J. Martyn Bailey

Lipid Metabolism in Cultured Cells

Abstract L-strain mouse fibroblasts grown on a lipid-free chemically denned medium (NCTC 109) derived cell lipids by synthesis from simpler components in the medium. By supplementing the medium with 14C-labelled precursors it was found that, under the conditions used, over half of the cell lipids were synthesized from glucose and about 20% from acetate. When serum was added to the growth medium, incorporation of [14C] acetate into cell lipids was almost completely (up to 98%) inhibited, and incorporation of glucose was also considerably (70%) reduced. The lipid composition of the cells, however, was essentially unchanged. The inhibiting effect of serum on acetate incorporation could be duplicated by addition of certain purified lipids, including cholesterol, to the synthetic growth medium. By fractionation of radioactive cell lipids on silicic acid columns it was shown that added serum depressed incorporation into all lipid classes to approximately the same extent. [2-14C]mevalonic acid added to synthetic growth medium was also utilized for synthesis of cellular lipids, most of the radioactivity being found in cholesterol. Addition of serum or purified lipids did not inhibit mevalonic acid incorporation. Cells adapted to growth on serum retained the ability to utilize glucose and acetate for lipid synthesis when transferred back to synthetic medium. The observations indicate that in cultured cells, a balance is maintained between synthesis de novo and utilization of lipid from the medium. This is brought about by a lipidinduced inhibition of glucose and acetate incorporation at some point early in the metabolism of these compounds. Some possible mechanisms for the process are suggested.

Altered lipoxygenase metabolism and decreased glutathione peroxidase activity in platelets from selenium-deficient rats

Abstract Washed platelets from selenium-deficient and control rats were incubated with [1- 14 C]-arachidonic acid and the lipoxygenase and cyclooxygenase products were identified by gas chromatography/mass spectrometry. Platelets from selenium-deficient rats showed a three to four-fold increased synthesis of the lipoxygenase-derived isomeric trihydroxy fatty acids, 8,9,12-trihydroxy-5,10,14-eicosatrienoic acid and 8,11,12-trihydroxy-5,9,14-eicosatrienoic acid. A major reduction in glutathione peroxidase activity was also observed in platelets from deficient rats. These results support the interpretation that these trihydroxy fatty acids arise from breakdown of the primary platelet lipoxygenase product L -12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE) under conditions in which its reduction to the L -12-hydroxy product (12-HETE) by a selenium-dependent glutathione peroxidase is limited. Further-more, these results indicate a specific function for selenium in platelet metabolism of essential fatty acids.

Corticosteroids suppress cyclooxygenase messenger RNA levels and prostanoid synthesis in cultured vascular cells

Prostacyclin synthesis by cultured vascular smooth muscle cells was inactivated by aspirin. Recovery required serum factors replaceable by EGF plus TGF-beta and was blocked by cycloheximide but not by actinomycin D. Recovery of cyclooxygenase activity was prevented by preincubation with dexamethasone (0.1 to 2 microM), which also suppressed basal enzyme activity by up to 70%. A full length 2.8 Kb cDNA hybridization probe for human cyclooxygenase identified a cyclooxygenase messenger RNA of approximately 2.8 Kb in these cells. Cyclooxygenase mRNA levels were enhanced by EGF/TGF-beta, but suppressed completely by corticosteroids. It is concluded that inhibition of prostanoid synthesis by corticosteroids is mediated by suppressing cyclooxygenase messenger RNA. These observations provide a new molecular mechanism for the anti-inflammatory activity of the corticosteroids.

