Origin of the arachidonic acid released post-mortem in rat forebrain.

To determine the origins of the arachidonic acid released post-mortem in brain tissue, [3H]arachidonic acid was injected by the intracerebro-ventricular route and radioactivity monitored in complex lipids and free arachidonic acid at various times after decapitation. The specific activity of the released arachidonic acid was close to that in the total phospholipid fraction and much lower than that of the neutral lipids. The phospholipid with the closest specific activity to the free arachidonic acid recovered at the end of the post-mortem period was phosphatidylinositol. Phosphatidylcholine showed a small but significant decrease in radioactivity post-mortem and could contribute 37% of the arachidonic acid released to the free fatty acid fraction. Arachidonic acid released in rat forebrain after decapitation thus comes from a mixture of phospholipids with phosphatidylinositol and phosphatidylcholine being the major source. Phosphatidylserine and phosphatidic acid did not make important contributions to the free arachidonic acid. In the microsomal fraction, the specific activity of the free arachidonic acid was very close to that in phosphatidylinositol.     

The role of thrombin in the regulation of the endothelial prostaglandin production

Prostaglandin synthesis in endothelial cells may be initiated by the addition of exogenous substrate (arachidonic acid) or by addition of thrombin or the CA2+-ionophore A23187, which leads to prostacyclin formation from endogenous substrates. We noticed that endothelial cells produce more than twice the amount of prostacyclin when incubated with thrombin and arachidonic acid together than with arachidonic acid alone. In addition, it was found that the thrombin-induced conversion of endogenous substrates was inhibited by exogenous arachidonic acid. This means that the conversion of exogenous added arachidonic acid to prostacyclin was stimulated by thrombin. This activation of the enzymes involved in prostacyclin synthesis lasted about 5 min and could be inhibited by phospholipase inhibitors such as mepacrine and p-bromophenyl-acylbromide but not by the cAMP analogue dibutyryl cAMP, an inhibitor of arachidonic acid release from cellular phospholipids. These data demonstrate that, in addition to causing release of endogenous substrate, thrombin and the Ca2+-ionophore also activate the enzyme system involved in the further transformation of arachidonic acid.     

Chronic N-methyl-D-aspartate administration increases the turnover of arachidonic acid within brain phospholipids of the unanesthetized rat

Whereas antibipolar drug administration to rats reduces brain arachidonic acid turnover, excessive N-methyl-d-aspartate (NMDA) signaling is thought to contribute to bipolar disorder symptoms and may increase arachidonic acid turnover in rat brain phospholipids. To determine whether chronic NMDA would increase brain arachidonic acid turnover, rats were daily administered NMDA (25 mg/kg, ip) or vehicle for 21 days. In unanesthetized rats, on day 21, [1-(14)C]arachidonic acid was infused intravenously and arterial blood plasma was sampled until the animal was euthanized at 5 min and its microwaved brain was subjected to chemical and radiotracer analysis. Using equations from our in vivo fatty acid model, we found that compared with controls, chronic NMDA increased the net rate of incorporation of plasma unesterified arachidonic acid into brain phospholipids (25-34%) as well as the turnover of arachidonic acid within brain phospholipids (35-58%). These changes were absent at 3 h after a single NMDA injection. The changes, opposite to those after chronic administration of antimanic drugs to rats, suggest that excessive NMDA signaling via arachidonic acid may be a model of upregulated arachidonic acid turnover in brain phospholipids.     

Arachidonic acid and its acyclic derivatives regulate the short-term plasticity of the cholinoreceptors of the neurons in the edible snail]

On identified Helix neurones RPa3 and LPa3 using the method of double-electrode clamp technique on the membrane the influence was shown of eicosanoids on the dynamics of inward current extinction caused by the repeated ionophoretic applications of acetylcholine to soma. Extracellular influence of arachidonic acid (50-100 microM) increased the extinction. Phospholipase A2 inhibitor quinacrine hydrochloride (100-600 microM) decreasing the content of arachidonic acid in the cell acted differently. Inhibitor of lipoxygenase oxidation of arachidonic acid (nordihydraquiaretic acid) (3-10 microM) weakened the extinction. Blockader of cyclooxygenase oxidation of arachidonic acid--indomethacin (10-50 microM) did not influence the extinction. All the studied composition decreased the amplitude of input current caused by acetylcholine. The obtained results allowed to suppose that arachidonic acid and its acyclic metabolites formed as a result of lipoxygenase oxidation regulated short-term plasticity of snail neurones cholinoreceptors. Cyclic eicosanoids formed at cyclooxygenase oxidation of arachidonic acid had no regulating influence on cholinoreceptors plasticity.     

