Eicosa-5,8,11,14-tetraynoic Acid Research Articles

Arachidonic acid modulates Na+ currents by non-metabolic and metabolic pathways in rat cerebellar granule cells

AA (arachidonic acid), which possesses both neurotoxic and neurotrophic activities, has been implicated as a messenger in both physiological and pathophysiological processes. In the present study, we investigated the effects of both extracellular and intracellular application of AA on the activity of Na(V) (voltage-gated Na(+) channels) in rat cerebellar GCs (granule cells). The extracellular application of AA inhibited the resultant I(Na) (Na(V) current), wherein the current-voltage curve shifted to a negative voltage direction. Because this effect could be reproduced by treating the GCs with ETYA (eicosa-5,8,11,14-tetraynoic acid) or a membrane-impermeable analogue of AA, AA-CoA (arachidonoyl coenzyme A), we inferred that AA itself exerted the observed modulatory effects on I(Na). In contrast, intracellular AA significantly augmented the elicited I(Na) peak when the same protocol that was used for extracellular AA was followed. The observed I(Na) increase that was induced by intracellular AA was mimicked by the AA cyclo-oxygenase metabolite PGE(2) (prostaglandin E(2)), but not by ETYA. Furthermore, cyclo-oxygenase inhibitors decreased I(Na) and quenched AA-induced channel activation, indicating that the effect of intracellular AA on Na(V) was possibly mediated through AA metabolites. In addition, the PGE2-induced activation of I(Na) was mimicked by cAMP and quenched by a PKA (protein kinase A) inhibitor, a G(s) inhibitor and EP (E-series of prostaglandin) receptor antagonists. The results of the present study suggest that extracellular AA modulates Na(V) channel activity in rat cerebellar GCs without metabolic conversion, whereas intracellular AA augments the I(Na) by PGE(2)-mediated activation of cAMP/PKA pathways. These observations may explain the dual character of AA in neuronal pathogenesis.

Regulation of Hepatic Δ-6 Desaturase Expression and Its Role in the Polyunsaturated Fatty Acid Inhibition of Fatty Acid Synthase Gene Expression in Mice

Dietary polyunsaturated fatty acids (PUFA) of the (n-6) and (n-3) families uniquely suppress the expression of lipogenic genes while concomitantly inducing the expression of genes encoding proteins of fatty acid oxidation. Although considerable progress has been made toward understanding the nuclear events affected by PUFA, the intracellular mediator responsible for the regulation of hepatic lipogenic gene expression remains unclear. On the basis of earlier fatty acid composition studies, we hypothesized that the Delta-6 desaturase pathway was essential for the production of the fatty acid regulator of gene expression. To address this hypothesis, male BALB/c mice (n = 8/group) were fed for 5 d a high glucose, fat-free diet (FF) or the FF plus 50 g/kg 18:2(n-6) with and without eicosa-5, 8,11,14-tetraynoic acid (ETYA) (200 mg/kg diet), a putative inhibitor of the Delta-6 desaturase pathway. ETYA had no effect on food intake or weight gain, but it completely prevented 18:2(n-6) from suppressing the hepatic abundance of fatty acid synthase mRNA. ETYA ingestion was associated with a decrease in the hepatic content of 20:4(n-6) and an increase in the amount of 18:2(n-6). The fatty acid composition changes elicited by ETYA were accompanied by a decrease in the enzymatic activity of Delta-6 desaturase. Interestingly, the hepatic abundance of Delta-6 desaturase mRNA was actually induced by ETYA one- to twofold. When the product of Delta-6 desaturase, i.e., 18:3(n-6), was added to the ETYA plus 18:2(n-6) diet, the hepatic content of 20:4(n-6) was normalized. In addition, 18:3(n-6) consumption reduced the level of hepatic Delta-6 desaturase mRNA by 50% and completely prevented the increase in fatty acid synthase mRNA that was associated with ETYA ingestion. Apparently, Delta-6 desaturation is an essential step for the PUFA regulation of the fatty acid synthase gene transcription. Finally, the suppression of Delta-6 desaturase by PUFA and its induction by ETYA suggest that the Delta-6 desaturase gene may be regulated by two different lipid-dependent mechanisms.

