Effect of phosphatidylethanolamine and its constituent base on the metabolism of linoleic acid in rat liver

Dietary phosphatidylethanolamine (PE) contributes the circulatory and hepatic free-ethanolamine in rats (Ikeda et al. (1987) Biochim. Biophys. Acta 921, 245). A role for circulatory ethanolamine has not been defined; however, our recent studies have shown that exogenous ethanolamine influences cholesterol and linoleic acid metabolism in rats (Imaizumi et al. (1983) J. Nutr. 113, 2403). In order to understand the role of dietary PE the effects of PE and its base on the hepatic metabolism of linoleic acid were investigated in vivo and in primary cultured hepatocytes in rats. Dietary PE increased the plasmic level of ethanolamine from 37 to 52 microM and decreased the ratio of arachidonate to linoleate in hepatic phospholipids. Activity of hepatic delta 6-desaturase decreased in rats given PE and the desaturation of [14C]linoleate in the cultured hepatocytes decreased by the addition of ethanolamine. Secretion [14C]linoleate labeled very-low-density lipoprotein from the cultured hepatocytes decreased by the addition of ethanolamine. Dietary PE caused an increased formation of CO2 from [14C]acetate by liver slices, and ethanolamine added to the hepatocytes caused an increased oxidation of [14C]linoleate and a suppression of fatty acid synthesis from [3H]serine. These results suggest that ethanolamine derived from the dietary PE plays a regulatory role in the linoleate metabolism in the liver.     

Effects of amino-acid composition of the medium and addition of fatty-acid derivatives on the induction of lipogenic enzymes in cultured hepatocytes

Effects of essential and non-essential amino acids on induction of lipogenic enzymes were investigated in cultured rat hepatocytes. Glucose-6-phosphate dehydrogenase was markedly induced by the addition of essential amino acids alone to the cultured medium, but was not induced by non-essential amino acids. Fatty-acid synthetase was also markedly induced by a combination of both amino-acid types (more than by either type of amino acid alone). However, acetyl-CoA carboxylase and malic enzyme were slightly induced by the addition of essential and/or non-essential amino acids. When various kinds of fatty acids were individually added to the medium, the lipid-dependent decreases in lipogenic enzyme inductions were in the following order: 18:2 greater than 20:4 greater than 18:1 greater than 16:0. When either linoleic acid, linoleoyl-CoA or trilinolein was added to the medium, linoleic acid was more effective as an inhibitor of the induction, without impairing the viability of cells.     

贵州地方芝麻种质资源品质性状的分析与评价

为探究贵州芝麻种质资源的品质特征,并对地方芝麻资源进行初步鉴定与评价,本研究对73份贵州芝麻种质资源的8个品质性状进行测试分析。结果表明:(1)贵州芝麻种质资源含油量介于41.45%~52.12%之间,平均含量为49.69%。在脂肪酸组成中,油酸、亚油酸等不饱和脂肪酸的平均含量分别为35.65%和50.66%;而棕榈酸、硬脂酸等饱和脂肪酸的平均含量仅为8.40%和4.79%。此外,贵州芝麻资源中芝麻素、芝麻林素和木质素的平均含量分别为5.03 mg/g、2.63 mg/g和4.79 mg/g。8个品质性状的变异系数介于3.69%~32.62%范围内,其中芝麻素含量变异系数最大,含油量变异系数最小。而芝麻素含量、芝麻林素含量及硬脂酸含量的变异系数均大于10%,表明这3个性状在芝麻样本间存在较大差异。(2)相关性分析结果显示:含油量与油酸、芝麻素含量呈极显著正相关,与亚油酸含量呈极显著负相关;油酸含量与芝麻素含量呈极显著正相关,与亚油酸含量呈极显负相关;亚油酸含量与芝麻素含量呈极显著负相关。表明品质性状间相关性大、关联程度较高,性状间相互影响较大。(3)主成分分析将8个品质性状综合为3个主成分,分别为油酸因子、含油量因子和芝麻素因子,3个主成分因子包含了贵州芝麻种质资源品质性状的绝大部分信息,累计贡献率达96%以上。(4)在欧氏距离D=9.75处将73份贵州芝麻资源划分为6个类群:第Ⅰ类群包含2份资源、第Ⅱ类群有7份、第Ⅲ类群有12份、第Ⅳ类群有5份、第Ⅴ类群有16份、第Ⅵ类群有31份。其中第Ⅵ类群油酸含量最高,且含油量、芝麻素含量较高。本研究探明了贵州芝麻品质的特征特性,可为芝麻种质资源的利用和创新提供依据,为芝麻品种选育和遗传改良提供参考。     

