Oxidant stress enhances Lyso-PAF-AcT activity by modifying phospholipase D and phosphatidic acid in aortic endothelial cells.

Oxidant stress, as a consequence of selenium (Se) deficiency, alters production of vasoactive compounds including platelet-activating factor (PAF). Recent studies report that enhanced PAF production during Se deficiency is a consequence of increased lyso-PAF:acetyl-coenzyme A acetyltransferase (Lyso-PAF-AcT) activity. To elucidate the mechanism behind increased Lyso-PAF-AcT activity during oxidant stress, phospholipase D (PLD) activity and phosphatidic acid (PA) production were examined. Increased PLD activity and PA production were exhibited in bovine aortic endothelial cells using a Se-deficient model of oxidant stress. The direct effects of PLD and PA on Lyso-PAF-AcT activity were assessed using selective inhibitors and repletion experiments. Following the inhibition of PLD and addition of exogenous PA, Lyso-PAF-AcT activity significantly decreased and increased, respectively. Therefore, Se deficiency enhances Lyso-PAF-AcT activity in part by modifying PLD and PA. This suggests a novel link between Se status and PAF production, providing potential upstream therapeutic targets for PAF regulation under conditions of oxidant stress.

Increased 15-HPETE production decreases prostacyclin synthase activity during oxidant stress in aortic endothelial cells.

Selenium (Se) is an integral component of glutathione peroxidase and is able to detoxify peroxides that can affect arachidonic acid (AA) metabolism, thereby influencing eicosanoid biosynthesis. This study investigated the effects of oxidant stress, a consequence of Se deficiency, on eicosanoid formation and important key enzyme expression in bovine aortic endothelial cells (BAEC). Bovine aortic endothelial cells cultured in Se-deficient media and stimulated with tumor necrosis factor alpha or H2O2 produced significantly less prostacyclin (PGI(2)) and more 15-hydroxyeicosatetraenoic acid, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), and thromboxane than Se-supplemented BAEC. Additionally, reverse transcription polymerase chain reaction and immunoblotting determined that the mRNA and protein levels of the eicosanoid forming enzymes cyclooxygenase-1 (COX1), cyclooxygenase-2 (COX2), and PGI synthase were not significantly changed. The addition of 15-HPETE to Se-supplemented BAEC inhibited the production of PGI(2) suggesting that the accumulation of lipid hydroperoxides during Se-deficiency may be the underlying factor in the altered eicosanoid production during Se deficiency. Furthermore, inhibition of COX and addition of PGH(2) to Se-deficient or Se-supplemented BAEC still resulted in lower PGI(2) formation by Se-deficient cells. Together, these results suggest that Se deficiency modifies eicosanoid production by affecting the activity of key enzymes, particularly PGI synthase, rather than their transcription or translation.

Selenium modulates 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) biosynthesis in bovine aortic endothelial cells.

Selenium (Se) deficiency has been reported to increase platelet-activating factor (PAF) production in human endothelial cells; however, the mechanism is unclear. This study demonstrated that tumor necrosis factor-alpha (TNF-alpha) stimulated Se-deficient bovine aortic endothelial cells (BAEC) produced significantly more PAF than Se-supplemented cells. Moreover, the increase in the level of PAF was associated with enhanced activity of two anabolic enzymes in the remodeling pathway: phospholipase A2 and Lyso-PAF:acetyl-coenzyme A acetyltransferase (Lyso-PAF-AcT). In contrast, the activity of the PAF catabolic enzyme, PAF-acetylhydrolase, was not affected by Se status. Interestingly, prostacyclin, a potent vasodilator and inhibitor of platelet aggregation, inhibited the activity of Lyso-PAF-AcT and reduced the PAF production in TNF-alpha-stimulated BAEC. Therefore, we conclude that Se deficiency alters PAF production in TNF-alpha-stimulated BAEC by altering the activity of anabolic enzymes involved in the remodeling pathway partially through the inhibition of prostacyclin production.

Altered eicosanoid biosynthesis in selenium-deficient endothelial cells.

Selenium (Se) is an integral part of the Se-dependent glutathione peroxidase (Se-GSH-Px) catalytic domain. By modulating the cellular levels of fatty acid hydroperoxides, Se-GSH-Px can influence key enzymes of arachidonic acid cascade, in this case cyclooxygenase (COX) and lipoxygenase (LOX). To investigate this phenomenon, the effects of cellular Se status on the enzymatic oxidation of arachidonic acid were investigated in bovine mammary endothelial cells (BMEC), which were cultured in either Se-deficient (-Se) or Se-adequate (+Se) media. When stimulated with calcium ionophore A23187, BMEC produced eicosanoids of both COX and LOX pathways. Compared with the Se-adequate cells, the production of prostaglandin I(2) (PGI(2)), prostaglandin F(2) (PGF(2alpha)), and prostaglandin E(2) (PGE(2)) was significantly decreased in Se-deficient cells, whereas the production of thromboxane A(2) (TXA(2)) was markedly increased in the -Se BMEC cultures. Although the enzymatic oxidation of arachidonic acid by the LOX pathway was found to be relatively less than by the COX pathway, the BMEC cultured in -Se media produced significantly more 15-hydroperoxyeicosatetraenoic acid (15-HPETE) than the +Se cells produced. Based on these results, we postulate that cellular Se status plays an important regulatory role in the enzymatic oxidation of arachidonic acid by the COX and LOX pathways. The altered eicosanoid biosynthesis, especially the overproduction of 15-HPETE, in -Se BMEC may be one of the underlying biochemical phenomena responsible for vascular dysfunction during Se deficiency.

Selenium deficiency alters the lipoxygenase pathway and mitogenic response in bovine lymphocytes.

We investigated the effect of altered selenium (Se) nutrition on arachidonic acid oxidation in immune cells. Experiments were conducted with peripheral blood lymphocytes obtained from dairy cattle fed diets either supplemented with or deficient in Se. The results indicate that the concanavalin A-stimulated lymphocyte proliferation was significantly lower in Se-deficient cows. When stimulated by calcium ionophore A23187, the lymphocytes derived from Se-deficient cows produced significantly less 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4) than those obtained from Se-supplemented cows. When included in cell cultures from animals fed +Se diets, 5-HETE and LTB4 elicited a partial reversal of the inhibition of lymphocyte proliferation by either hydrocortisone or nordihydroguaiaretic acid. Based on this information, we postulate that dietary Se status, which in turn determines tissue Se concentration, plays an important role in the regulation of arachidonate metabolism by way of the 5-lipoxygenase pathway. This may be one of the biochemical mechanisms underlying the inhibition of lymphocyte proliferation and the decrease in resistance to infectious diseases observed in Se-deficient animals.

Increased 12-HETE production in bovine lymphocytes during selenium deficiency.

When peripheral blood lymphocytes were incubated with arachidonic acid in the presence of Ca++ ionophore (A23187), the cells from the selenium-deficient dairy cows produced significantly greater quantities of 12-hydroxyeicosatetraenoic acid (12-HETE) than the cells from the selenium-supplemented animals. The major product of reaction was verified as 12-HETE by cochromatography with a 12-HETE standard on an HPLC and structural analysis by GC-MS. Additionally, concanavalin A-stimulated lymphocyte proliferation was significantly decreased in cells from the Se-deficient cows. Furthermore, 12-HETE generated by the A23187-stimulated lymphocytes inhibited lymphocyte proliferation when added to Se-supplemented cell cultures. These observations suggest that self-regulation of lymphocyte proliferation might be mediated by 12-HETE production, especially during an altered nutritional state such as Se deficiency.