Inhibitory Effect Of Arachidonic Acid Research Articles

Effect of arachidonic acid on twitch tension of the rat phrenic nerve-diaphragm.

Recent studies have demonstrated that unsaturated fatty acids are involved in the regulation of neuroeffector function. I have extended these studies by examining the effect of arachidonic acid on neuromuscular function in vitro using the rat phrenic nerve-diaphragm preparation. Arachidonic acid caused a time-and dose-dependent reduction in indirectly stimulated twitch tension, but had no effect on directly stimulated twitch tension. Linoleic acid and linolenic acid also reduced indirectly stimulated twitch tension, whereas stearic acid, oleic acid and arachidic acid had no effect. None of three blockers of arachidonic acid metabolism, the cyclooxygenase inhibitor indomethacin, the lipoxygenase inhibitor nordihydroguaiaretic acid or the cytochrome P-450 inhibitor ketoconazole, altered the effect of arachidonic acid on twitch tension. The free radical scavenger superoxide dismutase eliminated the inhibitory effect of arachidonic acid on twitch tension, suggesting that superoxide anion played a role in arachidonic acid's action.

Regulation of Ras-GAP and the Neurofibromatosis-1 Gene Product by Eicosanoids

Ras-GAP (GTPase activating protein) is a regulatory protein that stimulates the intrinsic guanosine triphosphatase (GTPase) activity of the proto-oncogene product p21ras. A domain of the neurofibromatosis gene product (NF1) that has sequence similarity to the catalytic domain of Ras-GAP and to yeast IRA gene products also has a specific stimulatory activity toward p21ras GTPase. Arachidonic acid and phosphatidic acid inactivate GAP, but no agents have been identified that stimulate GAP and thereby switch p21ras off. With the use of recombinant Ha-c-Ras and Ras-GAP, NF1, and GAP catalytic domains, it was found that prostaglandins PGF2 alpha and PGA2 stimulated Ras-GAP and that prostacyclin PGI2 inhibited Ras-GAP. The stimulatory effect of PGF2 alpha was saturable and structure-specific and competed with the inhibitory effect of arachidonic acid. Arachidonic acid also inhibited the catalytic activity of NF1, but prostaglandins were not stimulatory. These results suggest a mechanism for the allosteric control of Ras function through the modulation of arachidonate metabolism.

Possible Role for Arachidonic Acid in the Control of Steroidogenesis in Hen Theca1

Studies were conducted to evaluate if arachidonic acid (C20:4) could function as a second messenger within theca cells from the second largest preovulatory (F2) follicle from the ovary of the domestic hen. Arachidonic acid stimulated basal progesterone and androstenedione production, but inhibited LH-induced androstenedione production. The stimulatory effects of arachidonic acid were not altered by either cyclooxygenase or lipoxygenase pathway inhibitors (indomethacin and nordihydroguaiaretic acid, respectively), but were blocked by agents that prevented mobilization and/or efflux of calcium (TMB-8 and verapamil). The inhibitory effects of arachidonic acid on LH-stimulated steroidogenesis were determined to occur both prior and subsequent to cAMP formation. Fifty and 100 microM arachidonic acid attenuated LH- (10 ng) and forskolin- (0.2 microM) induced cAMP levels, and decreased androstenedione and estradiol production following treatment with 8-bromo-cAMP. Phospholipase A2 (PLA2) and the calcium ionophore, A23187, stimulated the release of 3H from theca cells prelabeled with [3H]arachidonic acid, and both PLA2 and the closely related fatty acid, eicosatrienoic acid (C20:3), could replicate the inhibitory effects of arachidonic acid on LH-stimulated androstenedione production. Finally, neither indomethacin nor nordihydroguaiaretic acid blocked the inhibitory effects of arachidonic acid on LH-promoted androstenedione production. We conclude that arachidonic acid can be released within theca cells in response to physiologic (PLA2) and pharmacologic agents (A23187), and accordingly, that it may act directly as a second messenger to modulate both basal and LH-stimulated steroid production.

