Regulation of arachidonic acid release by enzyme(s) of rat brain cortex

Brain cortex membranes labeled with [14C]arachidonic acid were used as the source of substrate and enzyme for the assay of arachidonic acid (AA) liberation. A significant amount of AA was released Ca2(+)-independently, mainly from phosphatidic acid, polyphosphoinositides and phosphatidylserine. Quinacrine, inhibitor of phospholipase A2 (PLA2), suppressed AA release by 60% and neomycin, inhibitor of phospholipase C (PLC) by about 30%. Both inhibitors applied together have an additive effect. Physiological calcium level elevated AA liberation by 50%, whereas 2 mM calcium enhanced this process by a further 30%. Carbachol, exclusively in the presence of calcium, activated AA release selectively from phosphatidylinositol and diglycerides. We suggest that Ca2(+)-independent PLA2 and PLC play an important role in AA liberation, and that physiological increments of calcium may have serious implications.     

Protein kinase C involved in zymosan-induced release of arachidonic acid and superoxide but not in calcium ionophore-elicited arachidonic acid release or formation of prostaglandin E2 from added arachidonate.

Zymosan and phorbol ester induced in liver macrophages the release of arachidonic acid, prostaglandin E2, and superoxide; the calcium ionophore A 23187 elicited a release of arachidonic acid and prostaglandin E2 but not of superoxide, and exogenously added arachidonic acid led to the formation of prostaglandin E2 only. The zymosan- and phorbol-ester-induced release of arachidonic acid, prostaglandin E2, and superoxide was dose-dependently inhibited by staurosporine and K252a, two inhibitors of protein kinase C, and by pretreatment of the cells with phorbol ester which desensitized protein kinase C. The release of arachidonic acid or prostaglandin E2 following the addition of A 23187 or arachidonic acid was not affected by these treatments. Zymosan and phorbol ester but not A 23187 or arachidonic acid induced a translocation of protein kinase C from the cytosol to membranes in intact cells. These results demonstrate an involvement of protein kinase C in the zymosan- and phorbol-ester-induced release of arachidonic acid, prostaglandin E2, and superoxide; the release of arachidonic acid and prostaglandin E2 elicited by A 23187 and the formation of prostaglandin E2 from exogenously added arachidonic acid, however, is independent of an activation of protein kinase C.     

Nitric oxide alters arachidonic acid turnover in brain cortex synaptoneurosomes

Nitric oxide (NO) and arachidonic acid (AA) and also its metabolites are very important inter- and intracellular second messengers. They are involved in mechanisms of learning and memory. However, liberated in excessive amount in brain ischemia, Parkinson and Alzheimer diseases they are responsible for cell degeneration and death. Previously, we could show that Alzheimer disease's amyloid-beta protein enhanced nitric oxide liberation. The role of NO in AA metabolism is till now not well understood. Therefore, the aim of the present study was to investigate the mechanisms of NO-evoked activation of AA release and inhibition of AA incorporation into phospholipids of cortical rat brain synaptoneurosomes. The studies were carried out using NO donors, butyryl-cGMP (b-cGMP) and H2O2. All these compounds enhanced AA liberation from phosphatydilinositol (PI) and phosphatidylcholine (PC). Protein kinase ERK1/2, protein kinase C (PKC), cGMP-dependent protein kinase G (PKG) were involved in basal and NO-induced cytosolic phospholipase A2 (cPLA2) activation. Moreover, NO donors, b-cGMP and hydrogen peroxide (H2O2) exerted inhibitory effect on AA incorporation into PI and PC influencing arachidonyl-CoA transferase (AA-CoA-T) activity. AA-CoA synthase (AA-CoA-S) activity did not change. Specific inhibitors of protein kinase ERK1/2 (UO126), PKC (GF109203X), PKG (KT5823) had no effect on NO-mediated lowering of AA incorporation into PI and PC but inhibited the basal AA-CoA-S activity. Our data indicated that AA (10 microM) itself markedly decreased AA incorporation by about 50% into phospholipids of synaptoneurosomes membranes. Increasing release of AA and its metabolites causes the lowering of AA incorporation evoked by NO, b-cGMP and H2O2. Antioxidant, Resveratrol (100 microM) prevented NO- and cGMP-evoked inhibition of AA incorporation. These results suggest that NO affects the intracellular level of AA through alteration of cPLA2 and AA-CoA acyltransferase activities and may have an important implication in alterations of nerve endings properties and function.     

