was enzymatically transformed into 12(L)-hydroxy-8,10-heptadecadienoic acid (HHD) and PGH3 into TxB3 and 12(L)-hydroxy-5,8,10,14-heptadecatetraenoic acid (delta 14-HHT) as earlier reported for solubilized and partially purified thromboxane synthase preparations. The ratio of thromboxane to C17 hydroxy fatty acid formation was 1:1 with PGG2, PGH2, and PGH3 as substrates. These results confirm and extend earlier observations with partially purified enzyme that the three products are formed in a common enzymatic pathway (Diczfalusy, U., Falardeau, P., and Hammarstr?m, S. (1977) FEBS Lett. 84, 271-274). A convenient spectrophotometric assay for thromboxane synthase activity measuring the ultraviolet light absorption of the C17 hydroxy acid formed (e.g., HHT) was developed. The validity of the assay was determined employing specific inhibitors for thromboxane synthase. The substrate specificity of thromboxane synthase was determined using this assay. PGG2 and PGH3 showed Vmax and KM values similar to those of PGH2. The KM value of PGH1 was also identical to that of PGH2 but the Vmax value PGH1 was more than twice as high as that of PGH2.     

Effect of tert-butyl hydroperoxide on cyclooxygenase and lipoxygenase metabolism of arachidonic acid in rabbit platelets

The effect of tert-butyl hydroperoxide (t-BOOH) on the formation of thromboxane (TX) B2, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) from exogenous arachidonic acid (AA) in washed rabbit platelets was examined. t-BOOH enhanced TXB2 and HHT formation at concentrations of 8 microM and below, and at 50 microM it inhibited the formation, suggesting that platelet cyclooxygenase activity can be enhanced or inhibited by t-BOOH depending on the concentration. t-BOOH inhibited 12-HETE production in a dose-dependent manner. When the platelets were incubated with 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) instead of AA, t-BOOH failed to inhibit the conversion of 12-HPETE to 12-HETE, indicating that the inhibition of 12-HETE formation by t-BOOH occurs at the lipoxygenase step. Studies utilizing indomethacin (a selective cyclooxygenase inhibitor) and desferrioxamine (an iron-chelating agent) revealed that the inhibitory effect of t-BOOH on the lipoxygenase is not mediated through the activation of the cyclooxygenase and that this effect of t-BOOH is due to the hydroperoxy moiety. These results suggest that hydroperoxides play an important role in the control of platelet cyclooxygenase and lipoxygenase activities.     

Effect of 13-hydroperoxy-9,11-octadecadienoic acid (13-hpode) on arachidonic acid metabolism in rabbit platelets

• 1.1. The effect of 13-hydroperoxy-9,11-octadecadienoic acid (13-HPODE) on the formation of thromboxane (TX) B₂, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) from exogenous arachidonic acid in washed rabbit platelets was examined.
• 2.2. 13-HPODE inhibited TXB₂ and HHT formation without affecting 12-HETE production.
• 3.3. 13-Hydroxy-9,11-octadecadienoic acid which was produced rapidly from 13-HPODE, did not suppress the formation of TXB₂ and HHT, indicating the requirement of the hydroperoxy moiety for the inhibitory effect of 13-HPODE on TXB₂ and HHT formation.
• 4.4. Experiments utilizing mannitol and dimethyl sulfoxide (hydroxy radical scavengers) revealed that the action of 13-HPODE is not due to hydroxy radicals which are expected to be formed from 13-HPODE.
• 5.5. These results suggest that 13-HPODE is a selective inhibitor of platelet cyclo-oxygenase and may have functional effects within platelets.

     

Metabolism of prostaglandin endoperoxide by microsomes from cat lung

It has been reported that the prostaglandin (PG) precursor, arachidonic acid, produces divergent hemodynamic responses in the feline pulmonary vascular bed. However, the pattern of arachidonic acid products formed in the lung of this species is unknown. In order to determine the type and activity of terminal enzymes in the lung, prostaglandin biosynthesis by microsomes from cat lung was studied using the prostaglandin endoperoxide, PGH2, as a substrate. The major products of incubations of PGH2 with microsomes were thromboxane (TX) B2 (the major metabolite of TXA2), 6-keto-PGF1 alpha (the breakdown product of PGI2) and 12L-hydroxy-5,8,10-heptadecatrienoic acid (HHT). Formation of TXB2 was markedly reduced by imidazole. Tranylcypromine decreased the formation of TXB2 and HHT and inhibited the formation of 6-keto-PGF1 alpha. At low PGH2 concentrations, equal production of TXB2 and 6-keto-PGF1 alpha was observed. However, as PGH2 concentration increased, 6-keto-PGF1 alpha production approached early saturation while TXB2 production increased in a linear fashion. These results suggest that enzymatic formation of TXA2 and PGI2 is a function of substrate availability in the lung. These findings provide a possible explanation for the divergent hemodynamic responses to arachidonic acid infusions at high and low concentrations in the feline pulmonary vascular bed.     