Arachidonic acid is a physiological activator of the ryanodine receptor in pancreatic beta-cells

Pancreatic beta-cells have ryanodine receptors but little is known about their physiological regulation. Previous studies have shown that arachidonic acid releases Ca(2+) from intracellular stores in beta-cells but the identity of the channels involved in the Ca(2+) release has not been elucidated. We studied the mechanism by which arachidonic acid induces Ca(2+) concentration changes in pancreatic beta-cells. Cytosolic free Ca(2+) concentration was measured in fura-2-loaded INS-1E cells and in primary beta-cells from Wistar rats. The increase of cytosolic Ca(2+) concentration induced by arachidonic acid (150microM) was due to both Ca(2+) release from intracellular stores and influx of Ca(2+) from extracellular medium. 5,8,11,14-Eicosatetraynoic acid, a non-metabolizable analogue of arachidonic acid, mimicked the effect of arachidonic acid, indicating that arachidonic acid itself mediated Ca(2+) increase. The Ca(2+) release induced by arachidonic acid was from the endoplasmic reticulum since it was blocked by thapsigargin. 2-Aminoethyl diphenylborinate (50microM), which is known to inhibit 1,4,5-inositol-triphosphate-receptors, did not block Ca(2+) release by arachidonic acid. However, ryanodine (100microM), a blocker of ryanodine receptors, abolished the effect of arachidonic acid on Ca(2+) release in both types of cells. These observations indicate that arachidonic acid is a physiological activator of ryanodine receptors in beta-cells.     

A biochemical analysis of the effects of arachidonic acid on sarcoplasmic reticulum function

Discontinuous sucrose gradients were used to determine the degree of association between arachidonic acid and sarcoplasmic reticulum vesicle membranes. Fraction analyses showed that arachidonic acid migrated to a different region of the sucrose gradient in the presence of sarcoplasmic reticulum membranes. This could suggest that arachidonic acid was complexed into the membranes. Arrhenius curves representing the temperature dependency of Ca2+-Mg2+-ATPase activity and calcium uptake in the presence and absence of arachidonic acid were constructed. The activation energy for ATPase did not change significantly due to the presence of arachidonic acid. The curve representing control calcium uptake did not show a discontinuity. However, the curve representing calcium uptake in the presence of arachidonic acid showed discontinuities at 18 degrees C and 21 degrees C. Activation energy increased sharply between these temperatures. The results suggest that arachidonic acid reached the critical micellar concentration between these temperatures. Enthalpy decreased in the presence of arachidonic acid. This observation could suggest a transition of the protein-phospholipid complex to a less rigid state since decreased order in the membrane would decrease the energy barrier for activation of ATPase.     

Effect of prolactin on the secretion of milk casein: metabolism of arachidonic acid

Mammary gland fragments were incubated in the presence of prolactin and arachidonic acid which stimulate casein secretion. The effects of these stimuli in the presence of agents that influence arachidonic acid metabolism were investigated. Chloroquine, a blocker of phospholipase A2 activity, decreased prolactin but not arachidonic acid stimulation of casein secretion. Phospholipase A2 markedly stimulated casein secretion. Nordihydroguaiaretic acid (NDGA), an antioxidant that inhibits lipoxygenase, blocked the stimulating effect of prolactin and arachidonic acid. Ultrastructural studies indicated that phospholipase A2-induced stimulation of secretion was comparable to that of prolactin but that arachidonic acid-induced stimulation did not involve the same Golgi membrane modifications. These studies suggest that prolactin and phospholipase A2 stimulate secretion by a common way, and that arachidonic acid interferes with secretion by metabolic products of the lipoxygenase pathway.     

