Docosahexaenoic acid synthesis from n-3 fatty acid precursors in rat hippocampal neurons.

Docosahexaenoic acid (DHA), the most abundant n-3 polyunsaturated fatty acid in the brain, has important functions in the hippocampus. To better understand essential fatty acid homeostasis in this region of the brain, we investigated the contributions of n-3 fatty acid precursors in supplying hippocampal neurons with DHA. Primary cultures of rat hippocampal neurons incorporated radiolabeled 18-, 20-, 22-, and 24-carbon n-3 fatty acid and converted some of the uptake to DHA, but the amounts produced from either [1-14C]alpha-linolenic or [1-14C]eicosapentaenoic acid were considerably less than the amounts incorporated when the cultures were incubated with [1-14C]22:6n-3.

Oxygenation of omega-3 fatty acids by human cytochrome P450 4F3B: effect on 20-hydroxyeicosatetraenoic acid production.

Cytochrome P450 (CYP) omega-oxidases convert arachidonic acid (AA) to 20-hydroxyeicosatetraenoic acid (20-HETE), a lipid mediator that modulates vascular tone. We observed that a microsomal preparation containing recombinant human CYP4F3B, which converts AA to 20-HETE, converted eicosapentaenoic acid (EPA) to 20-OH-EPA. Likewise, docosahexaenoic acid (DHA) was converted to 22-OH-DHA, indicating that human CYP4F3B also can oxidize 22-carbon omega-3 fatty acids.

20-Hydroxyeicosatetraenoic acid is a potent dilator of mouse basilar artery: role of cyclooxygenase.

20-Hydroxyeicosatetraenoic acid (20-HETE), an arachidonic acid (AA) metabolite synthesized by cytochrome P-450 omega-oxidases, is reported to produce vasoconstriction in the cerebral circulation. However, we find that like 14,15-epoxyeicosatrienoic acid (14,15-EET), 20-HETE produces dilation of mouse basilar artery preconstricted with U-46619 in vitro. Indomethacin inhibited the vasodilation produced by 20-HETE but not by 14,15-EET, suggesting a cyclooxygenase (COX)-dependent mechanism.

Omega-oxidation of 20-hydroxyeicosatetraenoic acid (20-HETE) in cerebral microvascular smooth muscle and endothelium by alcohol dehydrogenase 4.

20-Carboxyeicosatetraenoic acid (20-COOH-AA) is a bioactive metabolite of 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid that produces vasoconstriction in the cerebral circulation. We found that smooth muscle (MSMC) and endothelial (MEC) cultures obtained from mouse brain microvessels convert [3H]20-HETE to 20-COOH-AA, indicating that the cerebral vasculature can produce this metabolite. The [3H]20-COOH-AA accumulated primarily in the culture medium, together with additional radiolabeled metabolites identified as the chain-shortened dicarboxylic acids 18-COOH-18:4, 18-COOH-18:3, and 16-COOH-16:3.

Impaired arachidonic acid-mediated activation of large-conductance Ca2+-activated K+ channels in coronary arterial smooth muscle cells in Zucker Diabetic Fatty rats.

We studied the arachidonic acid (AA)-mediated modulation of large-conductance Ca2+-activated K+ (BK) channels in coronary arterial smooth myocytes from lean control and Zucker Diabetic Fatty (ZDF) rats. A total of 1 micromol/l AA enhanced BK current by 274% in lean and by 98% in ZDF rats. After incubation with 10 micromol/l indomethacin, 1 micromol/l AA increased BK currents by 80% in lean and by 70% in ZDF rats.

Impaired arachidonic acid-mediated dilation of small mesenteric arteries in Zucker diabetic fatty rats.

Arachidonic acid (AA) is a precursor of important vasoactive metabolites, but the role of AA-mediated vasodilation in Type 2 diabetes is not known. Using Zucker diabetic fatty (ZDF) rats, we examined the effects of AA in small mesenteric arteries preconstricted with endothelin. In ZDF rat mesenteric arteries, 1 microM AA produced only one-third the amount of dilation as in vessels from lean control animals.

