Activation and inactivation of cyclo-oxygenase in rat alveolar macrophages by aqueous cigarette tar extracts.

Cyclo-oxygenase (COX) activity and its level of expression, the release of arachidonic acid (AA), and the accumulation of prostaglandins (PGs) were determined in isolated rat pulmonary alveolar macrophages (PAM) exposed to aqueous cigarette tar (ACT) extracts. COX activity increased 3-fold above the initial activity within 2 h of incubation with ACT extracts and gradually decreased below the initial activity after 8 h of incubation. The increased COX activity after 2 h of incubation did not lead to increased accumulation of PGE2. Accumulated levels of PGE2 increased dramatically after 12 h of incubation despite decreased COX activity in cells incubated with ACT extracts. This increased accumulation of PGE2 was greater in cells derived from vitamin E deficient rats compared with control rats. Release of AA from cells was dramatically increased in cells incubated with ACT extracts in parallel to PG accumulation. Thus increased accumulation of PGE2 despite decreased COX activity after 12 h of incubation is likely the result of increased substrate availability. These results suggest that, contrary to earlier reports, cigarette smoke stimulates the formation of PGs in alveolar macrophages. Increased PG production may lead to suppressed immune response and enhanced risk of tumorigenesis in smokers' lungs.

Does vegetable oil attenuate the beneficial effects of fish oil in reducing risk factors for cardiovascular disease?

Contradictory reports on the protective effect of fish consumption on cardiovascular disease (CVD) risk could be due to variations in the intake of n-3 and n-6 polyunsaturated fatty acids (PUFAs). Metabolic competition between n-3 and n-6 PUFAs suggests that n-6 PUFAs in vegetable oils could attenuate the efficacy of n-3 PUFAs in fish oil to favorably alter endpoints relevant to CVD risk. We determined the effects of varying dietary amounts of fish oil on lipid and thrombotic endpoints relevant to risk factors for CVD and whether these effects were attenuated by vegetable oils. Two randomized, double-blind, placebo-controlled, parallel studies were conducted in human subjects fed varying amounts of n-3 and n-6 PUFAs; n-3 PUFA intake was varied by using fish or placebo oil capsules, and n-6 PUFA intake was modified by incorporating varying amounts of safflower oil into the diet. Endpoints included changes in membrane fatty acid composition, blood lipids, and thrombotic profile. The results indicated that absolute amounts of fish oil, and not the relative amounts of fish and vegetable oil (ratios of n-3 to n-6 PUFAs), determined the magnitude of the reduction of arachidonic acid and increase in eicosapentaenoic acid in phospholipids of plasma and platelets. The suppression of plasma triacylglycerols by fish oil was not affected by varying amounts of dietary n-6 PUFAs. Fibrinogen concentrations decreased with 15 g but not with 9 g fish oil/d fed at the same ratio of n-3 to n-6 PUFAs. The efficacy of fish oil in favorably modifying certain risk factors for CVD was not attenuated by vegetable oil.

Radicicol, a protein tyrosine kinase inhibitor, suppresses the expression of mitogen-inducible cyclooxygenase in macrophages stimulated with lipopolysaccharide and in experimental glomerulonephritis.