Prostaglandin endoperoxides. VII. Novel transformations of arachidonic acid in guinea pig lung

The following labeled compounds were isolated and identified after incubation of [1-¹⁴C]arachidonic acid with guinea pig lung homogenates: 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), the hemiacetal derivative of 8-(1-hydroxy-3-oxopropyl)-9,12-dihydroxy-5,10-heptadecadienoic acid (PHD), 12-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), PGE₂, PGF_2α, 11-hydroxy-5,8,12,14-eicosatetraenoic acid, and 15-hydroxy-5,8,11,13-eicosatetraenoic acid (in order of decreasing yield). Perfused guinea pig lungs released PHD (654–2304 ng), HHT (192–387 ng), HETE (66–111 ng), PGE₂ (15–93 ng), and PGF_2α (93–171 ng) following injection of 30 μg of arachidonic acid. Thus guinea pig lung homogenates as well as intact guinea pig lung converted added arachidonic acid predominantly into PHD and HHT, metabolites of the prostaglandin endoperoxide PGG₂, and to a lesser extent into the classical prostaglandins PGE₂ and PGF_2α.     

Metabolism of 7,10,13,16-docosatetraenoic to dihomo-thromboxane 14-hydroxy-7,10,12-nonadecatrienoic acid hydroxy fatty acids by human platelets

Human platelets metabolize 7,10,13,16-docosatetraenoic acid (22:4(n−6) into dihomo-thromboxane B₂ and 14-hydroxy-7,10,12-nonadecatrienoic acid at about twenty percent of the rate they convert arachidonic to thromboxane B₂ and 12-hydroxy-5,8,10-heptadecatrienoic acid. 14-Hydroxy-7,10,12,16-docasatetraenoic was the major metabolite produce via the lipoxygenase pathway. Several other hydroxy were also produced in small amounts via an indomethacin-insensitive pathway. Incubation of 20 μM arachidonic acid with various levels of 22:4(n−6) resulted In a dose-dependent inhibition of both thromboxane B₂ and 12-hydroxy-5,8,10-heptadecatrienoic acid production. Coversely, 12-hydroxy-5,8,10,14-eicosatetraenoic acid synthesis was stimulated because of substrate shunting to the lipoxygenase pathway. These results show that 22:4(n−6) may modify platelet function both by serving as a precursor for a 22-carbon thromboxane and by suppressing the synthesis of thromboxane A₂ from arachidonic acid. In addition, our results suggest that simultaneous release of 22:4(n−6) and arachidonic acid from platelet phospholipids will result in an elevation of both 12-hydroxy-5,8,10,14-eicosatetraenoic acid levels as well as simultaneous synthesis of 14-hydroxy-7,10,12,16-docosatetraenoic acid.     

The use of tritiated prostaglandins in metabolism studies. I: Evaluation of the kinetic isotope effect in the prostaglandin dehydrogenase reactions

Although numerous data exist concerning tritium kinetic isotope effect in enzymic reactions, little is related to the metabolism of tritiated prostaglandins. The present study reports an evaluation of the kinetic isotope effect which occurs during the oxidation of 15-hydroxyl group of tritium-labeled prostaglandins E2 and F2 alpha by the 15-hydroxyprostaglandin dehydrogenase and during the oxidation of 9-hydroxyl group of tritium-labeled prostaglandin F2 alpha by the 9-hydroxyprostaglandin dehydrogenase. The large kinetic isotope effect tends to limit the validity of the dehydrogenase assay using tritium-labeled prostaglandins as substrate. However these assays can be considered to be an indication of relative enzyme activity.     