Physiological effects of γ-linolenic acid and sesamin on hepatic fatty acid synthesis and oxidation

Interrelated effects of γ-linolenic acid (GLA) and sesamin, a sesame lignan, on hepatic fatty acid synthesis and oxidation were examined. Rats were fed experimental diets supplemented with 0 or 2 g/kg sesamin (1:1 mixture of sesamin and episesamin) and containing 100 g/kg of palm oil (saturated fat), safflower oil rich in linoleic acid, or oil of evening primrose origin containing 43% GLA (GLA oil) for 18 days. In rats fed sesamin-free diets, GLA oil, compared with other oils, increased the activity and mRNA levels of various enzymes involved in fatty acid oxidation, except for some instances. Sesamin greatly increased these parameters, and the enhancing effects of sesamin on peroxisomal fatty acid oxidation rate and acyl-CoA oxidase, enoyl-CoA hydratase and acyl-CoA thioesterase activities were more exaggerated in rats fed GLA oil than in the animals fed other oils. The combination of sesamin and GLA oil also synergistically increased the mRNA levels of some peroxisomal fatty acid oxidation enzymes and of several enzymes involved in fatty acid metabolism located in other cell organelles. In the groups fed sesamin-free diets, GLA oil, compared with other oils, markedly reduced the activity and mRNA levels of various lipogenic enzymes. Sesamin reduced all these parameters, except for malic enzyme, in rats fed palm and safflower oils, but the effects were attenuated in the animals fed GLA oil. These changes by sesamin and fat type accompanied profound alterations in serum lipid levels. This may be ascribable to the changes in apolipoprotein-B-containing lipoproteins.     

Interaction of Sesamin and Eicosapentaenoic Acid against Δ5 Desaturation and n-6/ n-3 Ratio of Essential Fatty Acids in Rat

Sesamin is a specific inhibitor of Δ5 desaturation, the conversion from dihomo-γ-linolenic acid (20: 3, n-6) to arachidonic acid (AA, 20: 4, n-6). Previously, we reported that sesamin inhibited Δ5 desaturation of n-6 fatty acids in rat hepatocytes but not that of n-3 fatty acids, from 20: 4 (n-3) to eicosapentaenoic acid (EPA, 20: 5, n-3). In this study, we investigated the interaction of sesamin and EPA on Δ5 desaturation of both series and the n-6/n-3 fatty acids ratio by measuring actural fatty acid contents in vivo. Rats were fed three types of dietary oils; 1) linoleic acid (LA, 18: 2, n-6): linolenic acid (LLA, 18: 3, n-3) = 3: 1, n-6/n-3 ratio of 3: 1 (LA group), 2) LA: LLA =1: 3, n-6/n-3 ratio of 1: 3 (LLA group), 3) LA: LLA: EPA =1: 0.5: 3, n-6/n-3 ratio of 1: 3.5 (EPA group) with or without sesamin (0.5% w/w) for 4 weeks. In all groups, sesamin administration increased the content of dihomo-γ-linolenic acid (20: 3, n-6) in the liver and decreased the Δ5 desaturation index of n-6 fatty acid, the ratio of 20: 4/20: 3 (n-6). On the contrary, the Δ5 desaturation index of n-3 fatty acid, the ratio of 20: 5 + 22: 5 + 22: 6/20: 4 (n-3), was increased by the administration of sesamin. These results suggest that sesamin inhibits the A5 desaturation of n-6 fatty acid, but not that of n-3 fatty acid in rat livers. Sesamin administration decreased incorporation of EPA (n-3) and simultaneously increased the AA (n-6) content in the liver. The n-6/n-3 ratio in the liver was increased by administering sesamin under n-3 rich conditions, i.e., the LLA and EPA groups.     