Two isoforms of cyclooxygenase (COX) have been identified in eukaryotic cells: a constitutively expressed COX-1 and mitogen-inducible COX-2, which is selectively expressed in response to various inflammatory stimuli. Thus, COX-2 instead of COX-1 is implicated to produce prostanoids mediating inflammatory responses. Major efforts have been focused on identifying nonsteroidal anti-inflammatory drugs (NSAIDS) which can selectively inhibit the enzyme activity of COX-2. Such NSAIDS would be more desirable anti-inflammatory agents in comparison to NSAIDS which inhibit both COX-1 and COX-2. Other than glucocorticoids, pharmacological agents which can selectively suppress the expression of COX-2 without affecting that of COX-1 have not been identified. We report here that radicicol, a fungal antibiotic, is a potent protein tyrosine kinase inhibitor, and that it inhibits the expression of COX-2 without affecting COX-1 expression in lipopolysaccharide (LPS)-stimulated macrophages with the IC50 value of 27 nM. Radicicol inhibited tyrosine phosphorylation of p53/56lyn, a Src family tyrosine kinase and one of the major tyrosine-phosphorylated proteins in LPS-stimulated macrophages. Radicicol also inhibited COX-2 expression in vivo in glomeruli of rats with experimental glomerulonephritis induced by the anti-glomerular basement membrane antibodies, in which COX-2 expression is known to be enhanced. The enzyme activity of COX-1 or COX-2 was not affected by radicicol in macrophages. Radiciciol also suppressed the COX-2 expression induced by IL-1 beta in rat smooth muscle cells. Other protein tyrosine kinase inhibitors suppressed the LPS-induced COX-2 expression in macrophages but at much higher concentrations than needed for radicicol. Radicicol did not inhibit the COX-2 expression induced by phorbol 12-myristate 13-acetate in macrophages. These results suggest that the activation of tyrosine-specific protein kinases is the proximal obligatory step in the LPS-induced signal transduction pathway leading to the induction of COX-2 expression in macrophages. The magnitude of the inhibition of COX-2 protein synthesis by radicicol was much greater than that of the steady state levels of COX-2 mRNA. These results suggest that radicicol inhibits COX-2 expression mainly at post-transcriptional steps.

Cloning two isoforms of rat cyclooxygenase: differential regulation of their expression.

Two isoforms of cyclooxygenase (COX) have been identified in eukaryotic cells: COX-1 encoded by a 2.8-kb mRNA, and a mitogen-inducible COX-2 encoded by a 4-kb mRNA. We have cloned the COX-1 and COX-2 cDNAs from the cDNA library constructed from lipopolysaccharide (LPS)-stimulated rat peritoneal macrophages. The deduced amino acid sequence showed that COX-1 contained 602 amino acids, whereas COX-2 contained 604 amino acids. There is 95% conservation of the nucleotide sequence in the open reading frame of COX-1 between the rat and the mouse, while the homology of the 3' untranslated region is 68% except for a 150 bp segment adjacent to the stop codon which is nonhomologous with the mouse. Transfection of both COX cDNAs into Cos-7 cells resulted in increased COX activity. In rat vascular smooth muscle cells, interleukin-1 beta selectively increased the expression of COX-2, but not that of COX-1, as assessed by enzyme activity, immunoprecipitation of COX proteins, and mRNA analysis. Only the brain among tissues tested exhibits basal expression of COX-2 as the major form of the enzyme. However, COX-2 mRNA was expressed in vivo in the lung and kidney, but not in the heart, after systemic administration of LPS, suggesting that COX-2 but not COX-1 plays a major role in producing COX-derived products of arachidonic acid during endotoxic shock. Thus, the two COX isoforms were differentially expressed, and COX-2 was selectively induced in response to inflammatory stimuli in rats.

Selective expression of mitogen-inducible cyclooxygenase in macrophages stimulated with lipopolysaccharide.

Two forms of cyclooxygenase are known to be present in eukaryotic organisms: a cyclooxygenase (COX-1) first purified from ram seminal vesicles encoded by a 2.8-kilobase mRNA, and a newly discovered mitogen-inducible cyclooxygenase (COX-2) encoded by a 4-kilobase mRNA. Expression of these two forms of the enzyme in rat alveolar macrophages stimulated with lipopolysaccharide was investigated by 1) determining the activity of newly synthesized enzyme after inactivating the endogenous enzyme with aspirin; 2) comparing levels of newly synthesized enzyme proteins in cells treated with or without lipopolysaccharide; and 3) assessing the expression of the mRNAs encoding COX-1 and COX-2. Levels of enzyme proteins were assessed by Western blot analysis and immunoprecipitation of 35S-labeled enzyme using two different antibodies, one specific for COX-2 and the other recognizing both forms of the enzyme but preferentially recognizing COX-1. We report here that the enhanced cyclooxygenase activity induced by the bacterial lipopolysaccharide in rat alveolar macrophages is caused by selective expression of the COX-2. Expression of COX-2 in macrophages stimulated by lipopolysaccharide was completely inhibited by dexamethasone, whereas COX-1 was unaffected. In resting unstimulated macrophages, only COX-1 but not COX-2 was detected. Levels of mRNA for the COX-2 in macrophages were increased, but those of the COX-1 were not affected by lipopolysaccharide as assessed by reverse transcription coupled with polymerase chain reaction. These results indicate that increased synthesis of prostaglandins and thromboxanes in lipopolysaccharide-stimulated macrophages results from selective expression of COX-2.