Evidence that Arachidonic Acid Influences Hen Granulosa Cell Steroidogenesis and Plasminogen Activator Activity by a Protein Kinase C-Independent Mechanism1

We recently proposed that arachidonic acid serves as a second messenger within granulosa cells from the largest preovulatory follicle of the hen. The present studies were conducted to determine whether the inhibitory effects of arachidonic acid on LH-induced cAMP accumulation and on the ability of cells to convert 25-hydroxycholesterol to progesterone are mediated via the protein kinase C pathway. Furthermore, we determined the effects of arachidonic acid on plasminogen activator activity in granulosa cells. In the first experiment, the putative protein kinase C inhibitor, staurosporine, completely reversed the inhibitory effects of phorbol 12-myristate 13-acetate (PMA) on LH-promoted cAMP formation, but failed to overcome the inhibitory effects of arachidonic acid. Prolonged pretreatment (18 h) with 1.6 microM PMA depleted granulosa cells of both cytosolic and membrane-associated protein kinase C, and subsequently attenuated the inhibitory effects of PMA on LH-induced progesterone production; however, such depletion did not alter the inhibitory effects of phospholipase A2 (PLA2; an agent that increases intracellular levels of arachidonic acid). PMA, but not arachidonic acid, caused a rapid (within 2 min) translocation of protein kinase C from the cytosol to the membrane (a characteristic of agents that activate protein kinase C). Finally, both arachidonic acid and PLA2 inhibit plasminogen activator (PA) activity in a dose-dependent fashion, whereas activation of protein kinase C with PMA stimulates PA activity. Taken together, the data suggest that the effects of arachidonic acid in granulosa cells can occur independently of protein kinase C activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effects of arachidonic acid on muscarinic current response in single pancreatic acinar cells of rats.

1. In single, enzymatically dissociated rat pancreatic acinar cells both acetylcholine (ACh) stimulation and IP3 (inositol 1,4,5-trisphosphate) injection evoke Ca2(+)-dependent transient responses. Exogenously applied arachidonic acid (AA) inhibits both responses in a dose-dependent manner. 2. Arachidonic acid oxidation inhibitors, indomethacin and nordihydroguaiaretic acid, cause no significant changes in ACh- and IP3-induced responses. The inhibitory effects of AA (50 microM) on IP3-induced responses are not influenced by the presence of these oxidation inhibitors. 3. An inhibitor of phospholipase A2 (PLA2), 4-bromophenacyl bromide (4-BPB; 10 microM), augments the ACh-induced response, and it potentiates the IP3-induced response by a factor of 10 to 20. The IP3-induced response, after its complete decay, is recovered by an administration of 4-BPB. 4. The results suggest that an increase in [Ca2+]i, induced by IP3 injection, activates PLA2, and that this resultant release of AA in turn inhibits IP3-dependent Ca2+ mobilization.

Modulation of phosphoinositide metabolism in rat brain slices by excitatory amino acids, arachidonic acid, and GABA

