Inhibition of prostaglandin biosynthesis by c-5,c-8,c-11-eicosatrienoic acid.

The enzyme prostaglandin H leads to E-isomerase (EC 5.3.99.3), which is present in sheep vesicular gland and needs glutathione as cofactor, is inhibited by c-5,c-8,c-11-eicosatrienoic acid, the fatty acid accumulating during essential fatty acid deficiency. The EFA-deficiency syndrome can partly be explained from a prostaglandin deficiency caused by lack of precursors. The present finding indicates that 5,8,11-eicosatrienoic acid could well be an additional factor in modifying the symptoms of EFA-deficiency.     

Oxygenation of 5,8,11-eicosatrienoic acid by prostaglandin H synthase-2 of ovine placental cotyledons: isolation of 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids

Prostaglandin H synthase-1 of ram vesicular glands metabolises 5,8,11-eicosatrienoic (Mead) acid to 13R-hydroxy-5,8,11-eicosatrienoic and to 11R-hydroxy-5,8,12-eicosatrienoic in a 5:1 ration. We wanted to determine the metabolism of this fatty acid by prostaglandin H synthase-2. Western blot showed that microsomes of sheep and rabbit placental cotyledons contained prostaglandin H synthase-2, while prostaglandin H synthase-1 could not be detected. Microsomes of sheep cotyledons metabolised [1-¹⁴C]5,8,11-eicosatrienoic acid to many polar metabolites and diclofenac (0.05 mM) inhibited the biosynthesis. The two major metabolites were identified as 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids. They were formed in a ratio of 3:2, which was not changed by aspirin (2 mM). 5,8,11-Eicosatrienoic acid is likely oxygenated by removal of the pro-S hydrogen at C-13 and insertion of molecular oxygen at either C-13 or C-11, which is followed by reduction of the peroxy derivatives to 13-hydroxy-5,8,11-eicosatrienoic and 11-hydroxy-5,8,12-eicosatrienoic acids, respectively. Prostaglandin H synthase-1 and -2 oxygenate 5,8,11-eicosatrienoic acid only slowly compared with arachidonic acid.     

Inhibition of prostaglandin biosynthesis by triynoic acids

Prostaglandin biosynthesis from eicosa-8,11,14-trienoic acid in microsomes from bovine seminal vesicles is inhibited by acetylenic acids. Octadeca-6,9,12-triynoic acid and eicosa-8,11,14-triynoic acid are the most potent inhibitors. These acids both contain an ω-8 methylene group. Within the 20-carbon acetylenic acid series, inhibition decreases in the sequence eicosa-8,11,14-triynoic acid > eicosa-7,10,13-triynoic acid > eicosa-5,8,11-triynoic acid. Furthermore, eicosa-8,11,14-triynoic acid is a more potent inhibitor of arachidonic acid induced platelet aggregation than either eicosa-7,10,13-triynoic acid or eicosa-5,8,11-triynoic acid. The ω-8 methylene group is not the only determinent of inhibitory potency since docosa-10,13,16-triynoic acid is less potent than its 18 and 20 carbon analogs and all of these acids have an ω-8 methylene group.     

Inhibition of intestinal tone, motility and prostaglandin biosynthesis by 5,8,11,14-eicosatetraynoic acid (TYA)

Microgram concentrations of 5,8,11,14-eicosatetraynoic acid (TYA) inhibited the spontaneous increases in tone which develop in isolated guinea-pig ileum and also inhibited intestinal motility in anesthetized guinea-pigs. TYA failed to block contractions of the ileum induced by various agonists including PGE₂. It did, however, inhibit both the spontaneous liberation of spasmogenic substances from isolated ileum and the biosynthesis of PGE₂ from arachidonic acid. It is concluded that the inhibitory effects of TYA were exerted through inhibition of PG biosynthesis. Studies with antagonist drugs (atropine, methysergide and pyribenzamine) confirmed that the effects of intestinal PGs are, in the guinea-pig, largely exerted through a cholinergic mechanism.     

High Yield of Methylophilus methylotrophus Cytochrome c″ by Coexpression with Cytochrome c Maturation Gene Cluster from Escherichia coli

Heterologous expression of c-type cytochromes in the periplasm of Escherichia coli often results in low soluble product yield, apoprotein formation, or protein degradation. We have expressed cytochrome c″ from Methylophilus methylotrophus in E. coli by coexpression of the gene encoding the cytochrome (cycA) with the host-specific cytochrome c maturation elements, within the ccmA-H gene cluster. Aerobic cultures produced up to 10 mg holoprotein per liter after induction with IPTG. In the absence of the maturation factors E. coli failed to produce a stable haem protein. Cytochrome c″ isolated from the natural host was compared with the recombinant protein. No structural differences were detected using SDS–PAGE, UV-Visible spectroscopy, differential scanning calorimetry, and ¹H-NMR spectroscopy. The success in expressing the mature cytochrome c″ in E. coli allows the engineering of the cycA gene by site-directed mutagenesis thereby providing an ideal method for producing mutant protein for studying the structure/function relationship.     