Possible inhibitory function of endogenous 15-hydroperoxyeicosatetraenoic acid on prostacyclin formation in bovine aortic endothelial cells

Arachidonic acid is metabolized via the cyclooxygenase pathway to several potent compounds that regulate important physiological functions in the cardiovascular system. The proaggregatory and vasoconstrictive thromboxane A2 produced by platelets is opposed in vivo by the antiaggregatory and vasodilating activity of prostacyclin (prostaglandin I2) synthesized by blood vessels. Furthermore, arachidonic acid is metabolized by lipoxygenase enzymes to different isomeric hydroxyeicosatetraenoic acids (HETE's). This metabolic pathway of arachidonic acid was studied in detail in endothelial cells obtained from bovine aortae. It was found that this tissue produced 6-ketoprostaglandin F1 alpha as a major cyclooxygenase metabolite of arachidonic acid, whereas prostaglandins F2 alpha and E2 were synthesized only in small amounts. The monohydroxy fatty acids formed were identified as 15-HETE, 5-HETE, 11-HETE and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT). The latter two compounds were produced by cyclooxygenase activity. Nordihydroguaiaretic acid (NDGA), a rather selective lipoxygenase inhibitor and antioxidant blocked the synthesis of 15- and 5-HETE. It also strongly stimulated the cyclooxygenase pathway, and particularly the formation of prostacyclin. This could indicate that NDGA might exert its effect on prostacyclin levels by preventing the synthesis of 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a potent inhibitor of prostacyclin synthetase. 15-HPETE could therefore act as an endogenous inhibitor of prostacyclin production in the vessel wall.     

Formation of 6-oxoprostaglandin F1 alpha, 6,15-dioxoprostaglandin F1 alpha, and monohydroxyicosatetraenoic acids from arachidonic acid by fetal calf aorta and ductus arteriosus

Particulate fractions and slices from fetal calf aorta convert arachidonic acid to 6-oxoprostaglandin F1 alpha (6-oxoPGF1 alpha), 6,15-dioxoPGF1 alpha, 12-hydroxy-5,8,10-heptadecatrienoic acid, 11-hydroxy-5,8,12,14-icosatetraenoic acid (11h-20:4), and 15-hydroxy-5,8,11,13-icosatetraenoic acid (15h-20:4). In some cases, small amounts of 12-hydroxy-5,8,10,14-icosatetraenoic acid (12h-20:4) were also detected. The products were all identified by gas chromatography-mass spectrometry after purification by normal phase and argentation high pressure liquid chromatography. Both 11h-20:4 and 15h-20:4 appeared to be formed by prostaglandin endoperoxide synthetase rather than by lipoxygenases, since their formation was inhibited by indomethacin but not by nordihydroguaiaretic acid. The formation of 12h-20:4, on the other hand, was stimulated by indomethacin, probably due to increased substrate availability. The formation of hydroxyicosatetraenoic acids was markedly stimulated by adrenaline. Substantial amounts of 6,15-dioxoPGF1 alpha were formed from arachidonic acid by particulate fractions from fetal calf blood vessels, especially in the presence of relatively high substrate concentrations. The formation of this product was stimulated by methemoglobin and inhibited by adrenaline, glutathione, and tryptophan. It would appear that particulate fractions from fetal calf aorta convert arachidonic acid to 15-hydroperoxyPGI2, which can either be reduced in the presence of various cofactors to form PGI2 or dehydrated to give 15-oxoPGI2. The formation of hydroperoxides from arachidonic acid could be an important factor in regulating PGI2 synthesis in aorta, since PGI2 synthetase is strongly inhibited by such intermediates.     

The use of tritiated prostaglandins in metabolism studies II: Kinetic isotopic effect: An useful tool to investigate catabolizing sequence of prostaglandins

Although numerous data exist concerning tritium kinetic isotope effect in enzymic reactions, little is related to the metabolism of tritiated prostaglandins. The present study reports an evaluation of the kinetic isotope effect which occurs during the oxidation of 15-hydroxyl group of tritium-labeled prostaglandins E₂ and F_2α by the 15-hydroxyprostaglandin dehydrogenase and during the oxidation of 9-hydroxyl group of tritium-labeled prostaglandin F_2α by the 9-hydroxyprostaglandin dehydrogenase. The large kinetic isotope effect tends to limit the validity of the dehydrogenase assay using tritium-labeled prostaglandins as substrate. However these assays can be considered to be an indication of relative enzyme activity.