Incorporation of different types of n-3 fatty acids into tissue lipids of poultry.

The objective of the present study was to compare the incorporation of different types of n-3 polyunsaturated fatty acids into thigh muscle lipids of poultry. Nine groups of broiler chicks were fed diets supplemented with three levels (1.0, 2.5, and 5.0%) of either corn, linseed, or menhaden oil. Birds supplemented with linseed oil, rich in linolenic acid (C18:3n-3), had significantly higher levels of n-3 fatty acids and higher n-3:n-6 ratios than those supplemented with the same level of menhaden oil, primarily due to an accumulation of C18:3n-3. Levels of eicosapentaenoic acid (C20:5n-3) were increased (P less than .05), compared with the controls fed the same level of corn oil, in the groups fed the two higher levels of linseed oil, and in all the groups fed menhaden oil. Linolenic acid is less susceptible to auto-oxidation, and is less likely to impart an off-flavor to the muscle. Thus, if it is desirable to increase the n-3:n-6 ratio in poultry, seed meals or oils with a high content of C18:3n-3 could be used in poultry feeds.

Dietary (n-3) fatty acids alter fatty acid composition and prostaglandin synthesis in rat testis.

The objective of this study was to determine the efficacy of linolenic acid [18:3(n-3)], compared with the long-chain (n-3) fatty acids in fish oil, in suppressing arachidonic acid [20:4(n-6)] metabolism in rat testis. Six groups of rats were fed three levels of 18:3(n-3) or fish oil, and the fatty acid composition of testis parenchyma lipids and prostaglandin (PG) I2 synthesis by tunica were determined after 12 wk. Levels of docosapentaenoic acid [22:5(n-6)], the major 22-carbon fatty acid in rat testis lipids, were significantly depressed compared with the control by both linolenic acid and fish oil; however, testis weights were not affected significantly. Arachidonic acid levels also were depressed significantly in testis lipids by dietary (n-3) fatty acids, but the decreases were not as pronounced as those observed in other tissues. The synthesis of PGI2 was significantly reduced compared with the control by (n-3) fatty acid feeding, but there were no differences among the experimental groups. Both 18:3(n-3) and the longer-chain (n-3) fatty acids from fish oil reduce levels of 20:4(n-6) and 22:5(n-6) in testis lipids and the capacity of the tunica to synthesize PGI2, but these fatty acids seem to cause no defect in testicular development as indicated by weight.

Lack of dose response by dietary n-3 fatty acids at a constant ratio of n-3 to n-6 fatty acids in suppressing eicosanoid biosynthesis from arachidonic acid.

This study evaluated whether it is the ratio of n-3 to n-6 fatty acids or the absolute amount of n-3 fatty acids in diets that determines the degree of inhibition of eicosanoid biosynthesis from arachidonic acid (AA). Rats were fed diets containing different doses of linolenic acid or menhaden oil for 3 mo. Constant ratios of n-3 to n-6 fatty acids were maintained by concomitant increases in safflower oil as the n-6 fatty acid source. Results showed that AA concentrations in liver, platelet, and lung phospholipids and concentrations of eicosanoids synthesized in tissues were significantly (P less than 0.05) suppressed both by linolenic acid and menhaden oil; however, there was a lack of a dose response within groups fed different amounts of the same dietary fat. These results indicate that the ratio of n-3 to n-6 fatty acids in the diets, rather than the absolute amount of n-3 fatty acids, is the determining factor in inhibiting eicosanoid biosynthesis from AA.

Dietary linolenic acid and longer-chain n-3 fatty acids: comparison of effects on arachidonic acid metabolism in rats.