Conjugated linoleic acid isomers modulate protein expression profile in rat hepatocytes

Conjugated linoleic acid (CLA) is a polyunsaturated fatty acid, which has been recently proven to be effective in reducing body fat mass, but brings as a side effect, the liver enlargement due to an increased lipid content. The in vivo lipogenic activity has been suggested to be due to the reduction in fat mass and to the consequent metabolism of blood glucose to fatty acid in the liver rather than in the adipose tissue. We investigated the ability of CLA to directly induce steatosis by modulating the expression pattern of hepatic proteins involved in lipid metabolism. To avoid interferences derived from CLA metabolism by other tissues, we used the in vitro model of freshly isolated rat hepatocytes incubated in the presence of different CLA isomers. The direct effect of CLA on lipid accumulation in hepatocytes was demonstrated by the altered expression pattern of several proteins involved in lipid metabolism, as assessed by two-dimensional gel electrophoresis and confirmed by Western blotting analysis. The CLA isomer c9,t11 was most effective in modulating the protein expression profile.     

Metabolism and short-term metabolic effects of conjugated linoleic acids in rat hepatocytes

Metabolic fate and short-term effects of a 1:1 mixture of cis-9,trans-11 and trans-10,cis-12-conjugated linoleic acids (CLA), compared to linoleic acid (LA), on lipid metabolism was investigated in rat liver. In isolated mitochondria CLA-CoA were poorer substrates than LA-CoA for carnitine palmitoyltransferase-I (CPT-I) activity. However, in digitonin-permeabilized hepatocytes, where interactions among different metabolic pathways can be simultaneously investigated, CLA induced a remarkable stimulatory effect on CPT-I activity. This stimulation can be ascribed to a reduced malonyl-CoA level in turn due to inhibition of acetyl-CoA carboxylase (ACC) activity. The ACC/malonyl-CoA/CPT-I system can therefore represent a coordinate control by which CLA may exert effects on the partitioning of fatty acids between esterification and oxidation. Moreover, the rate of oxidation to CO2 and ketone bodies was significantly higher from CLA; peroxisomes rather than mitochondria were responsible for this difference. Interestingly, peroxisomal acyl-CoA oxidase (AOX) activity strongly increased by CLA-CoA compared to LA-CoA. CLA, metabolized by hepatocytes at a higher rate than LA, were poorer substrates for cellular and VLDL-triacylglycerol (TAG) synthesis. Overall, our results suggest that increased fatty acid oxidation with consequent decreased fatty acid availability for TAG synthesis is a potential mechanism by which CLA reduce TAG level in rat liver.     

Comparative effect of sesamin and episesamin on the activity and gene expression of enzymes in fatty acid oxidation and synthesis in rat liver

Sesamin is one of the most abundant lignans in sesame seed. Episesamin, a geometrical isomer of sesamin, is not a naturally occurring compound and is formed during the refining process of non-roasted sesame seed oil. We compared the physiological activities of these compounds in affecting hepatic fatty acid metabolism in rat liver. Rats were fed either a control diet free of lignan or diets containing 0.2% of sesamin or episesamin for 15 days. These lignans increased the mitochondrial and peroxisomal palmitoyl-CoA oxidation rates. However, the magnitude of the increases was greater with episesamin than with sesamin. Sesamin caused 1.7- and 1.6-fold increases in mitochondrial and peroxisomal activity, respectively, while episesamin increased these values 2.3- and 5.1-fold. These lignans also increased the activity and gene expression of various fatty acid oxidation enzymes. Again, the increase was much more exaggerated with episesamin (1.5- to 14-fold) than with sesamin (1.3- to 2.8-fold). Diets containing sesamin and episesamin lowered the activity and gene expression of hepatic lipogenic enzymes to one-half of those obtained in the animals fed a lignan-free diet. However, no significant differences in these parameters were seen between rats fed sesamin and episesamin. Responses to sesamin and episesamin of hepatic lipogenesis are, therefore, considerably different from those observed in fatty acid oxidation. These results show that the physiological activity of the commercial sesamin preparation containing equivalent amounts of both sesamin and episesamin in increasing hepatic fatty acid oxidation observed previously was mainly ascribable to that of episesamin but not to sesamin.     

