Eicosanoid metabolism in cholesterol-enriched arterial smooth muscle cells: reduced arachidonate release with concomitant decrease in cyclooxygenase products.

A biochemical correlation between vascular cholesterol metabolism and eicosanoid biosynthesis has not been fully elucidated. To assess the effects of cholesteryl ester (CE) accretion on eicosanoid synthesis, we studied eicosanoid metabolism in cultured rabbit aortic smooth muscle cells (SMC) following lipid-enrichment by incubation with cationized LDL (cLDL). SMC exposed to cLDL synthesized 50% less immunoreactive 6-keto-PGF1 alpha than untreated cells when exposed to the calcium ionophore, A-23187. In addition, cLDL-treatment reduced arachidonate acid (AA)-induced prostacyclin (PGI2) production sevenfold. Components of cLDL decreased eicosanoid biosynthesis in the following rank-order: linoleate greater than cholesterol greater than apo-cLDL. Lipid-enriched cells incorporated amounts of [1-14C]AA into phosphatidylcholine and phosphatidylethanolamine equal to control cells, but subsequent exposure to ionophore released significantly less radioactivity as free arachidonate (AA), with proportionally less conversion to eicosanoids. Ionophore released equivalent amounts of AA from all phospholipids, suggesting specificity for uptake, but not release of AA by cellular phospholipases. Cells enriched in CE had an eightfold decrease in percentage of phospholipid-derived AA relative to linoleate as compared to controls. Taken together, our data demonstrate that SMC metabolism of cLDL leads to cholesterol and CE accretion concomitant with diminished production of eicosanoids. Potential mechanisms for this effect include competitive inhibition of eicosanoid production by linoleate derived from LDL, direct inhibition of phospholipase A2 activity by cholesterol, and decrease in cyclooxygenase activity. These findings may have pathophysiological significance in that a reduction in PGI2 synthetic capacity of arterial SMC may exacerbate CE deposition since PGI2 promotes intracellular CE hydrolysis.

Increased bioavailability of enzymes of eicosanoid synthesis in hepatic and extrahepatic tissues after portacaval shunting.

Metabolites of arachidonic acid have been attributed to severe circulatory, metabolic and hormonal alterations in patients with chronic liver disease. In order to study changes of the tissue-specific availability of enzymes of eicosanoid synthesis, we used portacaval-shunted rats, as this model exhibits many clinical and biochemical similarities to patients suffering from cirrhosis of the liver. Microsomal mass and maximal velocity of prostaglandin H synthase, the initial enzyme of prostaglandin synthesis, were markedly and permanently increased after shunting in both hepatic and extrahepatic tissues as compared to those of sham-operated rats. Maximal velocity of thromboxane synthase and prostacyclin synthase, two more peripheral enzymes of the arachidonic acid cascade, were tissue-specifically enhanced, whereas the apparent affinities (Km) remained unchanged. Determination of 5-lipoxygenase activity in tissue preparations disclosed a preferential increase in the liver, lung and renal cortex after portacaval shunting. Furthermore, exposure to endotoxin closely mimicked the shunting-induced changes. These results suggest that after portacaval shunting and possibly in patients with advanced liver disease, profound abnormalities at the level of local enzyme expression might play a pathophysiologically important role in the control of eicosanoid synthesis.

Differential effect of dexamethasone on the regulation of phospholipase A2 and prostanoid synthesis in undifferentiated and phorbolester-differentiated U937 cells.

The human undifferentiated histiocytic cell-line U937 can be induced to differentiate by incubation with 12-0-tetradecanoylphorbol-13-acetate (TPA) into macrophage-like cells. Dexamethasone reduced the prostaglandin production in TPA-differentiated U937 cells dose dependently, whereas undifferentiated U937 cells were dexamethasone insensitive. Concomitantly phospholipase A2, the enzyme liberating the prostaglandin precursor arachidonic acid, was inhibited by dexamethasone in TPA-differentiated but not in undifferentiated U937 cells. The activity of lysophosphatide acyltransferase, the key enzyme of fatty acid reacylation into phospholipids, remained unchanged both in undifferentiated and TPA-differentiated U937 cells. The data suggest that responsiveness to glucocorticoid-dependent regulation of prostanoid synthesis is acquired by cells of the monocyte-macrophage lineage late in differentiation.

The effect of a fish oil diet on the fatty acid composition of individual phospholipids and eicosanoid production by rat platelets.