In rat brain slices the synthesis of [3H]phosphoinositides and the production of [3H]inositol monophosphate (IP1) induced by norepinephrine (NE) were inhibited by glutamate. Calcium concentrations were varied to test if these inhibitory effects of glutamate were mediated by a calcium-dependent process. Although reducing calcium or addition of the calcium antagonist verpamil reduced the inhibitory effects of glutamate, these results were equivocal because reduced calcium directly decreased agonist-induced [3H]phosphoinositide synthesis. The inhibitory effects of glutamate were mimicked by quisqualate in a dose-dependent manner, but none of a variety of excitatory amino acid receptor antagonists modified the inhibition caused by quisqualate. It is suggested that glutamate activates a quisqualate-sensitive receptor (for which an antagonist is not available) and causes inhibition of phosphoinositide hydrolysis mediated in part by a direct or indirect inhibitory effect of calcium on phosphoinositide synthesis. Modulatory effects of arachidonic acid were examined because glutamate and calcium can activate phospholipase A2. Arachidonic acid caused a rapid and dose-dependent inhibition of [3H]phosphoinositide synthesis and of NE-stimulated [3H]IP1 production. A similar inhibition of the response to carbachol also occurred. The inhibition caused by arachidonic acid was unchanged by addition of inhibitors of cyclooxygenase or lipoxygenase. Activation of phospholipase A2 with melittin caused inhibitory effects similar to those of arachidonic acid. Inhibitors of phospholipase A2 were found to impair phosphoinositide metabolism, likely due to their lack of specificity for phospholipase A2. Further studies were carried out in slices that were prelabelled with [3H]inositol in an attempt to separate modulatory effects on [3H]phosphoinositide synthesis and agonist-stimulated [3H]IP1 production. Several excitatory amino acid agonists inhibited NE-stimulated [3H]IP1 production. This inhibitory interaction could be due to impaired synthesis of [3H]phosphoinositides because, even though the slices were prelabeled, addition of unlabelled inositol reduced NE-stimulated [3H]IP1 production, indicating that continuous regeneration of [3H]phosphoinositides is required. In contrast to the inhibitory effects of the excitatory amino acids, gamma-aminobutyric acid (GABA) enhanced the response to NE in cortical and hippocampal slices. GABA also enhanced the response to carbachol in hippocampal and striatal slices and to ibotenic acid in hippocampal slices. Baclofen potentiated the response to NE similarly to the effect of GABA and baclofen partially blocked the inhibitory effect of arachidonic acid but did not alter that of quisqualate.

Arachidonic Acid Inhibits Luteinizing Hormone-Stimulated Progesterone Production in Hen Granulosa Cells1

Arachidonic acid has been proposed to function as a hormone-induced second messenger in a variety of mammalian endocrine tissues. The present studies were conducted to evaluate whether arachidonic acid, either added exogenously or released endogenously following treatment with physiologic (phospholipase A2) or pharmacologic (melittin) agents, influences basal and/or luteinizing hormone (LH)-induced cyclic adenosine 3',5'-monophosphate (cAMP) and progesterone production in granulosa cells from domestic hens. Phospholipase A2 (PLA2) and melittin treatments failed to alter basal concentrations of progesterone, whereas arachidonic acid had a slight stimulatory effect (only at the 50-microM dose) on progesterone levels, and no effect on cAMP. By contrast, arachidonic acid, PLA2, and melittin each inhibited LH-promoted progesterone production in a dose-dependent fashion. The inhibitory effects of arachidonic acid on the progesterone response were determined to occur both prior and subsequent to cAMP formation since cAMP levels in arachidonic acid-treated cells were attenuated after treatment with 10 ng LH or 100 microM forskolin (at 10- to 100-microM doses of arachidonic acid), and progesterone production was decreased in the presence of 1 mM 8-bromo-cAMP (with 50 and 100 microM arachidonic acid). The post-cAMP mechanism of action is characterized by the inability of cells to convert 25-hydroxy-cholesterol, but not pregnenolone, to progesterone. The effects of arachidonic acid are probably direct, since pharmacologic inhibitors of the lipoxygenase (nordihydroguaiaretic acid) and cyclooxygenase (indomethacin) pathways of arachidonic acid metabolism failed to alter the suppression of

Analysis of the regulation of phosphatidylinositol-4,5-bisphosphate synthesis by arachidonic acid in exocrine pancreas

In pancreatic acinar cells prelabeled with either 32 P i or myo-[ 3H]inositol, arachidonic acid (10–50 μ m) rapidly decreased the steady-state levels of [ 32P]phosphatidylinositol 4′,5′-bisphosphate ([ 32P]PtdIns4,5P 2) and inhibited carbachol-stimulated accumulation of [ 3H]inositol trisphosphate ([ 3H]InsP 3). Both actions of arachidonic acid were rapidly reversed by bovine serum albumin (BSA). Indomethacin and nordihydoguaiaretic acid failed to block the inhibitory effects of arachidonic acid on [ 32P]PtdIns4,5P 2 levels. Arachidonic acid (10–50 μ m) also caused a prompt depletion of cellular ATP which was rapidly reversed by BSA. The ATP-depleting action of arachidonate paralleled in terms of concentration dependence and time course its inhibitory effects on [ 32P]PtdIns4,5P 2 and [ 3H]InsP 3 levels. Exposure of acinar cells to 50 μ m arachidonic acid produced an increase in oxygen consumption which exceeded that elicited by either carbachol or ionomycin. Arachidonic acid (10–50 μ m) also caused a concentration-dependent rise in cytosolic Ca 2+, which was partially obtunded by Ca 2+ deprivation. A proposed mechanism involving arachidonic acid as a negative feedback regulator of polyphosphoinositide turnover in exocrine pancreas is discussed.