Kinetic study of the activation of the neutrophil NADPH oxidase by arachidonic acid. Antagonistic effects of arachidonic acid and phenylarsine oxide

The O(2)(-) generating NADPH oxidase complex of neutrophils comprises two sets of components, namely a membrane-bound heterodimeric flavocytochrome b which contains the redox centers of the oxidase and water-soluble proteins of cytosolic origin which act as activating factors of the flavocytochrome. The NADPH oxidase can be activated in a cell-free system consisting of plasma membranes and cytosol from resting neutrophils in the presence of GTPgammaS and arachidonic acid. NADPH oxidase activation is inhibited by phenylarsine oxide (PAO), a sulfhydryl reagent for vicinal or proximal thiol groups. The site of action of PAO was localized by photolabeling in the beta-subunit of flavocytochrome b [Doussière, J., Poinas, A, Blais, C., and Vignais, P. V. (1998) Eur. J. Biochem. 251, 649-658]. Moreover, the spin state of heme b is controlled by interaction of arachidonic acid with the flavocytochrome b [Doussière, J., Gaillard, J., and Vignais, P. V. (1996) Biochemistry 35, 13400-13410]. Here we report that the promoting effect of arachidonic acid on the activation of NADPH oxidase is due to specific binding of arachidonic acid to flavocytochrome b. Elicitation of NADPH oxidase activity by arachidonic acid is in part associated with an increased affinity of flavocytochrome b for O(2), an effect that was counteracted by the methyl ester of arachidonic acid. On the other hand, the affinity for NADPH was not affected by arachidonic acid. We further demonstrate that PAO antagonizes the effect of arachidonic acid on oxidase activation by decreasing the affinity of the oxidase for O(2), but not for NADPH. PAO induced a change in the spin state of heme b, as arachidonic acid does, with, however, some differences in the constraints imposed to the heme. It is concluded that the opposite effects of arachidonic acid and PAO are exerted on the beta-subunit of flavocytochrome b at two different interacting sites.     

Control of the expression of interleukin-2 activity

Mitogen stimulation of lymphocytes activates phospholipase A₂, which in turn generates arachidonic acid by its action on phospholipids. Cyclooxygenases catalyze the conversion of arachidonic acid to prostaglandins and related cyclic compounds, whereas lipoxygenases direct the formation of straight-chain hydroxylated derivatives such as, for example, the leukotrienes. The studies in this report suggest a correlation between arachidonic acid metabolism and production of the lymphokine, interleukin-2 (IL-2). Inhibitors of phospholipase A₂ activation, mepacrine, tetracaine, glucocorticoids and estradiol, all inhibited the expression of IL-2 activity in concanavalin A-stimulated mouse spleen cells. Inhibition of cyclooxygenase and lipoxygenase activities also resulted in decreased IL-2 production. This was established by the use of the inhibitors 5,8,11,14-eicosatetraynoic acid (ETYA), indomethacin, and nordihydroguajaretic acid (NDGA). A more direct attempt at influencing the arachidonic acid metabolism by addition of the fatty acid to IL-2 production cultures demonstrated that arachidonic acid bound very tightly to IL-2. Extensive dialysis or partial purification of the lymphokine by reverse-phase high-performance liquid chromatography failed to remove the bound arachidonic acid. It was shown, however, that no covalent interactions were involved. In addition to an active arachidonic acid metabolism, continuous protein synthesis was required for expression of IL-2 activity. Thus incubation with puromycin inhibited IL-2 production.     

The effects of products and inhibitors of arachidonic acid metabolism on the hamster sperm acrosome reaction

The mammalian sperm acrosome reaction (AR) is a fusion and fenestration of sperm head membranes which is essential for fertilization. Our earlier work demonstrated that arachidonic acid could stimulate the AR 15 min after addition to hamster sperm capacitated by incubation for 4.5 h. The present study was undertaken to determine whether inhibitors of arachidonic acid metabolism could affect the stimulation of the AR by arachidonic acid and whether products of its metabolism could stimulate the AR. Phenidone or nordihydroguaiaretic acid, inhibitors of both the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism, and docosahexaenoic acid, a cyclo-oxygenase pathway inhibitor, inhibited the AR induced by arachidonic acid. PGE2, a product of the cyclo-oxygenase pathway of arachidonic acid metabolism and 5- or 12-hydroxyeicosatetraenoic acid (HETEs) products of the lipoxygenase pathway, stimulated the AR when added to sperm capacitated by incubation for 4.5 h. Prostaglandins not derived from arachidonic were also tested: PGE1 stimulated the AR, but PGF1 alpha and PGA2 did not. We suggest that arachidonic acid metabolites produced by the sperm and by the female reproductive tract are important for the mammalian sperm AR.     