Inhibition of the synthesis of eicosanoid-like substances in a human oral cancer cell line by interferon- γ and eicosapentaenoic Acid

The objectives were to examine the production of eicosanoids in a Chinese human oral cancer cell line (OEC-M1) and to test the effects of interferon- γ (IFN- γ), eicosapentaenoic acid (EPA) and enzyme inhibitors on this biosynthesis. The eicosanoids were identified by reverse phase-high performance liquid chromatography. Two predominant peaks appeared in the chromatograms. One compound (P-1) was identified by ultraviolet absorption at a λ max of 278 nm with shoulders at 272 and 284 nm. The other compound (P-2) was identified by ultraviolet absorption at a λ max of 284 nm with shoulders at 278 and 290 nm. The production of P-1 was significantly inhibited by the addition of IFN- γ (200 and 400 U/ml), and EPA (10 to 40 μM). It was only partially inhibited ( p<0.05) by indomethacin (INDO) (0.5 and 1 μM), nordihydroguaiaretic acid (NDGA) (30 and 60 μM/ml), and eicosa-5,8,11,14-tetraynoic acid (ETYA) (20–60 μM). It was almost completely inhibited by indomethacin (2 and 3 μM), and dexamethasone (0.6 and 6 μM). The production of P-2 was almost completely inhibited by IFN- γ (200 and 400 U/ml), and partially inhibited ( p<0.05) by EPA (10 and 20 μM), NDGA (30 and 60 μM), ETYA (20 and 40 μM), dexamethasone (0.6 and 6 μM). The production of both peaks was significantly reduced by excluding arachidonic acid (AA), and almost completely inhibited by heating at 100°C for 10 min during incubation. These results demonstrate that two eicosanoid-like compounds are synthesized by the OEC-M1 cell line and that their production can be modulated by IFN- γ, EPA, indomethacin, NDGA, ETYA, and dexamethasone.

Model experiments on squid axons and NG108-15 mouse neuroblastoma × rat glioma hybrid cells

Three types of ionic current essentially determine the firing pattern of nerve cells: the persistent Na + current, the M current and the low-voltage-activated Ca 2+ current. The present article summarizes recent experiments concerned with the basic properties of these currents. Keynes and Meves ( Proc R Soc Lond B (1993) 253, 61–68) studied the persistent or steady-state Na + current on dialysed squid axons and measured the probability of channel opening both for the peak and the steady-state Na + current (PF peak and PF ss) as a function of voltage. Whereas PF peak starts to rise at −50 mV and reaches a maximum at +40 to +50 mV, PF ss only begins to rise appreciably at around 0 mV and is still increasing at +100 mV. This differs from observations on vertebrate excitable tissues where the persistent Na + current turns on in the threshold region and saturates at around 0 mV. Schmitt and Meves ( Pflügers Arch (1993) 425, 134–139) recorded M current, a non-inactivating K + current, from NG108-15 neuroblastoma × glioma hybrid cells, voltage-clamped in the whole-cell mode, and studied the effects of phorbol 12,13-dibutyrate (PDB), an activator of protein kinase C (PKC), and arachidonic acid (AA). PDB and AA both decreased I M, the effective concentrations being 0.1–1 μM and 5–25 μM, respectively; while the PDB effect was regularly observed, the M current depression by AA was highly variable from cell to cell. The PKC 19–31 peptide, an effective inhibitor of PKC, in a concentration of 1 μM almost totally prevented the effects of PDB and AA on M current, suggesting that both are mediated by PKC. Schmitt and Meves ( Pflügers Arch (1994a) 426, Suppl R 59) measured low-voltage-activated (l-v-a) and high-voltage-activated (h-v-a) Ca 2+ currents on NG108-15 cells and investigated the effect of AA and PDB on both types of current. At pulse potentials > −20 mV, AA (25–100 μM) decreased l-v-a and h-v-a I Ca. The decrease was accompanied by a small negative shift and a slight flattening of the activation and inactivation curves of the l-v-a I Ca. The AA effect was not prevented by 50 μM eicosa-5,8,11,14-tetraynoic acid (ETYA), an inhibitor of AA metabolism, or PKC 19–31 peptide and not mimicked by 0.1–1 μM PDB. Probably, AA acts directly on the channel protein or its lipid environment. The physiological relevance of these three sets of observations is briefly discussed.