20-hydroxyeicosatetraenoic acid (20-HETE) metabolism in coronary endothelial cells.

We have investigated the role of endothelial cells in the metabolism of 20-hydroxyeicosatetraenoic acid (20-HETE), a vasoactive mediator synthesized from arachidonic acid by cytochrome P450 omega-oxidases. Porcine coronary artery endothelial cells (PCEC) incorporated 20-[(3)H]HETE primarily into the sn-2 position of phospholipids through a coenzyme A-dependent process. The incorporation was reduced by equimolar amounts of arachidonic, eicosapentaenoic or 8,9-epoxyeicosatrienoic acids, but some uptake persisted even when a 10-fold excess of arachidonic acid was available.

Effect of the delta6-desaturase inhibitor SC-26196 on PUFA metabolism in human cells.

The objective of this study was to determine the effect of 2,2-diphenyl-5-(4-[[(1 E)-pyridin-3-yl-methylidene]amino]piperazin-1-yl)pentanenitrile (SC-26196), a delta6-desaturase inhibitor, on PUFA metabolism in human cells. SC-26196 inhibited the desaturation of 2 microM [1-14C] 18:2n-6 by 87-95% in cultured human skin fibroblasts, coronary artery smooth muscle cells, and astrocytes. By contrast, SC-26196 did not affect the conversion of [1-14C]20:3n-6 to 20:4 in the fibroblasts, demonstrating that it is selective for delta6-desaturase.

Human coronary endothelial cells convert 14,15-EET to a biologically active chain-shortened epoxide.

Cytochrome P-450 epoxygenase-derived epoxyeicosatrienoic acids (EETs) play an important role in the regulation of vascular reactivity and function. Conversion to the corresponding dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolases is thought to be the major pathway of EET metabolism in mammalian vascular cells. However, when human coronary artery endothelial cells (HCEC) were incubated with (3)H-labeled 14,15-EET, chain-shortened epoxy fatty acids, rather than DHET, were the most abundant metabolites.

Comparison of 20-, 22-, and 24-carbon n-3 and n-6 polyunsaturated fatty acid utilization in differentiated rat brain astrocytes.

Astrocytes convert n-6 fatty acids primarily to arachidonic acid (20:4n-6), whereas n-3 fatty acids are converted to docosapentaenoic (22:5n-3) and docosahexaenoic (22:6n-3) acids. The utilization of 20-, 22- and 24-carbon n-3 and n-6 fatty acids was compared in differentiated rat astrocytes to determine the metabolic basis for this difference. The astrocytes retained 81% of the arachidonic acid ([(3)H]20:4n-6) uptake and retroconverted 57% of the docosatetraenoic acid ([3-(14)C]22:4n-6) uptake to 20:4n-6.

Docosahexaenoic acid synthesis from n-3 polyunsaturated fatty acids in differentiated rat brain astrocytes.

DHA, the main n-3 PUFA in the brain, is synthesized from n-3 PUFA precursors by astrocytes. To assess the potential of this process to supply DHA for the brain, we investigated whether the synthesis in astrocytes is dependent on DHA availability. Rat brain astrocytes differentiated with dibutyryl cAMP and incubated in media containing 10% fetal bovine serum synthesized DHA from alpha-linolenic acid ([1-(14)C]18:3n-3), docosapentaenoic acid ([3-(14)C]22:5n-3), tetracosapentaenoic acid ([3-(14)C]24:5n-3), and tetracosahexaenoic acid ([3-(14)C]24:6n-3).

Identification of a fatty acid delta6-desaturase deficiency in human skin fibroblasts.

Polyunsaturated fatty acid (PUFA) utilization was investigated in skin fibroblasts cultured from a female patient with an inherited abnormality in lipid metabolism. These deficient human skin fibroblasts (DF) converted 85;-95% less [1-14C]linoleic acid (18:2n-6) to arachidonic acid (20:4n-6), 95% less [3-14C]tetracosatetraenoic acid (24:4n-6) to docosapentaenoic acid (22:5n-6), and 95% less [1-14C]-linolenic acid (18:3n-3) and [3-14C]tetracosapentaenoic acid (24:5n-3) to docosahexaenoic acid (22:6n-3) than did normal human skin fibroblasts (NF). The only product formed by the DF cultures from [1-14C]tetradecadienoic acid (14:2n-6) was 18:2n-6.