Thromboxane formation from arachidonic acid and prostaglandin H2 in rabbit spleen

Incubation of [1-14C]arachidonic acid (AA) and [1-14C]prostaglandin (PG)H2 with rabbit spleen homogenate and microsomes resulted in the formation of a substance with the chromatographic properties of thromboxane (Tx)B2. The radiolabeled material was indistinguishable from authentic TxB2 on TLC in three solvent systems and on radiometric gas chromatography. The generation of TxB2-like material from AA and PGH2 was not observed after boiling of the homogenate and microsomes, and was completely inhibited by imidazole (5 mM). The transformation of AA into the TxB2-like material was not observed during incubation in the presence of indomethacin (28 microM). These results indicate that TxB2 is the principal product of arachidonic acid metabolism by the homogenate or microsomes of rabbit spleen.     

Heterologous desensitization of platelet-derived growth factor-mediated arachidonic acid release and prostaglandin synthesis.

Prolonged treatment of quiescent Swiss 3T3 cells with vasopressin induced heterologous desensitization of specific early signals stimulated by platelet-derived growth factor (PDGF). PDGF caused a striking dose-dependent release of [3H]arachidonic acid (EC50 = 2 ng/ml) and prostaglandin E2 (EC50 = 5 ng/ml). These responses are severely attenuated (greater than 85%) by prior exposure to vasopressin in a dose-dependent manner (IC50 = 1.5 nM). Maximal loss of responsiveness occurred after 40 h of vasopressin treatment with a half-maximal desensitization after 11-13 h. The desensitization is dependent upon binding to the V1 receptor, since it can be prevented by the antagonist [Pmp1,O-Me-Tyr2,Arg8]vasopressin. In contrast, stimulation of inositol phosphate accumulation and production of diacylglycerol and phosphatidic acid by PDGF are unchanged. Thus, the observed heterologous desensitization cannot be attributed to an inability to activate phospholipase C. Furthermore, prior exposure to vasopressin did not affect the ability of PDGF to evoke tyrosine phosphorylation of cellular substrates, demonstrating that vasopressin-induced heterologous desensitization causes a block at a point distal to activation of receptor tyrosine kinase activity. Other downstream responses including transient induction of c-fos expression and stimulation of DNA synthesis were attenuated by vasopressin pretreatment. The findings demonstrate a novel mechanism of heterologous cellular desensitization namely, persistent occupancy of a guanine nucleotide-binding protein-coupled receptor, like the V1 type vasopressin receptor, attenuates responsiveness to a polypeptide growth factor like PDGF that initiates responses through a tyrosine kinase receptor.     

Hydrocortisone inhibits prostaglandin production but not arachidonic acid release from cultured macrophages

We have investigated the action of hydrocortisosone on arachidonic acid mobilisation in cultures of mouse peritoneal macrophages, mouse L929 cells and the mouse macrophage-like cell line RAW264. Hydrocortisone inhibits both arachidonic acid release and prostaglandin production by L929 cells. However, prostaglandin production by macrophages or RAW264 cells is inhibited with a concomitant stimulation rather than inhibition of arachidonic acid release. These data suggest that hydrocortisone acts at the level of phospholipase activity in fibroblasts but at a later stage of prostanoid production in macrophages.     

Evidence of protein kinase C involvement in phorbol diester-stimulated arachidonic acid release and prostaglandin synthesis

Many stimulators of prostaglandin production are thought to activate the Ca2+- and phospholipid-dependent protein kinase first described by Nishizuka and his colleagues (Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T., and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695. In this paper we report evidence that the activation of protein kinase C caused by 12-O-tetradecanoylphorbol-13-acetate (TPA) is involved in the increased prostaglandin production induced by 12-O-tetradecanoylphorbol-13-acetate in Madin-Darby canine kidney (MDCK) cells. We have shown that TPA activates protein kinase C in MDCK cells with similar dose response curve as observed for TPA induction of arachidonic acid release in MDCK cells. Activation of protein kinase C was associated with increased phosphorylation of proteins of 40,000 and 48,000 daltons. We used two compounds (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OMe) and 1-(5-isoquinolinesulfonyl)piperazine) known to inhibit protein kinase C by different mechanisms to further examine if activation of protein kinase C was involved in the increased synthesis of prostaglandins in TPA-treated MDCK cells. We found that both compounds inhibited protein kinase C partially purified from MDCK cells and that ET-18-OMe inhibited the phosphorylation of proteins by protein kinase C in the intact cells. Addition of either compound during or after TPA treatment decreased both release of arachidonic acid from phospholipids and prostaglandin synthesis. Release of [3H]arachidonic acid from phosphatidylethanolamine in TPA-treated cells was blocked by ET-18-OMe or 1-(5-isoquinolinesulfonyl)piperazine addition. However, arachidonic acid release stimulated by A23187 is not blocked by Et-18-OMe. When assayed in vitro, treatment of cells with Et-18-OMe did not prevent the enhanced conversion of arachidonic acid into prostaglandins induced by pretreatment of cells with TPA. Our results suggest that the stimulation of phospholipase A2 activity by TPA occurs via activation of protein kinase C by TPA.     