Release of slow reacting substance (SRS) from human leucocytes by lymphokine

A human histamine releasing lymphokine (histamine releasing factor, HRF was studied for its ability to release slow reacting substance (SRS) alongside histamine from human leucocytes. HRF was found to release a substantial amount of SRS from human leucocytes. The SRS was identified by its slow contraction of isolated guinea pig ileum preparation in the presence of atropine and mepyramine, inhibition of this action by the SRS antagonist FPL 55712, the inhibition of its production by eicosa — 5,8,11,14 — tetraynoic acid (ETYA) but not indomethacin, and a final confirmation by a radioimmunoassay for leukotriene C 4 (LTC 4). Amounts of SRS ranging from a few nanograms to about 180 ng (eqt LTD 4) could be released from 10 8 leucocytes depending on the dose of HRF, and there is a good correlation between the amount of SRS produced and the percentage histamine release accompanying it. These findings suggest that the human lymphokine (HRF) may play an important role in acute hypersensitivity and inflammatory reactions in addition to its traditional role as mediator of delayed hypersensitivity reactions.

The role of prostaglandins in the stimulatory action of catecholamines on myometrial activity in term pregnant women.

The response of small strips of human myometrium to catecholamines was investigated using a superfusion technique. In term pregnant uteri, adrenaline (A) and noradrenaline (NA) caused contraction of both the upper and lower segments, but there was no significant change in response depending on whether or not the patient was in labor. Phenoxybenzamine blocked the excitatory effect of both A and NA, suggesting an alpha-adrenoceptor-mediated effect. Propranolol did not enhance the effect of A or NA, indicating that stimulation of beta-adrenoceptors does not contribute to the effect on the pregnant human uterus. Pretreatment of the tissue with indomethacin or eicosa-5,8,11,14-tetraynoic acid (ETA), drugs known to block endogenous prostaglandin synthesis, markedly reduced the effects of both A and NA. This suggests a specific role of prostaglandins in the mechanism of action of catecholamines on the human myometrium.

Bradykinin-stimulated electrolyte secretion in rabbit and guinea pig intestine. Involvement of arachidonic acid metabolites.

Bradykinin (BK) increases short-circuit current (Isc) when added to the serosal side of rabbit or guinea pig ileum or rabbit colon. Significant effects on Isc are seen at concentrations as low as 10(-10) M. Anion substitution experiments and unidirectional 36Cl flux measurements indicate that this effect of BK on Isc is due to Cl secretion. The effect of BK on Isc can be partially blocked (60-70% inhibition) by cyclooxygenase inhibitors (indomethacin and/or naproxen) and completely blocked by the phospholipase inhibitor, mepacrine. The combined cyclooxygenase/lipoxygenase inhibitors BW 755 and eicosa-5,8,11,14-tetraynoic acid (ETYA) also completely block the effect of BK on Isc but the slow-reacting substance of anaphylaxis (SRS-A) antagonist FPL 55712 has no effect. None of the above inhibitors diminish the effect on Isc of other exogenously added secretory stimuli such as vasoactive intestinal peptide (VIP), theophylline, or prostaglandin E2 (PGE2). Prior desensitization of rabbit ileum to PGE2 blocks the effect on Isc of BK but not those of VIP or theophylline. Conversely, prior desensitization of rabbit ileum to BK greatly reduces the effect of PGE2 on Isc. BK also stimulates the synthesis of PGE2 in rabbit ileal and colonic mucosa and this effect can be blocked by prior addition of either indomethacin or mepacrine. These effects of BK are similar to those of exogenously added arachidonic acid (AA). AA also stimulates Cl secretion and increases PGE2 synthesis and its effect on Isc can be inhibited by prior desensitization to PGE2 or by prior addition of indomethacin. The above results indicate that BK stimulates active Cl secretion in both small and large intestine and suggest that this effect is due to the intracellular release of AA. Although the prostaglandins appear to be the major products of AA metabolism contributing to the secretory response, lipoxygenase products may also play a role.