Inhibition of prostaglandin biosynthesis by sulphasalazine and its metabolites

Sulphasalazine (SZ) inhibits prostaglandin (PG) biosynthesis $\text{in vitro}$ with a potency comparable to that of aceylsalicylate. The metabolites of SZ, sulphapyridine and 5-aminosalicylic acid, were of considerably lower potency as inhibitors of PG biosynthesis in the synthetase preparations used. The inhibition of prostaglandin production by SZ could at least partly account for the clinical utility of sulphasalazine in ulcerative colitis. Sulphapyridine may help to maintain inhibitory concentrations of SZ by restraining bacterial breakdown of the active drug.     

Alpha-linolenic acid and its conversion to longer chain n−3 fatty acids: Benefits for human health and a role in maintaining tissue n−3 fatty acid levels

There is little doubt regarding the essential nature of alpha-linolenic acid (ALA), yet the capacity of dietary ALA to maintain adequate tissue levels of long chain n−3 fatty acids remains quite controversial. This simple point remains highly debated despite evidence that removal of dietary ALA promotes n−3 fatty acid inadequacy, including that of docosahexaenoic acid (DHA), and that many experiments demonstrate that dietary inclusion of ALA raises n−3 tissue fatty acid content, including DHA. Herein we propose, based upon our previous work and that of others, that ALA is elongated and desaturated in a tissue-dependent manner. One important concept is to recognize that ALA, like many other fatty acids, rapidly undergoes β-oxidation and that the carbons are conserved and reused for synthesis of other products including cholesterol and fatty acids. This process and the differences between utilization of dietary DHA or liver-derived DHA as compared to ALA have led to the dogma that ALA is not a useful fatty acid for maintaining tissue long chain n−3 fatty acids, including DHA. Herein, we propose that indeed dietary ALA is a crucial dietary source of n−3 fatty acids and its dietary inclusion is critical for maintaining tissue long chain n−3 levels.     

Inhibition of prostaglandin biosynthesis by 7-oxa- and 5-oxa-prostaglandin analogues (Short Communication)

Some oxaprostaglandin derivatives have been shown to inhibit prostaglandin biosynthesis from arachidonate by a particulate prostaglandin synthetase preparation. The most potent inhibitor was 5-oxaprost-13-trans-enoate, and inhibition by this compound appeared to be competitive. Certain structure-activity relationships were ascertained.     

Conversion of 8,11,14-eicosatrienoic acid to 11,12-epoxy-10-hydroxy-8-heptadecenoic acid by aorta

Particulate fractions from fetal calf aorta convert 8,11,14-eicosatrienoic acid to a number of products derived from 12-hydroperoxy-8, 10-heptadecadienoic acid, including 2 stereoisomers of 11,12-epoxy-10-hydroxy-8-heptadecenoic acid (11,12e-10h-17:1), which were identified by mass spectrometry. In the early stages of the reaction, considerable amounts of the epoxyhydroxy isomers were formed, but the amounts of these products decreased as the reaction continued. There was a concomitant increase in the formation of 10,11,12-trihydroxy-8-heptadecenoic acid (10,11,12th-17:1), which was present only in small amounts initially. Incubation of the 2 isomers of 11,12e-10h-17:1 with microsomal and cytosolic fractions resulted in their conversion to isomers of 10,11,12th-17:1. No hydrolysis of the epoxides occurred in the presence of boiled tissue fractions.     

Stimulation of prostaglandin biosynthesis by lipoic acid.

Enzyme preparations from sheep seminal vesicles display an enhanced ability to synthesize prostaglandins, particularly prostaglandin F from polyunsaturated fatty acids if alpha-lipoic acid is present in the incubation mixture prior to the addition of fatty acid. The stimulation by lipoate is reversible, time dependent, and involves modifications of V and Km for oxygenase activity. Product studies, structure vs. activity studies, and purification data indicate that lipoate exerts it effect by a mechanism distinct from a glutathione-like metabolism of the endoperoxide linkage in prostaglandin G and prostaglandin H. In addition, product studies suggest that lipoate is not a cofactor for the endoperoxide isomerase component of prostaglandin synthetase. Purification of the endoperoxide synthesizing activity by ion-exchange chromatography and isoelectric focusing yields preparations which are more responsive to lipoate than microsomal preparations.     

Human umbilical blood vessel converts all cis-5, 8, 11, 14, 17 eicosapentaenoic acid to prostaglandin I3

All cis-5, 8, 11, 14, 17 eicosapentaenoic acid (EPA) is presented being evaluated for dietary prophylactic use in thrombo-embolic disorders. EPA inhibits the production of TXA₂ and platelet aggregation. We here present results demonstrating that human umbilical arteries convert ¹⁴C- EPA to a substance that in aqueous solutions decomposes to 14C-δ¹⁷-6-keto-PGF_1α. The conversion rate in rat aortic tissue was found substantially lower. These results in combination with earlier data indicating that EPA does not influence the conversion of arachidonic acid (AA) into PGI₂ in human vascular tissue, encourage further research along the lines initiated by the findings of high EPA/AA ratio and low incidence of myocardial infarction in Greenland Eskimos.     