Platelet desensitization by arachidonic acid is associated with the suppression of endoperoxide/thromboxane A2 binding to the membrane receptor

The inhibitory mechanism of high levels of exogenously added arachidonic acid on activation of washed human platelets was investigated. While low levels of arachidonic acid (5-10 microM) induced aggregation, ATP secretion and increase in cytoplasmic free Ca2+ concentration (first phase of activation), these platelet responses did not occur significantly at high concentrations (30-50 microM). However, much higher concentrations than 80 microM again elicited these responses (second phase). The first phase of platelet activation was inhibited by cyclooxygenase inhibitor, indomethacin, whereas the second one was independent of such treatment. Thromboxane B2 was produced dose-dependently until reaching a plateau at arachidonic acid concentrations higher than 20 microM, irrespective of the lack of aggregation and secretion at high concentrations. After that the amount of free arachidonic acid which remained unmetabolized in platelets gradually increased. High concentrations of arachidonic acid as well as other polyunsaturated fatty acids caused desensitization of platelets in response to U46619, and also depressed the specific [3H]U46619-binding to the receptor as well as other polyunsaturated fatty acids. The amount free arachidonic acid needed in platelets to suppress [3H]U46619 binding corresponded to that needed to inhibit platelet aggregation. Furthermore, arachidonic acid dose-dependently induced fluidization of lipid phase of platelet membranes as detected by 1,6-diphenyl-1,3,5-hexatriene. These results suggest that the inhibition of platelet response by high levels of arachidonic acid can be attributed to interference with endoperoxide/thromboxane A2 binding to the receptor, probably due to perturbation of the membrane lipid phase due to excess amounts of free arachidonic acid remaining in the membranes.     

Incorporation of arachidonic acid into 1-acyl-2-lyso-sn-glycero-3-phosphocholine of the human neutrophil

In this study, the initial incorporation of arachidonic acid into human neutrophils has been examined. Neutrophils pulse labeled for 5 min with [3H]arachidonic acid rapidly incorporated this fatty acid into 1,2-diacylglycerophosphocholine. However, when neutrophils were pulse labeled with [3H]arachidonic acid for 5 min, washed, and allowed to incubate for an additional 120 min, the relative amount of [3H]arachidonic acid increased in alkylacylglycerophosphocholine molecular species. Similar, when neutrophils were pulse labeled, washed, and allowed to incubate in the presence of 30 microM unlabeled arachidonic acid for 120 min, [3H]arachidonic acid was also remodeled into alkylacylglycerophosphocholine. These results implied that the initial incorporation of [3H]arachidonic acid proceeded via a free fatty acid intermediate into 1,2-diacyl-GPC, while the subsequent remodeling of arachidonate-containing glycerophospholipids did not. This initial incorporation was further investigated in a number of cell-free systems. Disrupted neutrophils incubated with [14C]arachidonoyl-CoA incorporated [14C]arachidonic acid into 1,2-diacyl-GPC containing 16:0, 18:0, and 18:1 at their sn-1 position in a pattern similar to that seen when whole neutrophils were incubated with arachidonic acid for 5 min. A small percentage of [14C]arachidonate from [14C]arachidonoyl-CoA was incorporated into 1-alkyl-2-acyl-GPC. The enzymatic activity responsible was found predominately in the membrane fraction of the broken cell preparation. This selectivity of the CoA-dependent acyltransferase for 1-acyl-linked glycerophosphocholine was further examined by adding [14C]arachidonoyl-CoA and various 1-radyl-2-lyso-GPC to neutrophil membrane preparations. These studies provide evidence that the initial incorporation of arachidonic acid into sn-glycero-3-phosphocholine takes place by an arachidonoyl-CoA: lysophosphatidylcholine acyltransferase(s) which is selective for the 1-acyl-2-lyso-GPC.     