Effects of reduced glutathione on the 12-lipoxygenase pathways in rat platelets.

Arachidonic acid is converted into several more polar products in addition to 12-l-hydroperoxyeicosa-5,8,10,14-tetraenoic acid (12-HPETE) and 12-l-hydroxyeicosa-5,8,10,14-tetraenoic acid (12-HETE) by the cytosol fractions of rat platelets. The more polar products are formed via the lipoxygenase pathways in the same way as are 12-HPETE and 12-HETE, since their formation is not inhibited by indomethacin but by eicosa-5,8,11,14-tetraynoic acid (ETYA). The presence of 0.5-1.5mm-reduced glutathione (GSH) in the reaction mixture prevents the formation of the more polar products and produces 12-HETE as the only metabolite from arachidonic acid by the 12-lipoxygenase pathway. l-Cysteine has the same effect as GSH. However, oxidized glutathione (GSSG) and l-cystine are not able to prevent the formation of the more polar products. The results indicate that 12-HPETE peroxidase in the 12-lipoxygenase pathway is a GSH-dependent peroxidase and the more polar products might be formed from the non-enzymic breakdown of the primary 12-lipoxygenase product of 12-HPETE, owing to insufficient capability of the subsequent peroxidase system to completely reduce 12-HPETE to 12-HETE. Thus the presence of GSH in the reaction mixture offers a convenient and precise cell-free assay system for 12-lipoxygenase in rat platelets. Routine assays of 12-lipoxygenase are carried out in the presence of 1mm-GSH in the reaction mixture. The synthesis of 12-HETE by 12-lipoxygenase is linear during the first 4 min of incubation at 37 degrees C, and has a pH optimum of 7.7. The 12-lipoxygenase reaches half-maximal activity at an arachidonate concentration of 20mum. Fractionation of cell homogenates indicates that the cytosol fraction possesses almost all the 12-lipoxygenase activity, whereas the microsomal fraction exhibits little enzyme activity.

Effects of Arachidonic Acid and Its Metabolites on Antigen-Induced Histamine Release from Human Basophils in Vitro

Abstract We have studied the role of arachidonic acid (AA) metabolism in immediate hypersensitivity reactions. The AA analog, eicosa-5,8,11,14-tetraynoic acid (ETYA), which inhibits both the cyclo-oxygenase and lipoxygenase pathways of AA metabolism, was found to cause a dose-dependent inhibition of antigen-induced histamine release from human basophils; complete inhibition occurred at 10-4 M. In contrast, nonsteroidal anti-inflammatory drugs (NSAID) including indomethacin (10-9 to 10-6 M), meclofenamic acid (10-6 to 10-4 M), and aspirin (10-5 to 10-3 M), which block the cyclo-oxygenase pathway of AA metabolism, enhanced mediator release. AA (10-7 to 10-5 M) also enhanced basophil mediator release, an effect that was not affected by preincubation with indomethacin. Other fatty acids including linoleic, linolenic, and stearic acids were without effect. Indomethacin (and the other NSAID studied) reversed the inhibition caused by the AA metabolite, prostaglandin (PG) E2, in a dose-dependent fashion. This reversal of inhibition was not unique to PG, but was observed with all inhibitors of histamine release studied that act via activation of adenylate cyclase including isoproterenol and the H2 agonist, dimaprit. NSAID, however, did not reverse the inhibition caused by drugs that do not act on adenylate cyclase, i.e., dibutyryl cAMP, isobutylmethylxanthine, and 2-deoxyglucose. In the leukocyte preparations studied, the NSAID did not block the increase in the intracellular cAMP levels caused by agonists that act via adenylate cyclase. It appears that a product(s) of AA metabolism, generated via the lipoxygenase pathway, is involved in IgE-mediated histamine release and that the same or a different product(s) modulates the control mechanisms that are linked to adenylate cyclase.