Rats were fed graded amounts of purified 18:3n-3 or fish oil concentrate in the presence of a constant amount of 18:2n-6 to evaluate the ability of 18:3n-3 compared with longer-chain n-3 fatty acids to inhibit 20:4n-6 metabolism in platelets and lungs. Dietary 18:3n-3 at a ratio of 0.28 (n-3 to n-6 fatty acids) suppressed levels of 20:4n-6 in lung and plasma phospholipids and the capacity of the tissues to synthesize cyclooxygenase-derived products in a dose-dependent fashion. At similar ratios of n-3 to n-6 dietary fatty acids, longer-chain n-3 fatty acids, which are abundant in fish oil, appear to be more effective than 18:3n-3 in suppressing 20:4n-6 levels and the capacity of the tissues to synthesize cyclooxygenase-derived products. Much greater amounts of 12-hydroxyeicosapentaenoic acid (12-HEPE) and 5-HEPE than of 12-hydroxyeicosatetraenoic acid (12-HETE) and 5-HETE appeared to be formed in tissues of the group receiving the highest amount of fish oil. These results suggest that ingestion of fish oil leads to increased formation of lipoxygenase-derived products of longer-chain n-3 fatty acids.

Effects of chronic ethanol ingestion on arachidonic acid metabolism in rat tissues and in vitro effect of ethanol on cAMP in platelets.

Effects of chronic ethanol ingestion (3 weeks) on the capacity of peritoneal macrophages, lung and heart tissues, to metabolize endogenous arachidonic acid (AA) in rats, and the in vitro effect of ethanol on 3'-5' cyclic adenosine monophosphate (cAMP) levels in rat platelets were studied. Peritoneal resident macrophages were stimulated by calcium ionophore (A23187) and levels of 5-hydroxyeicosatetraenoic acid (5-HETE), leukotriene B4 (LTB4) and PGE2 were measured by radioimmunoassay. There were no differences in levels of the eicosanoids synthesized by macrophages between the ethanol treated and the control group. There were also no differences found in levels of the eicosanoids synthesized by heart or lung homogenate between the two groups. These results suggest that chronic ethanol ingestion does not alter the capacity to synthesize the eicosanoids from the endogenous precursor in tissues studied here. Preincubation of ethanol with platelet rich plasma resulted in a dose dependent increase in cAMP levels. The well documented inhibitory effects of ethanol in vitro on aggregation and AA metabolism in platelets may be due to the enhanced cAMP levels.

Effect of oral vitamin B6 supplementation on in vitro platelet aggregation.

A randomized, double-blind study was conducted with 12 healthy adult males to determine the effects of oral pyridoxine supplementation on in vitro platelet aggregation. Following a 4-wk baseline period, half the subjects received 100 mg/day of pyridoxine X HCl while the remaining subjects received a placebo for 6 wk. In vitro platelet responses to ADP and collagen and the plasma pyridoxal 5'-phosphate (PLP) concentrations were measured at biweekly intervals. Plasma PLP concentrations increased significantly (p less than 0.001) for those receiving the vitamin B6 compared to baseline values or compared to those receiving the placebo. However, there was no significant effect of increased levels of plasma PLP on collagen-stimulated platelet aggregation and only a slight effect on ADP-stimulated aggregation. Acute administration of 100 mg pyridoxine X HCl failed to alter the in vitro response of platelets to either ADP or collagen. Reevaluation of conclusions based solely on in vitro studies suggesting the use of pyridoxine as an effective in vivo antithrombotic agent may be warranted.

The effect of zinc deficiency on prostaglandin synthesis in rat testes.

The effect of zinc deficiency on prostaglandin synthesis in rat testes was determined by feeding three groups of rats egg white-based semipurified diets. One group (ZD) was fed a zinc-deficient diet and two control groups were pair-fed (PF) or fed ad libitum (AL) a zinc-sufficient diet. The concentration (nanograms/gram) of the prostacyclin metabolite, 6-keto-prostaglandin-F1 alpha (6-keto-PGF1 alpha), in the tunica homogenate was significantly lower in ZD than in PF and AL groups. However, there was no difference when 6-keto-PGF1 alpha concentration was expressed as nanograms/milligrams of tunica protein. Tunica PGE2 concentrations (nanograms/gram) were not significantly altered by zinc deficiency. Concentrations of prostaglandins (PGs) in testis parenchyma were slightly higher in ZD probably as a result of increased levels of the precursor, arachidonic acid (AA). There was a highly significant correlation between PGE2 and AA in parenchyma phospholipids. PG synthesis was much greater in the tunica than in the parenchyma and prostacyclin appeared to be the major PG synthesized in both the tunica and parenchyma. It was concluded that PG synthesis is altered in the testes of zinc-deficient rats probably due to changes in concentrations of protein in the tunica and AA levels in parenchyma lipids.