Pathways of epoxyeicosatrienoic acid metabolism in endothelial cells. Implications for the vascular effects of soluble epoxide hydrolase inhibition.

Epoxyeicosatrienoic acids (EETs) are products of cytochrome P-450 epoxygenase that possess important vasodilating and anti-inflammatory properties. EETs are converted to the corresponding dihydroxyeicosatrienoic acid (DHET) by soluble epoxide hydrolase (sEH) in mammalian tissues, and inhibition of sEH has been proposed as a novel approach for the treatment of hypertension. We observed that sEH is present in porcine coronary endothelial cells (PCEC), and we found that low concentrations of N,N'-dicyclohexylurea (DCU), a selective sEH inhibitor, have profound effects on EET metabolism in PCEC cultures.

12-lipoxygenase in porcine coronary microcirculation: implications for coronary vasoregulation.

Noncyclooxygenase metabolites of arachidonic acid (AA) have been proposed to mediate endothelium-dependent vasodilation in the coronary microcirculation. Therefore, we examined the formation and bioactivity of AA metabolites in porcine coronary (PC) microvascular endothelial cells and microvessels, respectively. The major noncyclooxygenase metabolite produced by microvascular endothelial cells was 12(S)-hydroxyeicosatetraenoic acid (HETE), a lipoxygenase product.

Conversion of epoxyeicosatrienoic acids (EETs) to chain-shortened epoxy fatty acids by human skin fibroblasts.

Epoxyeicosatrienoic acids (EETs), the eicosanoid biomediators synthesized from arachidonic acid by cytochrome P450 epoxygenases, are inactivated in many tissues by conversion to dihydroxyeicosatrienoic acids (DHETs). However, we find that human skin fibroblasts convert EETs mostly to chain-shortened epoxy-fatty acids and produce only small amounts of DHETs. Comparative studies with [5,6,8,9,11,12,14,15-(3)H]11,12-EET ([(3)H]11,12-EET) and [1-(14)C]11,12-EET demonstrated that chain-shortened metabolites are formed by removal of carbons from the carboxyl end of the EET.

Epoxide hydrolases regulate epoxyeicosatrienoic acid incorporation into coronary endothelial phospholipids.

Cytochrome P-450-derived epoxyeicosatrienoic acids (EETs) are avidly incorporated into and released from endothelial phospholipids, a process that results in potentiation of endothelium-dependent relaxation. EETs are also rapidly converted by epoxide hydrolases to dihydroxyeicosatrienoic acid (DHETs), which are incorporated into phospholipids to a lesser extent than EETs. We hypothesized that epoxide hydrolases functionally regulate EET incorporation into endothelial phospholipids.

Role of peroxisomal oxidation in the conversion of arachidonic acid to eicosatrienoic acid in human skin fibroblasts.

Human skin fibroblasts converted [5,6,8,9,11,12,14,15-3H]arachidonic acid ([3H]20:4) to eicosatrienoic acid (20:3), but appreciable amounts of radiolabeled 20:3 were not detected in corresponding incubations with [1-(14)C]20:4. This indicates that the main pathway for synthesizing 20:3 from arachidonic acid in the fibroblast involves oxidative removal of the carboxyl group of arachidonic acid. Fibroblasts deficient in long-chain acyl coenzyme A dehydrogenase (LCAD) converted [3H]20:4 to [3H]20:3.

13-(S)-hydroxyoctadecadienoic acid (13-HODE) incorporation and conversion to novel products by endothelial cells.

13(S)-Hydroxy-[12,13-3H]octadecadienoic acid (13-HODE), a linoleic acid oxidation product that has vasoactive properties, was rapidly taken up by bovine aortic endothelial cells. Most of the 13-HODE was incorporated into phosphatidylcholine, and 80% was present in the sn -2 position. The amount of 13-HODE retained in the cells gradually decreased, and radiolabeled metabolites with shorter reverse-phase high-performance liquid chromatography retention times (RT) than 13-HODE accumulated in the extracellular fluid.