Cysteine uptake and taurine biosynthesis in freshly isolated and primary cultured rat hepatocytes

Analysis of the uptake and metabolism of [14C]cysteine in rat liver was undertaken using freshly isolated hepatocytes and hepatocytes maintained in primary culture. The uptake of [14C]cysteine by freshly isolated hepatocytes was by means of both saturable and non-saturable transport systems and the former system was thought to involve facilitated diffusion. The uptake of [14C]cysteine by hepatocytes maintained in primary culture for 24 h also consisted of non-saturated and saturated transport mechanisms. The magnitude of the saturable transport system in cultured hepatocytes was, however, much greater than that found in freshly isolated hepatocytes, and was considered to be operated by active transport. Both freshly isolated and primary cultured hepatocytes had cysteine sulphinic acid decarboxylase activity, but this enzyme activity in the latter cells was noticeably reduced in comparison with that found in freshly isolated hepatocytes. Hepatocytes maintained in primary culture produced not only radiolabelled taurine, but also radiolabelled cysteine sulphinic acid, hypotaurine and alanine when incubated with [14C]cysteine. The present results indicate that cultured hepatocytes actively transport cysteine as well as metabolizing cysteine to taurine via cysteine sulphinic acid and hypotaurine.     

Metabolic changes in lipids of rat plasma and hepatocytes induced by 17 alpha-ethynylestradiol treatment

Cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol-treated rats were used to investigate the change of lipid metabolism induced by administration of 17 alpha-ethynylestradiol. Treatment with 17 alpha-ethynylestradiol caused a decrease of rat plasma lipids (free cholesterol, cholesterol ester, triacylglycerol and phosphatidylcholine). No difference in the ability of urea nitrogen synthesis could be demonstrated between cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol-treated rats and propylene glycol-treated rats (control). Total cholesterol and cholesterol ester contents of cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol-treated rats were increased in comparison with those of the control. Triacylglycerol content of cultured hepatocytes was not affected by 17 alpha-ethynylestradiol treatment. There was no difference in the composition of lipid content between liver tissues and cultured hepatocytes. These results suggest that hepatocytes isolated from livers maintain the character of livers treated with 17 alpha-ethynylestradiol or livers treated with propylene glycol. Free cholesterol and cholesterol ester synthesis from [14C]acetic acid by cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol-treated rats were decreased to about 30% of the control. Triacylglycerol and polar lipid (phospholipid) synthesis from [14C]acetic acid were not affected by 17 alpha-ethynylestradiol treatment. Microsomal hydroxymethylglutaryl-CoA reductase activity of rat liver treated with 17 alpha-ethynylestradiol was decreased to about 50% of control. The secretions of free cholesterol, cholesterol ester, triacylglycerol, phosphatidylcholine, apolipoprotein BL and BS by cultured hepatocytes isolated from livers of 17 alpha-ethynylestradiol treated rats were not decreased when compared with the control. Because lipid and apolipoprotein secretions from cultured hepatocytes treated with 17 alpha-ethynylestradiol were not decreased and cholesterol contents of liver tissues and cultured hepatocytes treated with 17 alpha-ethynylestradiol were increased and hepatic microsomal hydroxymethylglutaryl-CoA reductase activity was decreased by 17 alpha-ethynylestradiol treatment, it is suggested that the liver plays an important role in hypolipidemia induced by 17 alpha-ethynylestradiol by increasing the plasma lipid uptake mediated by an increased amount of lipoprotein receptors of liver membranes.     