Inhibition of fatty acid oxygenases by onion and garlic oils: Evidence for the mechanism by which these oils inhibit platelet aggregation

Abstract Inhibition of in vitro platelet aggregation by onion and garlic oils was accompanied by decreased formation of thromboxane B 2 (TXB 2 ) and 12-hydroxyheptadecatrienoic acid (HHT) from [1- 14 C]arachidonic acid (AA). At intermediate concentrations (10–30 μg ml ), these oils also induced a redistribution of the products of the lipoxygenase pathway; at higher concentrations (30–60 μg ml ), they completely suppressed the formation of all oxygenase products. Measurements of oxygen consumption in antimycin-treated platelets indicated a direct correlation between inhibition of platelet fatty acid oxygenases and the anti-aggregating activity of these oils. Onion and garlic oils also inhibited fatty acid oxygenases from sheep vesicular gland preparations as shown both by decreased oxygen consumption and decreased formation of prostaglandin E 2 (PGE 2 ) and PGD 2 from [1- 14 C]AA. Onion oil was approximately ten times more effective in inhibiting the platelet oxygenases than the oxygenases from the vesicular gland. In addition, these oils suppressed the soybean lipoxygenase-catalyzed oxygenation of AA. In all three enzyme systems, onion oil exhibited greater inhibitory activity than garlic oil. Many nonsteroidal anti-inflammatory and anti-thrombotic drugs act by inhibiting the cyclooxygenase component of the prostaglandin and thromboxane synthetases. Our findings thus suggest that onion and garlic contain compounds that may have serendipitous pharmacological effects.

Isolation of a new lipoxygenase metabolite of arachidonic acid, 8,11,12-trihydroxy-5,9,14-eicosatrienoic acid from human platelets

Washed human platelets incubated with 1-14C-arachidonic acid (1mM) produced a new metabolite which migrated on thin layer chromatography close to thromboxane B2, but which was identified by mass spectrometry as a trihydroxy fatty acid. The mass spectrum was consistent with the assigned structure, 8,11,12-trihydroxy-5,9,14-eicosatrienoic acid (THETE). Platelet THETE synthesis from arachidonate was not inhibited by preincubation with asprin or indomethacin but was blocked by 5,8,11,14-eicosatetraynoic acid. Therefore, THETE appears to arise via the platelet lipoxygenase pathway rather than via the prostaglandin cyclooxygenase. Two proposed structures, including a novel dihydro-hydroxy-pyran cyclic intermediate, which could give rise to THETE are presented.

Regulation of leukocyte and platelet lipoxygenases by hydroxyeicosanoids

During allergic and inflammatory reactions, arachidonic acid is oxidized by lipoxygenases to a variety of biologically active products, including leukotrienes. The mechanisms for regulation of the different lipoxygenase activities are not well defined. We report here that [14C]arachidonic acid metabolism by the 5- and 15-lipoxygenase activities in rabbit leukocytes and the 12-lipoxygenase in rabbit platelets is inhibited by various hydroxyeicosatetraenoic acids (HETEs). 15-HETE was the most effective inhibitor of the 5- and 12-lipoxygenases, whereas similar inhibitory potencies were observed for 5-HETE and 12-HETE acting on the 15-lipoxygenase. These three enzyme pathways were all least sensitive to their own products HETEs. To determine which structural characteristics of 15-HETE are essential for inhibition of the 5-lipoxygenase, various derivatives were prepared and purified by high pressure liquid chromatography, and their structures were confirmed by gas chromatography-mass spectrometry. The inhibitory potencies of 15-HETE analogs with different degrees of unsaturation were in the order of three double bonds greater than 4 greater than 2 greater than 0. 15-Hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) was four times more potent than 15-HETE. The 15-acetoxy, 15-keto and methyl ester derivatives were of comparable activity to 15-HETE, and the 15-acetoxy methyl ester derivative was less potent. Based upon the observed patterns of inhibition, we postulate that complex interregulatory relationships exist between the various lipoxygenases, and that cells containing these lipoxygenases may interact with each other via their lipoxygenase metabolites.