Effect of ethanol on the arachidonic acid metabolism in mouse peritoneal macrophages

Macrophages play an important role in the development of chronic inflammatory states. Ethanol has been shown to impair a number of membrane-linked phenomena. The synthesis and secretion of oxygenated metabolites of arachidonic acid is triggered at the cytoplasma membrane level. The present study was carried out in order to investigate the effect of ethanol on the arachidonic acid metabolism in mouse peritoneal macrophages. Two types of experiments were performed: with endogenous radiolabeled arachidonic acid and with exogenously added radiolabeled arachidonic acid. Our data show that ethanol in vitro activates the release of arachidonic acid from intracellular pools, while the proportion of endogenous substrate metabolized in the presence of ethanol is similar to that in controls. From the exogenous it seems clear that ethanol induces different effects depending whether the arachidonic acid is endogenous or added exogenously.     

On the importance of plasmalogen status in stimulated arachidonic acid release in the macrophage cell line RAW 264.7

We examined the dependence of stimulated arachidonic acid release on plasmalogens using the murine, macrophage cell line 264.7 and two plasmalogen-deficient variants, RAW.12 and RAW.108. All three strains responded to unopsinized zymosan to release arachidonic acid from phospholipid stores. Arachidonic acid release appeared to be dependent on calcium-independent phospholipase A(2) activation (iPLA(2)); bromoenol lactone, a specific inhibitor of calcium-independent iPLA(2), blocked arachidonic acid release with an IC(50) of approximately 2 x 10(-7)M. Propanolol, an inhibitor of phosphatidate phosphatase, and RHC-80267, an inhibitor of diglyceride lipase, had no effect on arachidonic acid release. Arachidonic acid release in the variants displayed similar magnitude, kinetics of response and sensitivity to the inhibitors when compared to the parent strain. Arachidonic acid was released from all major phospholipid head group classes with the exception of sphingomyelin. In wild-type cells, arachidonic acid released from the ethanolamine phospholipids was primarily from the plasmalogen form. However, in the plasmalogen-deficient cells release from the diacyl species, phosphatidylethanolamine, was increased to compensate. Restoration of plasmalogens by supplementation of the growth medium with the bypass compounds sn-1-hexadecylglycerol and sn-1-alkenylglycerol had no effect on arachidonic acid release. In summary, plasmalogen status appears to have no influence on the zymosan A stimulated release of arachidonic acid from the RAW 264.7 cell line.     

Possible role for arachidonic acid in the control of steroidogenesis in hen theca

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.     

Prostaglandin synthetase inhibitors modulate the effect of essential dietary arachidonic acid in the mosquito Culex pipiens

Effects of various inhibitors of prostaglandin metabolism on essential fatty acid function in Culex pipiens were examined by rearing the mosquito in synthetic dietary media containing arachidonic acid and putative prostaglandin inhibitors in various combinations. Both non-steroidal and steroidal anti-inflammatory drugs variously inhibited overall development and the arachidonic acid-dependent viability of newly emerged adults. In many cases such inhibitory effects could be counteracted by increased concentrations of dietary arachidonic acid, indicating that in the mosquito, as in mammals, these drugs interfered with arachidonic acid function specifically. In the cases of non-steroidal anti-inflammatorials (indomethacin, phenylbutazone and acetaminophen), which are known to inhibit enzymes of the prostaglandin synthetase complex, such inhibition is construed to indicate that prostaglandinogenesis may be among the physiological functions underlying the essentiality of arachidonic acid for the mosquito.     