When rats were fed a diet containing chow or fish oil for six weeks, the platelet phospholipid content and percent distribution were similar. In the fish oil fed animals there was a 54, 40, 41, and 24% reduction, respectively, in the levels of 20:4(n-6) in the choline-, ethanolamine-, inositol- and serine-containing glycerophospholipids. Dietary fish oil increased the total (n-3) polyunsaturated fatty acid content in all lipids. This effect was most pronounced in the ethanolamine glycerophospholipids which now contained 26, 11, and 4 nmols of 20:5(n-3), 22:5(n-3), and 22:6(n-3) in 10(9) cells. Ionophore A23187 stimulation of platelets from the chow fed rats resulted in the synthesis of 7, 64, and 3.5 nmols of 12-hydroxy-5,8,10-heptadecatrienoic acid, 12-hydroxy-5,8,10,14-eicosatetraenoic acid and 12-hydroxy-5,8,10,14,17-eicosapentaenoic acid, respectively, from 1 X 10(9) cells. The values from animals fed fish oil were 4, 18, and 27 nmol/10(9) platelets. It was not possible to detect any lipoxygenase products from 22:5(n-3) or 22:6(n-3), even though both acids are readily metabolized by lipoxygenase when added directly to platelets. These findings suggest that 22-carbon (n-3) fatty acids are not liberated when phospholipases are activated by calcium mobilization.

Lack of effects of trans fatty acids on eicosanoid biosynthesis with adequate intakes of linoleic acid.

The minimum requirement of linoleic acid to prevent effects of dietary C18 trans fatty acids on eicosanoid biosynthesis in rats was assessed. In a first experiment, six groups of animals were fed diets with a high content of trans fatty acids [20% of energy (en%)], and increasing amounts of linoleic acid (0.4 to 7.1 en%). In a second experiment, four groups of rats were fed diets designed to compare trans fatty acids with saturated and cis-monounsaturated fatty acids of the same chain length at the 2 en% linoleic acid level. After 9-14 weeks the biosynthesis of prostacyclin by pieces of aorta and the biosynthesis of hydroxy-heptadecatrienoic acid and 12-hydroxy-eicosatetraenoic acid by platelets were measured. The fatty acid compositions of aorta phospholipid and platelet lipid were also determined. Both the prostacyclin-production by aorta pieces and the production of hydroxy-heptadecatrienoic acid and 12-hydroxy-eicosatetraenoic acid by platelets appeared to be a linear function of the arachidonic acid level in aorta phospholipid and platelet lipid, irrespective of the trans fatty acid content in the diet. This indicates that trans fatty acids do not directly influence enzymes involved in eicosanoid biosynthesis. In a direct comparison with cis-monounsaturated or saturated fatty acids with 2 en% linoleic acid in the diet, only a moderate reduction in arachidonic acid level in aorta phospholipids in the group fed trans fatty acids was observed. The geometry of the double bond did not influence the arachidonic acid level in platelet lipid, although the diet rich in saturated fatty acids increased arachidonic acid levels significantly compared with all other diets.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of invading leukocytes in enhanced atrial eicosanoid production following rabbit left ventricular myocardial infarction.

The isolated perfused hearts of rabbits previously subjected to in vivo left ventricular myocardial infarction (LVMI) show a 5-10-fold increase in f-Met-Leu-Phe (FMLP) and bradykinin (BK)-stimulated eicosanoid metabolite production relative to noninfarcted hearts. This exaggerated arachidonate metabolism has been shown to occur primarily in the cardiac atria, a site remote from the zone of injury and to be associated with a 10-15-fold increase in atrial FMLP receptor number in the absence of atrial inflammation. All of these changes were temporally related to leukocyte infiltration into the infarct zone. To determine whether invading leukocytes mediate these responses, acute inflammatory cell influx was suppressed either by inducing leukopenia with nitrogen mustard or by administration of BW-755C, a mixed cyclooxygenase-lipoxygenase inhibitor. Both pharmacological manipulations resulted in a decrease in inflammatory cells in the infarct zone and a marked suppression (50-70%) of ex vivo agonist-stimulated eicosanoid metabolite production from perfused hearts and isolated atria. These manipulations also resulted in reversal of ex vivo FMLP-induced coronary vasoconstriction as well as augmentation of BK-induced coronary vasodilation. Further studies in nitrogen mustard-treated animals revealed a suppression of the LVMI-stimulated increase in atrial FMLP receptor number. These data show that suppression of leukocyte invasion after LVMI attenuates enhanced cardiac and atrial eicosanoid metabolite production, and results in marked changes in coronary vascular reactivity. An additional finding was that basal and stimulated LTB4 production was markedly increased in infarcted hearts. In vivo suppression of the increase in LTB4 production by BW-755C was associated with inhibition of inflammatory cell influx into the infarct zone. It therefore appears that LTB4 may be an important proinflammatory mediator of leukocyte invasion after LVMI.

n-3 fatty acids as precursors for active metabolic substances: dissonance between expected and observed events.