Species differences in metabolism of ketotifen in rat, rabbit and man: demonstration of similar pathways in vivo and in cultured hepatocytes

In vitro drug metabolism by cultured rat, rabbit and human adult hepatocytes has been studied, using ketotifen (ZADITEN) as a model substrate because it is biotransformed in vivo by various metabolic pathways in man and animals. The major in vivo pathways were demonstrated in vitro, namely oxidation in rat hepatocytes, oxidation, glucuronidation and sulfation in rabbit hepatocytes, reduction and glucuronidation in human hepatocytes. Human hepatocytes were the most stable in culture, displaying ketotifen biotransformation for at least one week. These results clearly demonstrated that cultured hepatocytes retain their in vivo specific drug metabolizing activities, including inter-species polymorphism, for a few days. Therefore, pure hepatocyte cultures represent a useful system suitable for drug metabolism studies.     

槲皮素对氧化应激大鼠肝细胞氨基酸代谢的影响

通过检测原代培养的大鼠肝细胞培养液中18种氨基酸含量的变化情况,探讨槲皮素对氧化应激大鼠肝细胞氨基酸代谢的影响。结果表明,与对照组相比,槲皮素组氨基酸质量浓度无明显变化;H2O2组、槲皮素+H2O2组培养液中丙氨酸、蛋氨酸和组氨酸的质量浓度显著减少(P<0.05),而牛磺酸的质量浓度显著增加(P<0.05),上述变化槲皮素+H2O2组较H2O2组更明显,槲皮素+H2O2组M et和H is的质量浓度显著低于H2O2组(P<0.05)。说明槲皮素可影响氧化应激肝细胞某些氨基酸,尤其是含硫氨基酸的代谢。这种影响可能是槲皮素抗氧化应激作用的机制之一。     

The effect of sodium butyrate on tyrosine aminotransferase induction in primary cultures of normal adult rat hepatocytes

Treatment of primary cultures of adult rat hepatocytes with 5 mM butyrate inhibited the spontaneous decrease in basal activity and mRNA levels of tyrosine aminotransferase (TAT) that occurred during culture (Staecker et al., submitted). We report here that butyrate treatment of primary cultures of rat hepatocytes initially inhibited the induction of TAT. This inhibition was followed by a period of accelerated TAT induction. TAT induction in butyrate-treated primary cultures of adult rat hepatocytes occurred only after metabolism of butyrate by the cultured hepatocytes. The accelerated induction of TAT in hepatocyte cultures treated with sodium butyrate was reflected by increased TAT activity and mRNA levels. Cultured hepatocytes rapidly metabolized butyrate, but the addition of more butyrate into cultures after its initial metabolism resulted in a rapid reduction in TAT activity. These findings indicate that butyrate treatment can affect the expression of TAT in primary hepatocyte cultures in both a positive (increased basal TAT expression) and a negative (inhibition of the induced expression of TAT) manner.     

The effects of unsaturated fatty acids on hepatic microsomal drug metabolism and cytochrome P-450

1. The effects of unsaturated fatty acids on drug-metabolizing enzymes in vitro were measured by using rat and rabbit hepatic 9000g supernatant fractions. 2. Unsaturated fatty acids inhibited the hepatic microsomal metabolism of ;type I' drugs with inhibition increasing with unsaturation: arachidonic acid>linolenic acid>linoleic acid>oleic acid. Inhibition was independent of lipid peroxidation. Linoleic acid competitively inhibited the microsomal O-demethylation of p-nitroanisole and the N-demethylation of (+)-benzphetamine. 3. The hepatic microsomal metabolism of ;type II' substrates, aniline and (-)-amphetamine, was not affected by unsaturated fatty acids. 4. The rate of reduction of p-nitrobenzoic acid and Neoprontosil was accelerated by unsaturated fatty acids. 5. Linoleic acid up to 3.5mm did not decelerate the generation of NADPH by rat liver soluble fraction, nor the activity of NADPH-cytochrome c reductase of rat liver microsomes. Hepatic microsomal NADPH oxidase activity was slightly enhanced by added linoleic acid. 6. No measurable disappearance of exogenously added linoleic acid occurred when this fatty acid was incubated with rat liver microsomes and an NADPH source. 7. The unsaturated fatty acids used in this study produced type I spectra when added to rat liver microsomes, and affected several microsomal enzyme activities in a manner characteristic of type I ligands.     