The negative coupling between serotonin and muscarinic receptor(s) for arachidonic acid and inositol phosphates release in brain cortex synaptoneurosomes. Effect of aging

In the present study we have investigated the relationship between serotonergic receptor(s) (5-HTR) and muscarinic cholinergic receptor (mAChR) activation, leading to the arachidonic acid (AA) release and inositol phosphates (IPs) formation in adult and aged brain cortex synaptoneurosomes. It was observed that serotonin (5-HT) almost completely inhibits carbachol-stimulated AA release in adult brain. This negative coupling between 5-HTR and mAChR probably depends on the direct stimulation of the acylation reaction coupled with 5-H_1AR. In the aged brain this type of interaction does not occur because aging eliminates carbachol and 5-HT effect. On the other hand, in aged brain, both 5-HT and carbachol more actively stimulate IPs accumulation than in adult brain. Serotonin inhibits carbachol-stimulated IPs release to the level observed during 5-HT₂R activation. Our study indicates for the first time, the negative coupling between 5-HTR and mAChR for AA liberation in adult brain and a lack of this kind of receptors interaction in senescent brain. In adult brain, 5-HT_1AR-stimulated AA incorporation is responsible for this coupling. Moreover, serotonin through the stimulation of 5-HT₂R, suppresses mAChR-dependent IPs liberation in adult and aged brain similarly. The interactions between these two neurotransmitter receptors lead to the modification of lipid mediators formation and may have important implications in alteration of signal transduction in adult and in aged brain.     

Triacylglycerol lipase mediated release of arachidonic acid for prostaglandin synthesis in rabbit kidney medulla microsomes

The effect of triarachidonin on the synthesis of prostaglandins in rabbit kidney medulla microsomes was examined. Medulla microsomes were incubated with triarachidonin in 0.1 M--Tris/HCl buffer (pH 7.0) containing reduced glutathione and hydroquinone and the formed prostaglandin E2, prostaglandin F2 alpha and prostaglandin D2 were measured by high-pressure liquid chromatography using 9-anthryldiazomethane for derivatization. The addition of triarachidonin (1-10 microM) stimulated prostaglandin formation in a dose-dependent manner. Under our incubation conditions rabbit kidney medulla was found to produce prostaglandin E2 mainly. When arachidonic acid, instead of triarachidonin, was added to the incubation mixture of microsomes, the identical profile of prostaglandin products was obtained. When the pH of the reaction mixture was changed from 7.0 to 8.0, the rate of triarachidonin-induced prostaglandin E2 formation was approximately 60% of that observed at pH 7.0. Studies utilizing Ca2+ and EGTA revealed that triacylglycerol lipase of kidney medulla is independent of Ca2+. The addition of epinephrine made the stimulatory effect of triarachidonin on prostaglandin E2 formation more pronounced. These results suggest that epinephrine-activated triacylglycerol lipase is present in the renomedullary microsomes, and this enzyme activity is a potential mediator of release of arachidonic acid for prostaglandin synthesis in the kidney medulla.     

Oxytocin stimulates the release of arachidonic acid and prostaglandin F2 alpha from human decidual cells

Oxytocin at a physiological concentration stimulated the immediate release of free arachidonic acid from dispersed human decidual cells in a perfusion system. This indicates that oxytocin activates phospholipase(s) thus enhancing prostaglandin synthesis. The effect of oxytocin on the release of [3H]-arachidonic acid from decidual cells of women in labor was significantly greater (1036 +/- 207, mean dpm +/- SEM, n = 23) than from those of women not-in-labor (505 +/- 121 dpm, n = 12) or with endometrial cells of non-pregnant women (711 +/- 210 dpm, n = 18), and correlates well with reported oxytocin receptor concentrations in these tissues. These new findings are consistent with a role for endogenous oxytocin in stimulating prostaglandin synthesis at the onset of parturition.     