It may be hypothesized that many diseases are associated with an overproduction of eicosanoids from the n-6 acid, arachidonic acid (20:4n-6), and the formation and function of these n-6 eicosanoids can be antagonized by dietary n-3 fats. This hypothesis provides a basis for evaluating the benefits and risks of including various amounts of n-3 and n-6 fats in the diet. Understanding the impact of dietary polyunsaturated fats leads inevitably to a reappraisal of what is 'normal' in terms of what is typical and what is desirable for the fatty acid composition of tissue lipids, the magnitude of eicosanoid-mediated responses, and the frequency and severity of certain diseases.

Endotoxin protection against pulmonary oxygen toxicity and its reversal by acetyl salicylic acid: role of eicosanoid production by broncho-alveolar lavage cells.

Small doses of endotoxin markedly increase the survival rate of adult rats exposed to 98% oxygen for periods that are normally lethal. The lysine salt of acetyl salicylic acid (L-ASA) partially reverses this protective effect of endotoxin. In this pilot study we investigated the level of eicosanoid production by broncho-alveolar lavage (BAL) cells and found that BAL cells of endotoxin protected rats, present in abundance, have an equal or increased capacity of HHT, 15-HETE, 12-HETE, LTB4 and 5-HETE production. These data suggest that production of the lipoxygenase products by BAL cells does not seem to play an important role in the pathogenesis of pulmonary oxygen toxicity. We did not find any indication for the occurrence of shunting of arachidonic acid metabolism to the lipoxygenase pathway as an explanation for the reversal of endotoxin's protective action by L-ASA.

Glomerular eicosanoid production in acute serum sickness nephritis.

To determine if the induction of immune-mediated glomerular injury influences the formation of glomerular cyclooxygenase products, we measured thromboxane B2 (TXB2), 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and prostaglandin E2 (PGE2) production by isolated glomeruli of rabbits induced with acute serum sickness nephritis by the administration of bovine serum ablumin (BSA). Animals were randomly assigned to one of three experimental groups: animals injected with BSA (BSA group; n = 11); animals injected with normal saline (control group; n = 11); and animals injected with BSA which were treated with the thromboxane synthetase inhibitor, OKY-046 (BSA + OKY-046; n = 6). Animals in the BSA and BSA + OKY groups developed severe proteinuria and glomerular histologic lesions of nephritis. No differences in proteinuria, serum creatinine and severity of histologic nephritis were observed between the two groups. Examination of glomerular eicosanoid production at the end of the experiment showed a marked reduction of glomerular PGE2 and 6-keto-PGF1 alpha production with a smaller reduction of glomerular TXB2 production in the BSA group. In the BSA + OKY-046 group, the production of TXB2 was significantly less than that in the BSA group; despite this, no effect on proteinuria could be discerned.

Synthesis of prostaglandins and eicosanoids by the mast cell secretory granule.

The identification of a non-bilayer phospholipid storage in the secretory granule and the linking of the eicosanoid production with the release of histamine have prompted us to examine whether the secretory granule may also serve as both the source as well as the site of prostaglandin synthesis during exocytosis. By exposing the contents of purified granules to exogenous arachidonic acid at neutral pH, we observed the rapid formation of many eicosanoids. The presence of prostaglandins E2, D2 and F2a were identified. The kinetics of E2 formation was also followed. The localization of the arachidonic acid cascade to the secretory granule explains why the production of eicosanoids is so intimately tied to the process of granule exocytosis.

Eicosanoid synthesis in 7,12-dimethylbenz(a)anthracene-induced mammary carcinomas in Sprague-Dawley rats fed primrose oil, menhaden oil or corn oil diet.