Effects of postdecapitation ischemia on the metabolism of [14C]arachidonic acid and [14C]palmitic acid in the mouse brain

The effect of postdecapitation ischemia on the labeling of the free fatty acid pool and their incorporation in lipids was examined during the first 10 min after decapitation in mouse brain that had been injected intracerebrally with either [1-14C]arachidonic acid or [1-14C]palmitic acid. One min after decapitation, animals injected with labeled arachidonic acid exhibited a greatly reduced incorporation of label in brain phospholipids, diglycerides, and triglycerides. When radioactive palmitic acid was used, brain lipids exhibited considerably less inhibition of label. However, a similar degree of inhibition was observed 10 min after decapitation with both fatty acids. At this time, free arachidonic acid had decreased 84% as compared to the 24% decrease observed in the controls, and about 77% of the free palmitic acid remained in the free fatty acid fraction as compared with 30% in the controls. This decreased labeling may reflect ATP shortage that affects the fatty acid activation-reacylation reactions or the enzymes involved. Alternatively, the enhanced endogenous free arachidonic acid may compete with the radiolabeled arachidonic acid resulting in an inhibition of lipid labeling. Inhibition of label may have been greater in radiolabeled arachidonic acid than palmitic because of the larger accumulation of the former endogenous fatty acid during early ischemia.     

Arachidonic acid activation of the rat cardiovascular system]

The hypotensive activity of arachidonic acid is more important by intraaortic than by intravenous injection, in the rat. The evisceration of the animal abolishes this difference and reduces the activity of arachidonic acid. This action is not accompanied by thrombopenia and is only observed with high doses of arachidonic acid in this species. The hypotensive activity is inhibited by indomethacin but not by tranylcypromine.     

In vitro study of frog adrenal function--IX. Evidence against the involvement of lipoxygenase metabolites in the control of steroid production

The possible role of arachidonic acid metabolites of the lipoxygenase pathway in the regulation of steroidogenesis was studied in vitro using perifused frog interrenal (adrenal) glands. Graded doses of arachidonic acid (10(-6)-10(-4)M) increased the production of corticosterone and aldosterone in a dose-dependent manner. In the presence of indomethacin (5 X 10(-6)M), the effect of arachidonic acid on steroid secretion was totally abolished. Nordihydroguaiaretic acid (NDGA: 10(-6)M), a lipoxygenase inhibitor, did not alter the spontaneous secretion of corticosteroids and did not impair the stimulatory effect of arachidonic acid. In the presence of NDGA, both ACTH and angiotensin II were still able to stimulate corticosteroid production. Our data support the view that arachidonic acid metabolites play an important role in the regulation of amphibian steroidogenesis. Moreover, the results show that the lipoxygenase pathway is not involved in the spontaneous secretion of corticosteroids and in angiotensin II- or ACTH-induced steroidogenesis.     

Activation of the phosphatidylinositol 3-kinase-Akt/protein kinase B signaling pathway in arachidonic acid-stimulated human myeloid and endothelial cells: involvement of the ErbB receptor family

Although arachidonic acid has been demonstrated to stimulate a wide variety of cellular functions, the responsible mechanisms remain poorly defined. We now report that arachidonic acid stimulated the activity of class Ia phosphatidylinositol 3-kinase (PI3K) in human umbilical vein endothelial cells, HL60 cells, and human neutrophils. Pretreatment of endothelial cells with AG-1478, an inhibitor of the ErbB receptor family, resulted in the suppression of PI3K activation by arachidonic acid. The fatty acid enhanced the tyrosine phosphorylation of ErbB4 but not of ErbB2 or ErbB3. The ability of arachidonic acid to stimulate PI3K activity in neutrophils was suppressed by indomethacin and nordihydroguaiaretic acid, inhibitors of the cyclooxygenases and lipoxygenases, respectively, but not by 17-octadecynoic acid, an inhibitor of omega-hydroxylation of arachidonic acid by cytochrome P450 monooxygenases. Consistent with this, the activity of PI3K in neutrophils was stimulated by 5-hydroxyeicosatetraenoic acid. Arachidonic acid also transiently stimulated the phosphorylation of Akt on Thr-308 and Ser-473. Although PI3K was not required for the activation of the mitogen-activated protein kinases, ERK1, ERK2, and p38, in arachidonic acid-stimulated neutrophils, the fatty acid acted via PI3K to stimulate the respiratory burst. These results not only define a novel mechanism through which some of the actions of arachidonic acid are mediated but also demonstrate that, in addition to ErbB1 (epidermal growth factor receptor), ErbB4 can also be transactivated by a non-epidermal growth factor-like ligand.