Stereoselective actions of hepoxilins A3 and B3 and their cyclopropane analogs (HxdeltaA3 and HxdeltaB3) on bradykinin and PAF-evoked potentiation of vascular leakage in rat skin.

Native hepoxilins (Hx) A3 and B3 as well as their synthetic cyclopropane analogs, HxdeltaA3 and HxdeltaB3 are inactive on their own in causing changes in vascular permeability in rat skin measured by leakage of plasma-bound Evans Blue dye. Several of these compounds, however, were observed to potentiate the leakage of dye evoked by bradykinin (BK) and platelet-activating factor (PAF). The syn epimer of HxA3 was effective in potentiating dye leakage evoked by BK but not by PAF. The syn epimer of HxB3, on the other hand, was capable of potentiating both BK- and PAF-evoked plasma protein leakage. The anti epimer of both hepoxilins was inactive. In contrast, the anti epimer of the cyclopropane analog HxdeltaA3 potentiated only the BK-evoked changes, whereas the anti epimer of HxdeltaB3 potentiated only the PAF-evoked changes in dye leakage. The corresponding other epimer of each compound was inactive. Our findings indicate that the hepoxilin cyclopropane analogs appear to mimic the actions of the native compounds.

Hepoxilins raise circulating insulin levels in vivo.

We have demonstrated over a decade ago that hepoxilins cause the release of insulin from isolated pancreatic islets of Langerhans in vitro. However, no studies are available so far to indicate whether these compounds are active in vivo. The present study is the first to our knowledge which demonstrates that hepoxilins administered intra-arterially in the anaesthetized rat cause the release of insulin in the circulation. This release is dependent on the glucose status of the rat. Hence, animals fasted overnight do not respond to hepoxilin administration, while animals that have had free access to food respond to hepoxilins with a rise in insulin concentrations in blood. The hepoxilin effect is rapid and varies with different hepoxilins, the most potent of which is hepoxilin A(3) (HxA(3)) (both the 8S and the 8R enantiomers). Administration of 100 microg HxA(3) produces a rise in blood insulin equivalent to that caused by the administration of 5 mg glucose. In view of earlier evidence showing that these compounds cause a rise in intracellular calcium levels in vitro at a <1 microg/ml concentration through a receptor-mediated mechanism, we speculate that the actions of hepoxilins in causing the release of insulin from the pancreas may be due to alterations in calcium levels within the beta-cell. We believe that hepoxilins may represent new lead compounds as therapeutics in type II diabetes mellitus.

Hepoxilin signaling in intact human neutrophils: biphasic elevation of intracellular calcium by unesterified hepoxilin A3.

We have previously shown that the methyl ester of hepoxilin A3 causes a receptor-induced rise in intracellular calcium through the release from intracellular stores in suspended human neutrophils. The corresponding free acid was devoid of activity. We now report that the action of the free acid form of hepoxilin A3 is dependent on the type of vehicle used, i.e. it is active in releasing calcium when used in an ethanol vehicle but not in DMSO. The methyl ester is equally active in either vehicle. The pattern of calcium release between the free acid and the methyl ester is qualitatively different. Both compounds show a biphasic pattern, i.e. an initial rapid phase followed by a slow decline in calcium levels but never reaching pre-hepoxilin A3 baseline levels. The methyl ester appears slightly more potent in the initial phase of calcium release than the free acid (methyl = 188+/-14 S.D., free acid = 135+/-11 S.D. nM, P < 0.0005). Both compounds appear to reach the same calcium levels at the plateau of the second prolonged phase (methyl = 88+/-8 S.D., free acid = 107+/-15 S.D. nM, not significant). Lanthanum chloride (an inhibitor of calcium influx) interfered with the second phase of the curve causing calcium levels to return to normal pre-hepoxilin levels for both compounds. Addition of lanthanum chloride prior to the hepoxilin addition or carrying out the experiments in calcium-free medium, eliminated the second phase completely, with the calcium peak returning rapidly to normal baseline levels, suggesting that the second phase is due to calcium influx. Again the methyl ester is more active than the free acid (methyl, 189+/-12; free acid, 145+/-6 S.D. nM, P<0.005). Additional experiments with tritium-labelled methyl ester of hepoxilin A3 demonstrated that the compound is hydrolyzed into the free acid intracellularly. These experiments demonstrate that DMSO interacts with hepoxilin free acid, interfering with its entry into the cell while ethanol does not. Once inside the cell, hepoxilin interacts with its own receptor to release calcium rapidly from stores, but it also causes a more prolonged influx of calcium from the extracellular milieu.