Anti-inflammatory drugs in experimental atherosclerosis

Groups of New Zealand white male rabbits were fed atherogenic diets containing 1% cholesterol. The diets of experimental groups were supplemented additionally with either aspirin, phenylbutazone, mefenamic acid, flufenamic acid, oxyphenylbutazone or aminopyrine. Blood cholesterol and phospholipids were measured at 3--4 week intervals. After 12 weeks the animals were sacrificed and the severity of atherosclerosis in the thoracic aorta was measured. In separate experiments, rabbit platelets were incubated with each of the drugs individually and conversion of [14C]arachidonic acid to thromboxanes and related compounds was assayed. Inhibition of collagen and arachidonic acid-induced platelet aggregation by each drug was also measured. All drugs inhibited thromboxane synthesis and platelet aggregation in varying degrees with flufenamate and aspirin being most and aminopyrine least effective. The pattern of metabolite formation from [14C]arachidonate was consistent with a block in the cyclooxygenase reaction. Phenylbutazone, flufenamic acid and oxyphenylbutazone produced significant reductions in atherosclerotic plaque formation without major changes in blood cholesterol levels or blood cholesterol--phospholipid ratios. Aspirin and aminopyrine were ineffective. The results indicate that the effectiveness of anti-inflammatory drugs as inhibitors of thromboxane synthesis and platelet aggregation in vitro does not afford a sufficient predictive index of their anti-atherogenicity in vivo. The significance of these findings is discussed in terms of the possible involvement of cyclooxygenase derivatives in atherogenesis.

Formation of 15-hydroxyeicosatetraenoic acid (15-HETE) as the predominant eicosanoid in aortas from Watanabe Heritable Hyperlipidemic and cholesterol-fed rabbits

Atherosclerotic plaque formation is accompanied by hyperproliferative events which have many features of an inflammatory response. A high-performance liquid chromatography procedure was developed to analyze the inflammatory prostaglandins, leukotrienes and hydroxyeicosatetraenoic acids (HETEs) produced by aortic segments. Normal rabbit aortas incubated with tritiated arachidonic acid synthesized 12-HETE as the principal lipoxygenase metabolite, and prostacyclin as the major cyclooxygenase product. In contrast, atherosclerotic aortas from both cholesterol-fed and Watanabe Heritable Hyperlipidemic rabbits showed major increases in synthesis of lipoxygenase-derived 15-HETE, which became the predominant eicosanoid in the aortas of both types of rabbit. No leukotrienes or other 5-lipoxygenase products were detected to the detection limit of 0.5 pmol/cm aorta. 15-HETE, which is chemotactic for smooth muscle cells, mitogenic for endothelial cells, and an inhibitor of prostacyclin synthesis may thus play a role in atherogenesis.

Differential separation of thromboxanes from prostaglandins by one and two-dimensional thin layer chromatography.

[14C]-labelled thromboxane B2 and hydroxy fatty acids were isolated using thin layer and gas chromatographic procedures from human platelets incubated with [1-14C]-arachidonic acid. A number of TLC solvent systems were evaluated for differential separation of thromboxanes and hydroxy fatty acids from prostaglandins E2, A2, D2 and F2alpha. Chromatographic properities in nine different solvent systems are tabulated. Two dimensional TLC procedures suitable for complete resolution of mixtures of these compounds on a single plate were developed. The systems were used to demonstrate conversion of [1-14C]-arachidonic acid to thromboxane B2 and prostaglandin E2 by human lung fibroblasts in tissue culture.

Inhibition of interleukin-2-dependent cytotoxic T-lymphocyte growth by gangliosides.

Previous work has shown that exogenous gangliosides suppress lectin-induced T-lymphocyte mitogenesis in mixed populations of immune cells. As one potential cellular site of this inhibition, the influence of gangliosides on interleukin-2-dependent T-cell proliferation was tested, using cultures of cytotoxic T cells (strain CT-6). Incubation of CT-6 cells with mixed bovine brain gangliosides resulted in a dose-dependent inhibition of cell proliferation. Cell viability was unaffected by gangliosides, and the inhibition was totally reversed when the gangliosides were removed. Individual purified gangliosides were tested and GM2 was most inhibitory (I50 = 15 microM). GD1a and GT1b were somewhat less potent, whereas GM1 and GM3 were only weakly inhibitory. Various nonpolar lipids, sulfatides, and sialic acid did not inhibit CT-6 cell growth. The results suggest that a primary mechanism whereby gangliosides inhibit lectin-induced lymphocyte mitogenesis is by inhibition of the interleukin-2-stimulated proliferation of T cells in these cultures.