Inhibitory effect of fatty acids on the binding of androgen receptor and R1881.

Unsaturated long chain fatty acids are known to inhibit the binding between estrogen and estrogen receptor, or progesterone and progesterone receptor in rat uterus. The effects of long chain fatty acids on the binding between androgen receptor of castrated rat prostate and 3H-R1881 were studied. The binding was not affected by saturated fatty acids such as palmitic acid (16:0) or stearic acid (18:0). But unsaturated fatty acids such as oleic acid (18:1), arachidonic acid (20:4) and docosahexaenoic acid (22:6) inhibited the binding between androgen receptor and 3H-R1881. The inhibitory effect of arachidonic acid was dose dependent. Scatchard analysis showed that the addition of arachidonic acid markedly decrease the number of binding sites of androgen receptor. But the dissociation constant was not affected. The inhibitory effect of arachidonic was not a competitive one.

Inhibitory mechanism of opioid binding to receptor by phospholipase A 2

1. 1. The inhibition of [ 3H]naloxone binding to the opioid receptor upon short-term incubation with phospholipase A 2 (Plase A 2) was abolished by treatment with BSA, but not after long-term incubation. 2. 2. In contrast to the restorative effect of BSA on the strong inhibition occurring with Plase A 2, BSA only partially abolished the inhibitory effect of arachidonic acid or lysophosphatides, even though the degree of inhibition was slight. 3. 3. These results suggest that Plase A 2 products only become associated with hydrophobic receptor sites or with phospholipids near to the receptor, thus reversibly inhibiting opioid binding, and that irreversible inhibition occurs through release of the phospholipid necessary for receptor binding.

Effect of arachidonic acid, fatty acids, prostaglandins, and leukotrienes on volume regulation in Ehrlich ascites tumor cells.

Arachidonic acid inhibits the cell shrinkage observed in Ehrlich ascites tumor cells during regulatory volume decrease (RVD) or after addition of the Ca ionophore A23187 plus Ca. In Na-containing media, arachidonic acid increases cellular Na uptake under isotonic as well as under hypotonic conditions. Arachidonic acid also inhibits KCl and water loss following swelling in Na-free, hypotonic media even when a high K conductance has been ensured by addition of gramicidin. In isotonic, Na-free medium arachidonic acid inhibits A23187 + Ca-induced cell shrinkage in the absence but not in the presence of gramicidin. It is proposed that inhibition of RVD in hypotonic media by arachidonic acid is caused by reduction in the volume-induced Cl and K permeabilities as well as by an increase in Na permeability and that reduction in A23187 + Ca-induced cell shrinkage is due to a reduction in K permeability and an increase in Na permeability. The A23187 + Ca-activated Cl permeability in unaffected by arachidonic acid. PGE2 inhibits RVD in Na-containing, hypotonic media but not in Na-free, hypotonic media, indicating a PGE2-induced Na uptake. PGE2 has no effect on the volume-activated K and Cl permeabilities. LTB4, LTC4 and LTE4 inhibit RVD insignificantly in hypotonically swollen cells. LTD4, moreover, induces cell shrinkage in steady-state cells and accelerates the RVD following hypotonic exposure. The effect of LTD4 even reflects a stimulating effect on K and Cl transport pathways. Thus none of the leukotrienes show the inhibitory effect found for arachidonic acid on the K and Cl permeabilities. The RVD response in hypotonic, Na-free media is, on the other hand, also inhibited by addition of the unsaturated oleic, linoleic, linolenic and palmitoleic acid, even in the presence of the cationophor gramicidin. The saturated arachidic and stearic acid had no effect on RVD. It is, therefore, suggested that a minor part of the inhibitory effect of arachidonic acid on RVD in Na-containing media is via an increased synthesis of prostaglandins and that the major part of the arachidonic acid effect on RVD in Na-free media, and most probably also in Na-containing media, is due to the inhibition of the volume-induced K and Cl transport pathways, caused by a nonspecific detergent effect of an unsaturated fatty acid.