Possible Role of Arachidonic Acid and Its Metabolites in Mediator Release from Rat Mast Cells

Abstract The role of arachidonic acid (AA) metabolism in the release of inflammatory mediators from rat mast cells was studied. Eicosa-5,8,11,14-tetraynoic acid (ETYA), an acetylenic analog of AA, was found to inhibit histamine release induced by anti-IgE, concanavalin A (Con A), or the ionophore A-23187 with ID50 values of 65, 50, and 17 µM, respectively. Mediator release was not affected by aspirin or indomethacin in concentrations up to 60 µM. Addition of free AA (0.1 to 100 µM) to unstimulated mast cells did not initiate noncytotoxic mediator release. Preincubation of mast cells with 1 to 10 µM AA inhibited subsequent release induced by anti-IgE or Con A (up to 38%). This inhibition of release was blocked if aspirin (10 µM) or indomethacin (10 µM) was present, suggesting that AA inhibition was mediated by cyclo-oxygenase products. When AA was added after secretion was initiated by anti-IgE or Con A, a modest potentiation of release was noted. These studies suggest that AA metabolism by enzyme systems other than cyclo-oxygenase is an integral part of the mast cell secretory process. Availability of free AA, however, does not appear to be a sufficient condition to initiate secretion by otherwise unstimulated cells. Activation of mast cells by secretory signals appears to lead to altered AA metabolism which in turn appears to be involved in the secretion of inflammatory mediators.

Formation and action of prostaglandin endoperoxides in the isolated human umbilical artery.

The effects on the isolated human umbilical artery (HUA) of the recently isolated endoperoxide intermediates in prostaglandin (PG) biosynthesis, PGG2 and PGH2, were studied. Both endoperoxides were potent contractors of the artery strips, the threshold concentrations being 3 (1-4) ng/ml for PGG2 and 1 (1-12) ng/ml for PGH2, as compared with 200 (40-400) ng/ml for PGE2. The hemiacetal derivative of 8-(1-hydroxy-3-oxopropyl)-9,12L-dihydroxy-5,10-heptadecadienoic acid (thromboxane B2), a metabolite of PGG2, appeared in the bath medium indicating PG and thromboxane generation in the isolated HUA. The formation of thromboxane B2 was inhibited by indomethacin (8-40 mug/ml) and by eicosa-5,8,11,14-tetraynoic acid (ETA) (25 mug/ml), ETA also inhibited the contractile responses to both endoperoxides. The results support the view that local generation of PGs might be involved in the closure of the HUA at birth.

Reduction of the tone of the isolated human umbilical artery by indomethacin, eicosa-5,8,11,14-tetraynoic acid and polyphloretin phosphate.

The effects of 2 prostaglandin synthetase inhibitors, indomethacin and eicosa-5,8,11,14-tetraynoic acid (ETA) and of the prostaglandin antagonist, polyphloretin phosphate (PPP), on the tone of the isolated human umbilical artery and on the responses of this preparation to 5-hydroxytryptamine (5-HG) and prostaglandin F2alpha (PGF2alpha) were investigated. Indomethacin (8 mug/ml), ETA (5 mug/ml) or PPP (40 mug/ml) reduced the tone of human umbilical arteries but had no influence on the responses to 5-HT. In these concentrations ETA and PPP but not indomethacin antagonized the action of PGF2alpha. When the concentration of indomethacin or PPP was increased 5-fold both 5-HT- and PGF2alpha-induced contractions were antagonized indicating a non-specific inhibition at these concentration levels. A 10-fold increase in the concentration of ETA had no antagonizing action on 5-HT-induced contractions suggesting a more selective inhibition of the PGF2alpha action than displayed by the other compounds. The effects on the tone of the human umbilical artery of the compounds studied might reflect inhibition of prostaglandin biosynthesis and/or antagonism of the action of formed prostaglandins. The findings are compatible with the view that intramural synthesis of prostaglandins contributes to the maintenance of the tone of the isolated human umbilical artery.