Inhibition of prostaglandin biosynthesis by sulphasalazine and its metabolites.

Sulphasalazine (SZ) inhibits prostaglandin (PG) biosynthesis in vitro with a potency comparable to that of aceylsalicylate. The metabolites of SZ, sulphapyridine and 5-aminosalicylic acid, were of considerably lower potency as inhibitors of PG biosynthesis in the synthetase preparations used. Th inhibition of prostaglandin production by SZ could at least partly account for the clinical utility of sulphasalazine in ulcerative colitis. Sulphapyridine may help to maintain inhibitory concentrations of SZ by restraining bacterial breakdown of the active drug.     

Inhibition of abscisic acid biosynthesis inCercospora rosicola by triarimol

The fungicide triarimol was tested for its effect on abscisic acid (ABA) accumulation in growing culturesof Cercospora rosicola. ABA accumulation was reduced by approximately 50% with 10^–8 M triarimol. Growth ofC. rosicola, as measured by dry weight accumulation, was inhibited by triarimol concentrations at or greater than 10^–7 M. These results are compared with those obtained with clomazone, ancymidol, and paclobutrazol, which inhibit ABA accumulation by 50% at concentrations of 5 × 10^–5, 5 × 10^–6, and 5 × 10^–7 M, respectively. Triarimol, therefore, is among the most potent inhibitors of ABA biosynthesis reported to date. Feeding studies with [¹⁴C]mevalonic acid confirmed the inhibition of ABA biosynthesis by 5 × 10^–8 M triarimol. These results support previous suggestions that one or more of the steps in the ABA biosynthetic pathway from mevalonic acid is catalyzed by cytochrome P-450. Feeding studies with 1-deoxy-[²H]-ABA in resuspended cultures ofC. rosicola show that the conversion of this substrate is not inhibited by triarimol.     

Human umbilical blood vessel converts all cis-5, 8, 11, 14, 17 eicosapentaenoic acid to prostaglandin I3

All cis-5, 8, 11, 14, 17 eicosapentaenoic acid (EPA) is presently being evaluated for dietary prophylactic use in thrombo-embolic disorders. EPA inhibits the production of TXA2 and platelet aggregation. We here present results demonstrating that human umbilical arteries convert 14c-EPA to a substance that in aqueous solutions decomposes to 14C-delta 17-6-keto-PGF1 alpha . The conversion rate in rat aortic tissue was found substantially lower. These results in combination with earlier data indicating that EPA does not influence the conversion of arachidonic acid (AA) into PGI2 in human vascular tissue, encourage further research along the lines initiated by the findings of high EPA/AA ratio and low incidence of myocardial infarction in Greenland Eskimos.     

De novo biosynthesis of arachidonic acid and 5,11,14-eicosatrienoic acid in the cricket Teleogryllus commodus

The de novo biosynthesis of 5,11,14-eicosatrienoic acid (5,11,14-20:3), arachidonic acid (20:4(n - 6] and eicosadienoic acid (20:2(n - 6] and the elongation/desaturation of linoleic acid (18:2(n - 6] to 20:4(n - 6) and alpha-linolenic acid (18:3(n - 3] to eicosapentaenoic acid (20:5(n - 3] were demonstrated in adult males of the field cricket Teleogryllus commodus. Sodium [1-14C]acetate, [1-14C]18:2(n - 6) and [1-14C]18:3(n - 3) were injected into adult male crickets and after an incubation period, the testes and remaining tissues were extracted and the methyl esters obtained from the phospholipid and triacylglycerol fractions were analyzed. After 5 days of daily injections of [1-14C]acetate, the methyl esters of the triene and tetraene fatty acids from the testicular phospholipid fraction were purified by AgNO3-TLC and HPLC and analyzed by GLC, radio-HPLC, and radio-GLC of ozonolysis products. The results demonstrate the de novo biosynthesis of 20:2(n - 6), 20:4(n - 6) and an isomer of 20:3(n - 6) with double bonds in the 5,11,14 positions. the elongation/desaturation of 18:2(n - 6) to 20:4(n - 6) and 18:3(n - 3) to 20:5(n - 3) was demonstrated by analysis of the methyl esters derived from the testicular phospholipid fraction by radio-HPLC after injecting crickets with radiolabeled substrates.     

ACTH-induced prostaglandin biosynthesis from 3H-arachidonic acid by adrenocortical cells

Prostaglandins biosynthesized from ³H-arachidonic acid by trypsin-dispersed cat adrenocortical cells were isolated by silicic acid and thin layer chromatography. PGE, PGF, and a third component with mobility properties indistinguishable from either PGA or PGB were identified both in cortical cell homogenates and incubation medium. Concentrations of ACTH (125–250 μU) which stimulate steroidogenesis enhanced the conversion of labeled precursor to all three of these prostaglandins. These findings provide further evidence for the proposal that prostaglandins function as a critical link in ACTH-induced steroidogenesis.