Regulation of linoleic acid Δ6-desaturation by a cytosolic lipoprotein-like fraction in isolated rat liver microsomes

A peripheral component of the Δ 6-fatty acid-desaturase system of rat liver microsomes has been isolated from the cytosol by ultracentrifugation at a saline density of 1.26 g/ml. It exhibited lipoprotein characteristics with an approximate protein/lipid ratio of 1.22 and free fatty acids and phosphatidylcholine as its main lipid components. Linoleic acid desaturation activity diminished in washed microsomes, since they lost the adsorbed cytosolic fraction. Addition of the factor reactivated the reaction and the recovery was dependent on the concentration of the factor in the medium. Linoleic acid and linoleyl-CoA were bound by the cytosolic fraction. However, the transport of substrate to the desaturase was not apparently a main function of the cytosolic fraction, since transport occurred equally in the absence of the factor. Moreover, the solubilization of linoleyl-CoA was not enhanced and the free monomeric concentration was not altered by the presence of the cytosolic fraction. In addition, the factor did not divert Δ 6-desaturase substrate to or from other metabolic pathways such as esterification to phospholipids. γ-Linolenic acid produced by Δ 6-desaturation of linoleic acid in the microsomes inhibited the desaturase, but it was removed by the factor from the membrane towards the cytosol, preventing the inhibition. The anti-inhibitory effect of the cytosolic factor was blockaded by addition of columbinic acid or γ-Linolenic acid to the factor. Moreover, the inhibitory effect of arachidonic acid was not prevented by addition of the cytosolic fraction. These results suggest that the cytosolic fraction studied would optimize the Δ 6-desaturation of linoleic acid in vitro in rat liver microsomes by removal of the product, γ-linolenic acid, as it is formed.

The influence of arachidonic acid metabolites on cell division in the intestinal epithelium and in colonic tumors

Various metabolites of arachidonic acid are now known to influence cell division. In this paper the effects on cell proliferation of arachidonic acid, some inhibitors of arachidonic acid metabolism and some analogs of arachidonic acid metabolites is described. The epithelial cell proliferation rate in the jejunum, in the descending colon and in dimethylhydrazine-induced tumors of rat colon was measured using a stathmokinetic technique. Administration of arachidonic acid resulted in retardation of cell proliferation in each of the tissues examined. A cyclooxygenase inhibitor (Flurbiprofen) prevented this effect of arachidonic acid in the jejunal crypts and in colonic tumors, but not in colonic crypts. In contrast, inhibitors of both cyclooxygenase and lypoxygenase (Benoxaprofen and BW755c) prevented the effect of arachidonic acid in the colonic crypts and reduced its effect on colonic tumours but did not alter its effect on the jejunum. An inhibitor of thromoboxane A 2 synthetase (U51,605) was also able to prevent the inhibitory effect of arachidonic acid on colonic tumors. Treatment with 16,16-dimethyl PGE 2 inhibited cell proliferation in jejunal crypts and in colonic tumors, as did a thromboxone A 2 mimicking agent, U46619. Nafazatrom, an agent that stimulates prostacyclin synthesis and inhibits lypoxygenase, promoted cell proliferation in the jejunal crypts and colonic crypts, but inhibited cell proliferation in colonic tumours.