Serum-induced arachidonic acid release and prostaglandin biosynthesis are potentiated by oxygenated sterols in NRK 49F cells

Fetal calf serum is able to activate arachidonic acid release from phospholipids in NRK 49F cells. We showed that this phenomenon can be potentiated by adding oxysterols to the culture medium. The oxysterol effect was dose-dependent and was not observed in the absence of fetal calf serum. Greater amounts of prostaglandin E2 and prostaglandin F2 alpha were released into the medium in the presence of oxysterols without apparent modification of the cyclooxygenase activity. The most effective oxysterols, in descending order, were the following: calcitriol greater than 7 alpha-hydroxycholesterol greater than 7 beta-hydroxycholesterol greater than 25-hydroxycholesterol. Cholesterol and 7-ketocholesterol were unable to activate phospholipase activity. The mechanism of this activation by oxysterols is still unknown.     

The regulation of prostaglandin and arachidonoyl-CoA formation from arachidonic acid in rabbit kidney medulla microsomes by palmitoyl-CoA

Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl-CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). In the present study, we investigated the effects of palmitic acid (PA) and palmitoyl-CoA (PA-CoA) on the PG and AA-CoA formation from high and low concentrations of AA (60 and 5 microM) in rabbit kidney medulla microsomes. The kidney medulla microsomes were incubated with 60 or 5 microM [14C]-AA in 0.1 M-Tris/HCl buffer (pH 8.0) containing cofactors of COX (reduced glutathione and hydroquinone) and cofactors of ACS (ATP, MgCl2 and CoA). After incubation, PG (as total PGs), AA-CoA and residual AA were separated by selective extraction using petroleum ether and ethyl acetate. PA (10-100 microM) had no effect on the PG and AA-CoA formation from either 60 or 5 microM AA. PA-CoA (10-100 microM) was without effect on the PG and AA-CoA formation from 60 microM AA, whereas it markedly decreased the PG formation (6-40%) and increased the AA-CoA formation (1.1-2.3-fold) from 5 microM AA, showing that the effects of PA-CoA on the PG and AA-CoA formation change depending on the AA concentration. These results suggest that PA-CoA, but not PA, may regulate the PG and AA-CoA formation at low substrate concentrations (close to the physiological concentration of AA), and that this in-vitro method using 5 microM AA may be useful for clarifying the homeostatic control of the metabolic fate of AA into these two enzymatic pathways.     

Eicosapentaenoic acid metabolism in brain microvessel endothelium: effect on prostaglandin formation

Mouse brain microvessel endothelial cells convert eicosapentaenoic acid (EPA) to prostaglandin (PG) E3, PGI3, and several hydroxy fatty acid derivatives. Similar types of products are formed by these microvessel endothelial cells from arachidonic acid. The formation of PGI2 and PGE2 is reduced, however, when the brain microvessel endothelial cultures are incubated initially with EPA. Exposure to linolenic or docosahexaenoic acid also decreased the capacity of these microvessel endothelial cells to form PGI2 and PGE2, but the reductions were smaller than those produced by EPA. Like the endothelial cultures, intact mouse brain microvessels convert EPA into eicosanoids, and incubation with EPA reduces the subsequent capacity of the microvessels to produce PGI2 and PGE2. Brain microvessel endothelial cells took up less EPA than arachidonic acid, primarily due to lesser incorporation into the inositol, ethanolamine, and serine glycerophospholipids. By contrast, considerably more EPA than arachidonic acid was incorporated into triglycerides. These findings suggest that the microvessel endothelium may be a site of conversion of EPA to eicosanoids in the brain and that EPA availability can influence the amount of dienoic prostaglandins released by the brain microvasculature. Furthermore, the substantial incorporation of EPA into triglyceride suggests that this neutral lipid may play an important role in the processing and metabolism of EPA in brain microvessels.     

Correlation between cyclic AMP and LH release following prostaglandin E2 administration.