Hepoxilin A3 is oxidized by human neutrophils into its omega-hydroxy metabolite by an activity independent of LTB4 omega-hydroxylase.

Hepoxilin A3-methyl ester is taken up by intact human neutrophils where it is first hydrolyzed into the free acid which is subsequently converted into a single major metabolite. The structure of this metabolite was determined through mass spectral analysis of several derivatives, and through identity with an authentic compound prepared by chemical synthesis. The metabolite was identified as omega-hydroxy-hepoxilin A3 showing that the epoxide functionality of the parent hepoxilin is not opened during incubation with human neutrophils. All attempts to investigate hepoxilin metabolism in broken cells, despite the presence of protease inhibitors (Aproteinin, PMSF, DFP) and supplementation with NADPH were unsuccessful. Metabolism of hepoxilin A3 required the intact cell, while parallel experiments with LTB4 as substrate demonstrated that this eicosanoid was metabolized into its omega-hydroxy metabolite regardless of whether intact or broken cell preparations were used provided that NADPH was present in the latter. Hepoxilin metabolism in intact cells was inhibited dose-dependently by CCCP (0.01-100 microM), a mitochondrial uncoupler, whereas LTB4 metabolism was unaffected by CCCP. This data suggests that metabolism of hepoxilin A3 occurs in intact human neutrophils through omega-oxidation, is likely located in the mitochondrial compartment of the cell (inhibition by CCCP) and is carried out by an activity that is independent of the well characterized, relatively stable microsomal LTB4 omega-hydroxylase.

Chemical synthesis and actions of 11,12-thiirano-hepoxilin A3.

A novel analog of hepoxilin A3 has been chemically synthesized in which the 11,12-epoxide group has been altered to a thiirano group. This has been accomplished through allylic rearrangement of unnatural (11 R, 12 R)-hepoxilin B3 under Mitsunobu conditions, first into unnatural (11 R, 12 R)-hepoxilin A3, followed by conversion of this compound with inversion of the epoxide centers into the thiirano-hepoxilin A3 having the natural 11 S, 12 S configuration. We also report herein evidence showing that thiirano-hepoxilin A3 raises intracellular calcium concentrations in intact human neutrophils.

Hepoxilins: a review on their enzymatic formation, metabolism and chemical synthesis.

This article reviews published evidence describing the enzymatic and nonenzymatic formation and the routes of metabolism of the hepoxilins. Also treated are the major approaches used for the chemical synthesis of these compounds and for some of their analogs.

[A comparison of the effects of 5-hydroxyeicosatetraenoic acid and hepoxilin A3 on the plasticity of the snail neuronal cholinoreceptors]. / Sravnenie éffektov 5-gidroksiéikozatetraenovoi kisloty i gepoksilina A3 na plastichnost' kholinoretseptorov neironov vinogradnoi ulitki.

The effects of two acyclic derivatives of arachidonic acid which are formed under the action of 5- and 12-lipoxygenases 5(S)-hydroxy-(6,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE) and (8R/S)-hydroxy-(11S,12S)-epoxy-5Z,9E,14Z-eicosatrienoi c acid (hepoxilin A3) on extinction of inward current evoked by local acetylcholine (ACh-current) application on soma of Helix lucorum RPa3 and LPa3 neurons were studied by the double-electrode voltage clamp technique. It was shown an increase in ACh-current extinction by 5-HETE. Hepoxilin A3 did not influence cholinoreceptor plasticity. The present results confirm earlier assumptions concerning the regulation of cholinoreceptor plasticity by acyclic eicosanoids which were formed from arachidonic acid under the influence of 5-lipoxygenase and the lack of 12-lipoxygenase metabolites in this regulation.