14,15-Epoxyeicosatrienoic acid inhibits prostaglandin E2 production in vascular smooth muscle cells.

14,15-Epoxyeicosatrienoic acid (EET), a cytochrome P-450 epoxygenase product of arachidonic acid (AA), reduced PGE2 formation by 40-75% in porcine aortic and murine brain microvascular smooth muscle cells. The inhibition was reversed 6-10 h after removal of 14,15-EET from the medium and was regioisomeric specific; 8,9-EET produced a smaller effect, whereas 11,12- and 5,6-EET were ineffective. Although the cells converted 14,15-EET to 14, 15-dihydroxyeicosatrienoic acid (14,15-DHET), 14,15-DHET did not inhibit PGE2 formation, and the 14,15-EET-induced inhibition was potentiated by 4-phenylchalcone oxide, an epoxide hydrolase inhibitor.

Conversion of eicosapentaenoic acid to chain-shortened omega-3 fatty acid metabolites by peroxisomal oxidation.

Human skin fibroblasts can convert arachidonic acid to 14- and 16-carbon polyunsaturated fatty acid products by peroxisomal beta-oxidation. The purpose of this study was to determine whether similar products are formed from eicosapentaenoic acid (EPA) and whether EPA and arachidonic acid compete for utilization by this oxidative pathway. Three radiolabeled metabolites with shorter retention times than EPA on reverse-phase high-performance liquid chromatography accumulated in the medium during incubation of fibroblasts with [5,6,8,9,11,12,14,15,17,18-3H] EPA ([3H]EPA).

Cytochrome P450 metabolites of arachidonic acid: rapid incorporation and hydration of 14,15-epoxyeicosatrienoic acid in arterial smooth muscle cells.

Arachidonic acid is converted to epoxyeicosatrienoic acids (EETs) by cytochrome P450 monooxygenases. EETs produce arterial vasodilatation, and recent evidence suggests that they are endothelium-derived hyperpolarizing factors. In porcine coronary arteries contracted with a thromboxane mimetic agent, we find that relaxation is rapidly initiated by exposure to 14,15-EET.

Potentiation of endothelium-dependent relaxation by epoxyeicosatrienoic acids.

Epoxyeicosatrienoic acids (EETs) are potent endothelium-derived vasodilators formed from cytochrome P-450 metabolism of arachidonic acid. EETs and their diol products (DHETs) are also avidly taken up by endothelial cells and incorporated into phospholipids that participate in signal transduction. To investigate the possible functional significance of EET and DHET incorporation into cell lipids, we examined the capacity of EETs and DHETs to relax porcine coronary arterial rings and determined responses to bradykinin (which potently activates endothelial phospholipases) before and after incubating the rings with these eicosanoids.

Conversion of arachidonic acid to tetradecadienoic acid by peroxisomal oxidation.

Human skin fibroblasts convert [5,6,8,9,11,12,14,15-3H]arachidonic acid to two radiolabeled polar metabolites that accumulate in the culture medium. Previous studies identified the most abundant of these products as 4,7,10-hexadecatrienoic acid (16:3). We have now identified the second metabolite as 5,8-tetradecadienoic acid (14:2).

Fatty acid binding proteins reduce 15-lipoxygenase-induced oxygenation of linoleic acid and arachidonic acid.

Free fatty acids in plasma and cells are mainly bound to membranes and proteins such as albumin and fatty acid binding proteins (FABP), which can regulate their biological activities and metabolic transformations. We have investigated the effect of FABP and albumin on the peroxidation of linoleic acid (18:2) and arachidonic acid (20:4) by 15-lipoxygenase (15-LO). Rabbit reticulocyte 15-LO produced a rapid conversion of [1-14C]18:2 to 13-hydroxyoctadecadienoic acid (13-HODE) and [3H]20:4 to 15-hydroxyeicosatetraenoic acid (15-HETE).