Fatty acid composition of the testes of zinc-deficient rats: the effect of docosapentaenoic acid supplementation.

To determine if low levels of docosapentaenoic acid (22:5 omega 6), observed in the testis lipids of zinc-deficient rats, are related to some of the symptoms of zinc deficiency in rat testes, four groups of rats were fed egg white- and corn oil (CO)-based semipurified diets. One group was fed a zinc-deficient diet (ZDCO) and a control group was pair-fed a zinc-supplemented diet (PFCO). Two additional groups were treated in an identical fashion, but their diets were supplemented with 1% testis lipid (TL) containing 10-12% 22:5 omega 6 (ZDTL and PFTL). Testes weights and sperm counts were slightly higher in ZDTL compared to ZDCO, but these trends were not statistically significant. Testis zinc concentrations were significantly higher in both TL groups compared to the corresponding CO group. Dietary 22:5 omega 6 appeared to be accumulated in liver lipids; however, there was no difference between TL and CO groups in 22:5 omega 6 level in testes phospholipids or testes total lipids. These results indicate that 22:5 omega 6 concentration and spermatogenesis in the testes of zinc-deficient rats do not appear to be affected by the dietary supplementation of 22:5 omega 6 at the level used in this study.

Zinc deficiency affects neither platelet arachidonic acid metabolism nor platelet aggregation in rats.

The effects of zinc deficiency on arachidonic acid (AA) metabolism and platelet aggregation were studied in rats. Concentrations of AA metabolites synthesized by collagen-stimulated platelets were greater in rats fed a zinc-deficient diet (ZD group) than pair-fed control rats (PF group). This difference appeared to be due to increased platelet number in the ZD group. Preincubation of platelet-rich plasma of ZD group with various concentrations of zinc sulfate, prior to the aggregation induced by collagen suspension, Affected neither concentrations of AA metabolites nor the degree of platelet aggregation. Contrary to a report by other investigators, ADP-induced platelet aggregation was not impaired in rats fed the zinc-deficient diet for 1 week or 5 days as compared to the pair-fed controls. Secondary phase aggregation was not observed during ADP-induced platelet aggregation regardless of concentrations of ADP, types and concentrations of anticoagulants used, routes of blood collection or types of dietary protein in this study. These results indicated that zinc deficiency does not seem to affect arachidonic acid metabolism or platelet aggregation in rats in relatively short-term studies.

Effects of dietary 9-trans, 12-trans linoleate on arachidonic acid metabolism in rat platelets.

In order to determine the minimal amount of dietary 9-trans, 12-trans-linoleate which can decrease endoperoxide metabolites synthesized and their precursor in rat platelets, graded amounts (0, 0.1, 0.5, 1.0, 2.5%) of the trans-linoleate were fed to rats with a constant amount of all-cis-linoleate (2.5%) for 12 weeks. Arachidonic acid levels in platelet phospholipids of groups receiving the trans-linoleate at 2.5 and 1.0% were significantly (p less than 0.01) lower than that of the control receiving no trans-linoleate. Concentrations of TXB2 and PGF2 alpha in sera of the group receiving 2.5% trans-linoleate were significantly (p less than 0.05) lower than those of the control; however, there was no difference between the group receiving 1.0% trans-linoleate and the control. To determine whether the difference in serum concentration of endoperoxide metabolites could be manifested if rats were fed for longer period of time, 2 groups of rats were again fed diets containing 0 and 1.0% trans-linoleate, respectively, for 16 weeks. Arachidonic acid in platelet phospholipids of the group receiving the trans-linoleate was again significantly (p less than 0.01) lower than that of the control group. Concentrations of TXB2 and PGF2 alpha, and 12-hydroxyeicosatetraenoic acid formed in platelets, were smaller in the group receiving trans-linoleate than the control group; however, the difference was not statistically significant. These results indicated that all-trans-linoleate can reduce arachidonic acid metabolites formed in rat platelets when its dietary level is equal to or exceeds the level of all-cis-linoleate.