[Inactivation of prostaglandin H synthase from sheep vesicular glands with acetylenic fatty acids]. / Inaktivatsiia prostaglandin-H-sintazy iz vezikuliarnykh zhelez barana atsetilenovymi zhirnymi kislotami.

Acetylenic fatty acids, 8,11,14-eicosatrienoic, 5,8,11,14-eicosatetraenoic and 5,8,11,14,17-eicosapentaenoic acids, were compared with respect to their effects on the particulate prostaglandin H synthase. All the acetylenic acids inactivated the enzyme in a time-dependent manner. The eicosatriynoic and eicosatetraynoic acids exhibited comparable efficacies, whereas the eicosapentaenoic acid proved to be a weaker inactivator. The time-courses of inactivation fitted a kinetic model suggest an enzyme-catalysed conversion of the compounds via alternative suicidal and non-suicidal routes. The characteristics of inactivation of prostaglandin H synthase differ from those of lipoxygenases.

[Biosynthetic paths of acyclic lipoxygenase metabolites of polyenic acids]. / Puti biosinteza atsikicheskikh lipksigenaznykh metabolitov polienovykh kislot.

The review deals with the basic features of the fatty acid lipoxygenation resulting in the formation of leukotrienes, hydroxy fatty acids, lipoxins, hepoxylins. The biological role, prospects for the further studies of this class of bioregulators are discussed.

[Short and long latent effects of 15-hydroxyeicosatetraenoic acid on the extinction of the acetylcholine-induced inward current in neurons of the Helix lucorum]. / Korotko- i dlinnolatentnye éffekty 15-gidroksiéikozatetraenovoi kisloty na ugashenie vyzvannogo atsetilkholinom vkhodiashchego toka neironov vinogradnoi ulitki.

The impact of a non-cyclized arachidonic acid derivative 15S-hydroxy-5Z, 8Z, 11Z, 13E-eicosatetraenoic acid (15-HETE) upon the dynamics of the inward current extinction, caused by repeated ion-tophoretic acetylcholine applications on the soma, was studied through a double electrode voltage clamp technique on the Helix lucorum identified neurons RPa3 and LPa3. The extracellular effect of 15-HETE (4-16 microM) was found to have a two phase influence on the inward current extinction, depending on the time of exposure to the compound. The short-latent effect (up to 60-80 min) displays itself as an extinction decrease, whereas the long-latent effect (after 60-80 min) - as an enhanced extinction. The effects caused by 15-HETE are irreversible. The short-latent one was probably due to the earlier described inhibition of 5- and 12-lipoxygenase enzymes by 15-HETE while the long-latent one - due to its intrinsic function.

[Synthesis of 5,8,11,14,17-eicosapentaenic acid]. / Sintez 5,8,11,14,17-éikozopentainovoi kisloty.

A synthetic route to 5,8,11,14,17-eicosapentaynoic acid, an acetylenic precursor of 5Z,8Z,11Z,14Z,17Z-eicosapentaenoic acid, has been developed based on condensation of a propargyl synthon with omega-acetylenic fragments. Physico-chemical constants and spectral characteristics (IR, 1H-NMR, and mass-spectrometry) of the target and intermediate compounds are given.

[Synthesis of (8Z,11Z,14Z)-eicosatrien-5-ynoic (5,6-dehydroarachidonic) acid and its transformation into [5,6-3H]arachidonic acid and [5,6-3H]prostaglandins E2 and F2 alpha]. / Sintez (8Z,11Z,14Z)-éikozatrien-5-inovoi (5,6-degidroarakhidonovoikisloty i ee prevrashchenie v [5,6-3H]arakhidonovuiu kislotu, [5,6-3H]prostaglandiny E2 i F2 alpha.

8Z,11Z,14Z-Eicosatriene-5-ynoic acid and its tritium-labelled analogue, [5,6-3H]arachidonic acid, have been synthesized on the basis of acetylenic compounds. [5,6-3H]Arachidonic acid has been used as substrate for the enzymatic synthesis of [5,6-3H]PGE2 and [5,6-3H]PGF2 alpha.