Limitation of theophylline elimination by reduced oxygen availability in mouse hepatocytes and rat isolated livers

The effect of oxygen availability on theophylline metabolism by mouse hepatocytes and rat isolated livers was examined. The elimination of theophylline by mouse hepatocytes and the metabolism of theophylline to dimethyluric acid by isolated, perfused rat livers was seriously impaired when the gas mixture supplied contained less than 28% oxygen. The correlation coefficients relating oxygen supply and the concentration of theophylline remaining in mouse hepatocyte suspensions were -0.74 to -0.84. In the isolated, perfused rat liver experiments, the correlation coefficient relating oxygen availability and dimethyluric acid production was 0.87. These observations are interpreted as supporting the hypothesis that oxygen availability per se is an important factor in determining the rate of theophylline metabolism.     

Mechanisms of the protective effect of L-alanine to D-galactosamine-induced hepatocellular injury: comparative studies of L-alanine and pyruvate

The addition of L-alanine reduced lactate dehydrogenase leakage from primary cultured rat hepatocytes treated with galactosamine (D-gal), while D-alanine and other amino acids did not. However, the mechanisms have not yet been entirely clarified. In this study, we used various inhibitors of metabolism, i.e., aminooxyacetate, oligomycin, and quinolinic acid, to examine the relation between this protective effect and the metabolism of L-alanine. Quinolinic acid (10 mM) did not affect the hepatoprotective effect of L-alanine, while oligomycin (0.1 mug/ml) and aminooxyacetate (1 mM) eliminated the hepatoprotective effect of L-alanine. L-Alanine also increased the albumin secretion by cultured hepatocytes treated with D-gal, while pyruvate had little effect. It was revealed that the intracellular content of pyruvate did not increase as a result of addition of L-alanine. These results are consistent with the hypothesis that L-alanine metabolism is important for hepatoprotection, but pyruvate cannot be used as a substitute for L-alanine.     

New actions of melatonin on tumor metabolism and growth

Melatonin is an important inhibitor of cancer growth promotion while the essential polyunsaturated fatty acid, linoleic acid is an important promoter of cancer progression. Following its rapid uptake by tumor tissue, linoleic acid is oxidized via a lipoxygenase to the growth-signaling molecule, 13-hydroxyoctadecadienoic acid (13-HODE) which stimulates epidermal growth factor (EGF)-dependent mitogenesis. The uptake of plasma linoleic acid and its metabolism to 13-HODE by rat hepatoma 7288CTC, which expresses both fatty acid transport protein and melatonin receptors, is inhibited by melatonin in a circadian-dependent manner. This inhibitory effect of melatonin is reversible with either pertussis toxin, forskolin or cAMP. While melatonin inhibits tumor linoleic acid uptake, metabolism and growth, pinealectomy or constant light exposure stimulates these processes. Thus, melatonin and linoleic acid represent two important environmental signals that interact in a unique manner to regulate tumor progression and ultimately the host-cancer balance.     

Regulation of hepatic glucose metabolism by leptin in pig and rat primary hepatocyte cultures

Direct effects of leptin on gluconeogenesis in rat hepatocytes are equivocal, and model systems from other species have not been extensively explored in assessing the regulation of glucose metabolism by leptin. Therefore, the goal of the present study was to compare the effects of leptin on gluconeogenesis in pig and rat hepatocyte cultures as well as to investigate an underlying mechanism of action at the level of phosphoenolpyruvate carboxykinase (PEPCK). In rat hepatocytes, leptin exposure (3 h, 50 and 100 nM) attenuated glucagon-stimulated hepatic gluconeogenesis by 35 and 38% (P < 0.05), respectively. However, leptin did not produce any significant acute effect in pig hepatocytes. Leptin exposure for 24 h failed to produce any significant effect on gluconeogenesis in either rat or pig hepatocytes cultured in the presence of glucagon or dexamethasone. Mechanistically, there was a 25-35% decrease (P < 0.05) in glucagon-induced PEPCK mRNA levels in rat but not pig hepatocytes cultured with leptin. This effect on PEPCK mRNA was not due to an alteration in the relative abundance of the leptin receptor or the ability of PEPCK to respond to cAMP. The nonuniformity of the effects of leptin on gluconeogenesis in pig and rat hepatocytes indicates differences in leptin action between species. Furthermore, the unique action of leptin in porcine hepatocytes points to the utility of this model system for biomedical research and also underscores the value of comparative studies.     