Five min following a single iv injection of PGE2 into ovariectomized mature rats pretreated with estrogen and progesterone, plasma LH and plasma and pituitary cyclic AMP levels were raised significantly. A close correlation was observed between increased pituitary cyclic AMP contents and release of plasma LH. The average level of cyclic AMP in the anterior pituitary and plasma cyclic AMP increased significantly, while the circulating plasma LH level was not changed at 1 min after PGE2 injection. Plasma LH le-el increased at 2 min after PGE2 and reached a maximum level at the above-mentioned time. This is consistent with hypothesis that increased release of hormone is a consequence of increased pituitary cyclic AMP content.     

The interaction of ethanol and exogenous arachidonic acid in the formation of leukotrienes and prostaglandin D2 in mastocytoma cells

The present studies were undertaken to examine the hypothesis that ethanol could affect cellular biosynthesis in the murine mastocytoma cell of prostaglandins and leukotrienes, oxidative metabolites of arachidonic acid, at concentrations that could be encountered in vivo as well as during in vitro experiments. The effects of ethanol which encompass these concentration ranges (200-1000 mg%) can be summarized as follows: first in the absence of exogenous arachidonic acid, ethanol caused a dose dependent decrease in the production of leukotrienes which was statistically significant at 200 mg%. At 1000 mg%, ethanol caused a 20-50% decrease in leukotrienes and a 21% decrease in the amount of prostaglandin D2 (PGD2) formed in these cells. Secondly, when cells were incubated with exogenous arachidonic acid (14 micrograms/ml), large increases in both PGD2 and leukotrienes occurred. Under these conditions, ethanol caused a further increase in the amount of leukotrienes and a small increase in the amount of PGD2 formed. This stimulatory effect was specific for ethanol since neither t-butanol nor n-butanol caused the enhanced production of leukotrienes with exogenous arachidonic acid. Thus, these experiments suggest that ethanol affects metabolism of arachidonic acid at reasonably low doses (200-400 mg%) of ethanol in a manner dependent on the free arachidonic acid in the tissue. Also, in vitro experiments in which ethanol is used as a solvent for arachidonic acid could be greatly affected by high levels of ethanol (500-1000 mg%) which are frequently utilized.     

Mechanism of thrombin-induced arachidonic acid release in osteoblast-like cells

In a previous study, we have reported that thrombin stimulates phosphatidylcholine hydrolysis by phospholipase (PL) D, but has little effect on phosphoinositide hydrolysis by PLC in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the mechanism of the thrombin-induced arachidonic acid (AA) release in MC3T3-E1 cells. Thrombin stimulated AA release dose dependently in the range between 0.1 and 1 U/ml. Quinacrine, a PLA₂ inhibitor, suppressed the thrombin-induced AA release. In addition, quinacrine also suppressed the thrombin-induced prostaglandin E₂ synthesis in these cells. On the other hand, propranolol, which is known to inhibit phosphatidic acid phosphohydrolase, did not affect the thrombin-induced AA release. 1(6-((17β-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-d |ione (U-73122), a PLC inhibitor, had no effect on the AA release by thrombin. In addition, 1,6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC-80267), a selective inhibitor of diacylglycerol lipase, had little effect on the thrombin-induced AA release. Neither propranolol, U-73122 nor RHC-80267 affect the thrombin-induced prostaglandin E₂ synthesis. These results strongly suggest that thrombin induces AA release not by phosphatidylcholine hydrolysis by PLD nor phosphoinositide hydrolysis by PLC but mainly by PLA₂ in osteoblast-like cells.     

A carbon-centered free radical intermediate in the prostaglandin synthetase oxidation of arachidonic acid. Spin trapping and oxygen uptake studies

The ESR spin-trapping technique has been used to identify a free radical involved in the oxygenation of arachidonic acid by ram seminal vesicle microsomes. The ESR spectrum of the radical adduct indicates that a carbon-centered arachidonic acid free radical has been observed. The formation of this species is inhibited by indomethacin, but not by phenol, and it is probably the first intermediate formed during the prostaglandin synthetase-catalyzed oxidation of arachidonic acid. The chemical identity of the trapped radical was substantiated with an independent synthesis of a closely related radical adduct.     

Phagocytosis-induced release of arachidonic acid from human neutrophils

The phospholipids of human neutrophils were labeled with [³H] arachidonic acid and [¹⁴C] palmitic acid. Phagocytosis of opsonized zymosan resulted in rapid release of free arachidonic acid but not of palmitic acid. Arachidonic acid was not released when the cells were exposed to unopsonized zymosan, zymosan-activated serum, or phorbol myristate acetate. These observations suggest that phagocytosis of opsonized zymosan results in the activation of a phospholipase A₂.