The comparative effects of high-fat diets (20%, w/w) on eicosanoid synthesis during mammary tumor promotion in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rats were studied using diets containing 20% primrose oil (PO), 20% menhaden oil (MO) or 20% corn oil (CO). Sprague-Dawley rats fed the PO or MO diet had 21% of 24% fewer adenocarcinomas, respectively, than rats fed the CO diet. Histologically (i.e., mitotic figures, inflammatory cell infiltration and necrosis), the CO-fed rats exhibited the highest frequency of changes within tumors. Plasma fatty acid composition was significantly altered by diet, reflecting the composition of the oils which were being fed. Only the plasma of PO-fed rats contained detectable levels of gamma-linolenic acid (GLA). Arachidonic acid (AA) levels were significantly higher (p less than 0.05) in PO-fed than in CO- or MO-fed rats. MO-fed rats had significantly higher levels of plasma palmitic acid, while palmitoleic, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids were detected only in MO-fed rats. As expected, linoleic acid (LA) and AA levels were lower (p less than 0.05) in the MO-fed rats than in PO- or CO-fed groups. The plasma of the CO-fed rats contained significantly higher levels of oleic acid. Eicosanoid synthesis in mammary carcinomas of rats fed the 20%-fat diets was 2-10 times higher than in mammary fat pads of control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of anti-inflammatory drugs on eicosanoid formation in a chronic model of inflammatory bowel disease in the rat.

1. The effects of anti-inflammatory drugs on eicosanoid formation and colonic damage in a chronic model of inflammatory bowel disease (IBD) in the rat were investigated. 2. A single colonic instillation of the hapten, trinitrobenzene sulphonic acid (TNB) resulted in ulceration and inflammation which persisted for 3 weeks. 3. The macroscopic colonic damage, present 3 weeks after TNB, was correlated with an increase in immunoreactive 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and leukotriene B4 (LTB4) synthesis by the rat colon. 4. Anti-inflammatory drugs were administered 2 weeks after TNB, when there was substantial colonic damage, and continued for a week. The experimental drug BW755C inhibited the increased formation of 6-keto-PGF1 alpha and LTB4 by the inflamed colon. Indomethacin and aspirin markedly inhibited prostanoid formation in both inflamed and control colon. Sulphasalazine or prednisolone also inhibited the formation of 6-keto-PGF1 alpha but the effects were less marked. 5. None of the anti-inflammatory drugs significantly reduced the colonic damage induced by TNB. 6. The results suggest that eicosanoids, including LTB4, have only a minor role in maintaining the chronic macroscopic damage induced in the rat colon by TNB. The role of such eicosanoids in the underlying infiltration and activity of inflammatory cells in this model of IBD, however, is not known.

The influence of n-6 and n-3 fatty acids on kidney phospholipid composition and on eicosanoid production in aging rats.

Changes in eicosanoid production may contribute to some of the complications of the aging process such as atherosclerosis and glomerular sclerosis. Polyunsaturated fatty acids of the n-6 and n-3 series are precursors of eicosanoids. We fed diets containing safflower oil as a source of n-6 fatty acids, fish oil as a source of n-3 fatty acids or beef tallow as a source of saturated fats to three groups of normal rats from 2-18 months of age. We demonstrated incorporation of the n-3 fatty acids, 20:5n-3 and 22:6n-3 into kidney phospholipids. Feeding of the diet containing n-3 fatty acids was associated with a markedly decreased glomerular production of PGE, 6-keto-PGF1 alpha and TXB2. It also decreased the aortic production of 6-keto-PGF1 alpha and platelet production of TXB2. No significant effect of n-6 fatty acids on dienoic eicosanoid production was observed. There were no adverse effects on kidney function as measured by urinary protein excretion and serum creatinine levels or on renal morphology by any diet. A diet enriched in n-3 fatty acids for 18 months remains effective in decreasing dienoic eicosanoids in the aging rat.

Labelling of lipids and phospholipids with [3H]arachidonic acid and the biosynthesis of eicosanoids in U937 cells differentiated by phorbol ester.