Essential role of intracellular glutathione in controlling ascorbic acid transporter expression and function in rat hepatocytes and hepatoma cells

Although there is in vivo evidence suggesting a role for glutathione in the metabolism and tissue distribution of vitamin C, no connection with the vitamin C transport systems has been reported. We show here that disruption of glutathione metabolism with buthionine-(S,R)-sulfoximine (BSO) produced a sustained blockade of ascorbic acid transport in rat hepatocytes and rat hepatoma cells. Rat hepatocytes expressed the Na(+)-coupled ascorbic acid transporter-1 (SVCT1), while hepatoma cells expressed the transporters SVCT1 and SVCT2. BSO-treated rat hepatoma cells showed a two order of magnitude decrease in SVCT1 and SVCT2 mRNA levels, undetectable SVCT1 and SVCT2 protein expression, and lacked the capacity to transport ascorbic acid, effects that were fully reversible on glutathione repletion. Interestingly, although SVCT1 mRNA levels remained unchanged in rat hepatocytes made glutathione deficient by in vivo BSO treatment, SVCT1 protein was absent from the plasma membrane and the cells lacked the capacity to transport ascorbic acid. The specificity of the BSO treatment was indicated by the finding that transport of oxidized vitamin C (dehydroascorbic acid) and glucose transporter expression were unaffected by BSO treatment. Moreover, glutathione depletion failed to affect ascorbic acid transport, and SVCT1 and SVCT2 expression in human hepatoma cells. Therefore, our data indicate an essential role for glutathione in controlling vitamin C metabolism in rat hepatocytes and rat hepatoma cells, two cell types capable of synthesizing ascorbic acid, by regulating the expression and subcellular localization of the transporters involved in the acquisition of ascorbic acid from extracellular sources, an effect not observed in human cells incapable of synthesizing ascorbic acid.     

Role of 12-lipoxygenase in hypoxia-induced rat pulmonary artery smooth muscle cell proliferation

The 12-lipoxygenase (12-LO) pathway of arachidonic acid metabolism stimulates cell growth and metastasis of various cancer cells and the 12-LO metabolite, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], enhances proliferation of aortic smooth muscle cells (SMCs). However, pulmonary vascular effects of 12-LO have not been previously studied. We sought evidence for a role of 12-LO and 12(S)-HETE in the development of hypoxia-induced pulmonary hypertension. We found that 12-LO gene and protein expression is elevated in lung homogenates of rats exposed to chronic hypoxia. Immunohistochemical staining with a 12-LO antibody revealed intense staining in endothelial cells of large pulmonary arteries, SMCs (and possibly endothelial cells) of medium and small-size pulmonary arteries and in alveolar walls of hypoxic lungs. 12-LO protein expression was increased in hypoxic cultured rat pulmonary artery SMCs. 12(S)-HETE at concentrations as low as 10(-5) microM stimulated proliferation of pulmonary artery SMCs. 12(S)-HETE induced ERK 1/ERK 2 phosphorylation but had no effect on p38 kinase expression as assessed by Western blotting. 12(S)-HETE-stimulated SMC proliferation was blocked by the MEK inhibitor PD-98059, but not by the p38 MAPK inhibitor SB-202190. Hypoxia (3%)-stimulated pulmonary artery SMC proliferation was blocked by both U0126, a MEK inhibitor, and baicalein, an inhibitor of 12-LO. We conclude that 12-LO and its product, 12(S)-HETE, are important intermediates in hypoxia-induced pulmonary artery SMC proliferation and may participate in hypoxia-induced pulmonary hypertension.