Lamb ductus arteriosus: Effect of prostaglandin synthesis inhibitors on the muscle tone and the response to prostaglandin E 2

The prostaglandin synthesis inhibitors, indomethacin and eicosa-5,8,11, 14-tetraynoic acid (ETA), have been tested on the isolated lamb ductus arteriosus at low and high PO 2 levels. Both compounds produced a gradual contraction of the hypoxic vessel, and at equal doses the effect of indomethacin was stronger. The maximal tension output of the hypoxic tissue under indomethacin was equal to that of the oxygen-contracted control. ETA- and indomethacin-treated preparations contracted further upon transfer from a low to a high oxygen environment, and the response under indomethacin exceeded significantly control values. Control preparations were relaxed markedly by PGE 2 in low oxygen but showed little or no response in high oxygen. In contrast, preparations pretreated with the inhibitors retained their sensitivity to PGE 2 during exposure to high oxygen. The data are consistent with the idea that E-type prostaglandins play a role in the regulation of the intrinsic tone of the ductus arteriosus during foetal life. It is also suggested that the sensitivity of ductal muscle to E-type prostaglandins is controlled by the rate of endogenous prostaglandin formation.

The effects of indomethacin and eicosa-5,8,11,14-tetraynoic acid on the response of the rabbit portal vein to electrical stimulation.

1 The effects of indomethacin and eicosa-5,8,11,14-tetraynoic acid (ETYA) on the contractile response of the transmurally stimulated rabbit portal vein were studied in vitro.2 When the veins were stimulated for 240 pulses at 1 and 2 Hz, the responses were potentiated by indomethacin and ETYA. However, responses to 4 and 8 Hz were not potentiated. The responses to continuous electrical stimulation at 2 Hz were also potentiated by indomethacin and ETYA. This potentiating effect was attenuated when the veins were pretreated with alpha-methyl-p-tyrosine. The responses of the veins to noradrenaline were not altered by either indomethacin or ETYA.3 Prostaglandin E(2) inhibited the responses of the portal vein to electrical stimulation. The magnitude of this inhibition was inversely related to the frequency of stimulation. The responses of the vein to noradrenaline were not altered by prostaglandin E(2).4 It is concluded that potentiation by indomethacin and ETYA of the response of the isolated portal vein is due to an increased release of newly synthesized noradrenaline as a result of inhibition of prostaglandin synthesis.

Effect of prostaglandin precursors, dihomo-γ-linolenic acid and arachidonic acid on the vasoconstrictor response to norepinephrine in the dog paw

The present investigation was undertaken to provide further evidence for the possible role of prostaglandins in the regulation of blood flow in the dog paw. The effect of an intraarterial infusion of dihomo-γ-linolenic acid (DHL) and arachidonic acid (AA), the immediate precursors of prostaglandin E 1 and E 2, respectively on the vasoconstrictor response to norepinephrine was examined in the paw under the condition of constant flow. Infusion of AA (55 or 110 μg/min) and DHL (55 or 110 μg/min) to a lesser extent depressed the vasoconstrictor response to norepinephrine. Intraarterial infusion of the prostaglandin synthesis inhibitors eicosa-5,8,11,14 tetraynoic acid (ETA) or indomethacin antagonized the depressant effect of DHL on the vasoconstrictor response to norepinephrine. ETA had no effect on the depressant activity of AA. In a second series of experiments, utilizing the lower infusion rate of AA (55 μg/min), administration of indomethacin in addition to ETA antagonized the depressant effect of AA; however, this antagonism was less than found with indomethacin alone. Alpha linolenic acid which is not a prostaglandin precursor had no depressant effect on the response to norepinephrine. The demonstration that DHL and AA can depress the vasoconstrictor response to norepinephrine and that this can be antagonized by inhibitors of prostaglandin synthesis suggests the presence in the dog paw of an enzymatic system capable of generating E prostaglandins.