Phorbol esters induce morphologic and biochemical differentiation in U937 cells, a monocyte/macrophage-like line derived from a human histiocytic lymphoma. We are interested in the phorbol ester-stimulated release of arachidonic acid from cellular membranes and the subsequent synthesis of eicosanoids, as it may prove to correlate with the induced cellular differentiation. Undifferentiated log-phase U937 cells released little recently incorporated [3H]arachidonic acid, but phorbol 12-myristate 13-acetate increased its apparent rate of release to that of cells differentiated by exposure to phorbol myristate acetate for 3 days. Exposure of washed differentiated cells immediately prelabelled with [3H]arachidonic acid to additional phorbol myristate acetate did not augment the release of [3H]arachidonic acid. The basal release of nonradioactive fatty acids from differentiated cells was 5-10 times that of undifferentiated cells, and phorbol myristate acetate increased their release from both types of cell 2- to 3-fold. Differentiated cells immediately prelabelled with [3H]arachidonic acid exhibited greater incorporation into phosphatidylinositol and phosphatidylcholine, and contained more radioactive free arachidonic acid, compared with undifferentiated cells. Undifferentiated cells contained more radioactivity in phosphatidylserine, phosphatidylethanolamine and neutral lipids. Phorbol myristate acetate caused differentiated cells to release [3H]arachidonic acid from phosphatidylinositol, phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine, but release from neutral lipids was reduced, and the content of [3H]arachidonic acid increased. In undifferentiated cells incubated with phorbol myristate acetate, radioactivity associated with phosphatidylserine, phosphatidylethanolamine and neutral lipid was reduced and [3H]arachidonic acid was unchanged. Synthesis of cyclooxygenase products exceeded that of lipoxygenase products in both differentiated and undifferentiated cells. Phorbol myristate acetate increased the synthesis of both types of product, cyclooxygenase-dependent more than lipoxygenase-dependent, especially in differentiated cells. The biological significance of these changes in lipid metabolism that accompany phorbol myristate acetate-induced differentiation are yet to be established.

Wy-48,252 (1,1,1-trifluoro-N-[3-(2-quinolinylmethoxy)phenyl]membrane sulfonamide), an orally active leukotriene antagonist: effects on arachidonic acid metabolism in various inflammatory cells.

The LTD4 antagonist, Wy-48,252 (1,1,1-trifluoro-N-[3-(2-quinolinylmethoxy)phenyl]methanesulfonamide), was assessed for its ability to modulate arachidonic acid metabolism in several inflammatory cells. In A23187-stimulated rat neutrophils, Wy-48,252 effectively inhibited the conversion of exogenous [14C]arachidonic acid to radiolabeled 5-hydroxyeicosatetraenoic acid (5-HETE) and thromboxane B2 (TxB2) (IC50 = 2 and 9.1 microM, respectively). Synthesis of immunoreactive leukotriene B4 (LTB4) (IC50 = 4.6 microM) and TxB2 (IC50 = 3.3 microM) from endogenous substrate by these cells in the absence of [14C]arachidonic acid was similarly reduced. Wy-48,252 also reduced leukotriene C4 (LTC4) and PGE2 synthesis by zymosan-activated mouse peritoneal macrophages (IC50 = 4.4 and 4.3 microM, respectively). 5-Lipoxygenase (5-LO) catalyzed reactions in human neutrophils, lung mast cells and basophils activated by various stimuli were dose dependently inhibited by Wy-48,252 while PGD2 synthesis by lung mast cells was inhibited at 100 microM. By contrast, 12-LO, 15-LO, phosphodiesterase activity and histamine release from mast cells and basophils were unaffected by Wy-48,252. These data suggested that the LTD4 antagonist, Wy-48,252, also inhibited the synthesis of eicosanoids, a feature that may contribute to its pharmacological actions in vivo.

Thromboxane receptor-mediated bronchial and hemodynamic responses in ovine endotoxemia.

The role of thromboxane A2 in sheep endotoxemia, an animal model of the adult respiratory distress syndrome, was investigated by a combined biochemical and pharmacological approach. Endogenous thromboxane biosynthesis was assessed by gas chromatographic-mass spectrometric analysis of urinary (thromboxane B2, 2,3-dinor-thromboxane B2) and plasma (11-dehydrothromboxane B2) metabolites that demonstrated a significant stimulation by endotoxin. The functional relevance of thromboxane A2 was probed with a specific thromboxane-prostaglandin endoperoxide receptor antagonist, SQ 29548. The antagonist significantly blunted the increase in pulmonary arterial pressure, pulmonary vascular resistance, lung lymph flow, and lymph protein clearance induced by endotoxin. Whereas the reduction in lung compliance caused by endotoxin was abolished, the augmented airway resistance was unaffected. From the simultaneous increase in thromboxane biosynthesis and effects of receptor blockade, it was concluded that thromboxane A2 mediates the early pathophysiological changes of sheep endotoxemia. Thromboxane receptor antagonism may offer a potential therapeutic approach to patients at risk of the adult respiratory distress syndrome.