An inhibitory effect of arachidonic acid on subsequent responses of canine cerebral arteries to serotonin and other agonists

Arachidonic acid (AA) at 10 −4M and 10 −3M produced a phasic contraction in isolated canine basilar arteries that peaked rapidly and then slowly declined. This contraction was evidently due to the conversion of AA to prostanoids because it was blocked by cyclooxygenase inhibitors and because 11, 14, 17 eicosatrienoic acid (10 −3M), which is not a cyclooxygenase substrate, failed to produce a contraction. When the artery was exposed to 10 −3M AA for 20 min and washed, subsequent contractile responses to 10 −6M serotonin (5-HT) were only 10% of control. Contractions produced by prostaglandin E 2 (10 −5M), uridine triphosphate (10 −4M) and potassium (5.5×10 −4M) were inhibited to a lesser degree than 5-HT, the response to potassium being the least affected (66% of control). This damaging effect of 10 −3M AA did not occur if the artery was washed at peak contraction nor with 10 −4M AA. Autooxidation products were evidently not responsible for the damage because prior oxygenation (90 min) of 10 −4M AA had no such effect. Pretreatment with superoxide dismutase or ascorbate did not prevent the inhibition, suggesting that free radical reactions were not involved. Pretreatment with indomethacin (3×10 −4M) or meclofenamate (10 −4M) also failed to prevent the inhibitory phenomenon. Saponin, a detergent, produced similar inhibitory effects but 11, 14, 17 eicosatrienoic acid or oleate (10 −3M) did not. The arteries partially recovered from the inhibition with time. In conclusion, AA produced contraction in basilar arteries by inducing prostaglandin synthesis but can produce secondarily by an unidentified mechanism an inhibition of the contractile responses evoked by various agonists that is both time and concentration dependent.

Effect of exogenous and endogenous prostacyclin on the contractility of rabbit splenic capsular smooth muscle.

1. Using sensitive and specific radioimmunoassays release of prostaglandins (PGs) E2, F2α, 6-keto-F1α and thromboxane B2 (TXB2) from rabbit splenic capsular strips was determined. 2. Contraction of the strips by noradrenaline increased the release of 6-keto-PGF1α, PGE2, PGF2α and TXB2 as compared to basal conditions. Indomethacin inhibited synthesis and release of PGs and TXB2 and simultaneously increased noradrenaline-induced contractions. 3. Arachidonic acid and dihomo-γ-linolenic acid, both substrates for the enzyme fatty acid cyclooxygenase, dose-dependently antagonized noradrenaline-induced contractions. On the other hand, fatty acids, which are not substrates for the cyclooxygenase, had no effect. Pretreatment of the smooth muscle strips with indomethacin inhibited the effect of arachidonic acid and dihomo-γ-linolenic acid. 4. The PG endoperoxides PGG2 and PGH2 as well as their derivative prostacyclin (PGI2) were more effective than arachidonic acid as inhibitors of noradrenaline-induced contractions. The stable degradation product of PGI2, 6-keto-PGF1α, did not influence the contractions. The effects of the PG endoperoxides and PGI2 were not inhibited by indomethacin. 5. The 6(9)oxycyclase inhibitor 9,11-azoprosta-5,13-dienoic acid antagonized the inhibitory effects of arachidonic acid and PGG2 on the contractions, but did not modify the effect of PGI2. 6. Inhibitors of PGI2 synthesis such as 15-hydroperoxyarachidonic acid and 9,11-azoprosta-5,13-dienoic acid increased, like indomethacin, noradrenaline-induced contractions of the rabbit splenic capsular strips in the absence of exogenous PG precursors. On the other hand, a specific inhibitor of TX synthesis, 11,9-epoxyiminoprosta-5,13-dienoic acid, was without effect on the contractions. 7. The results are compatible with the view that endogenous PGI2 modulates the contractile response of rabbit splenic capsular smooth muscle to noradrenaline. The hypothetical PGI2 receptor on the smooth muscle seems to resemble in its specificity the PGI2 receptor on thrombocytes of various species as only PGE1 in higher concentrations has effects comparable to PGI2. 8. Besides the presynaptic inhibition of noradrenaline release by PGE the postsynaptic inhibition of noradrenaline-induced smooth muscle contraction by endogenous PGI2 might represent, at least in vitro, another feed-back mechanism to attenuate the effects of sympathetic nerve stimulation. Synthesis and release of antiaggregatory and contraction-inhibiting PGI2 by splenic tissue might play a role in the function of the spleen as a storage site for thrombocytes.