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. By contrast, 68% of the eicosapentaenoic acid ([(3)H]20:5n-3) uptake was elongated, and only 9% of the [3-(14)C]22:5n-3 uptake was retroconverted to 20:5n-3. Both tetracosapentaenoic acid ([3-(14)C]24:5n-3) and tetracosatetraenoic acid ([3-(14)C]24:4n-6) were converted to docosahexaenoic acid (22:6n-3) and 22:5n-6, respectively. Therefore, the difference in the n-3 and n-6 fatty acid products formed is due primarily to differences in the utilization of their 20- and 22-carbon intermediates. This metabolic difference probably contributes to the preferential accumulation of docosahexaenoic acid in the brain.

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). When DHA was added to media containing a 5 microM concentration of these (14)C-labeled n-3 PUFA, radiolabeled DHA synthesis was reduced but not completely suppressed even when the DHA concentration was increased to 15 microM. Radiolabeled DHA synthesis also was reduced but not completely suppressed when the astrocytes were treated with 30 microM DHA for 24 h before incubation with 5 microM [1-(14)C]18:3n-3.These findings indicate that although the DHA synthesis in astrocytes is dependent on DHA availability, some synthesis continues even when the cells have access to substantial amounts of DHA. This suggests that DHA synthesis from n-3 PUFA precursors is a constitutive process in the brain and, therefore, is likely to have an essential function.

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. However, they produced 50;-90% as much 20:4n-6 as the NF cultures from [1-14C]hexadecatrienoic acid (16:3n-6), [1-14C]gamma-linolenic acid (18:3n-6), and [1-14C]dihomo-gamma-linolenic acid (20:3n-6), PUFA substrates that contain Delta6 double bonds. DF also contained 80% more 18:2n-6 and 25% less 20:4n-6. These results suggested that DF are deficient in Delta6 desaturation. This was confirmed by Northern blots demonstrating an 81;-94% decrease in Delta6-desaturase mRNA content in the DF cultures, whereas the Delta5-desaturase mRNA content was reduced by only 14%. This is the first inherited abnormality in human PUFA metabolism shown to be associated with a Delta6-desaturase deficiency. Furthermore, the finding that the 18- and 24-carbon substrates are equally affected suggests that a single enzyme carries out both Delta6 desaturation reactions in human PUFA metabolism.

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. Treatment with 3 microM DCU reduced cellular conversion of 14,15-EET to 14,15-DHET by 3-fold after 4 h of incubation, with a concomitant increase in the formation of the novel beta-oxidation products 10,11-epoxy-16:2 and 8,9-epoxy-14:1. DCU also markedly enhanced the incorporation of 14,15-EET and its metabolites into PCEC lipids. The most abundant product in DCU-treated cells was 16,17-epoxy-22:3, the elongation product of 14,15-EET. Another novel metabolite, 14,15-epoxy-20:2, was present in DCU-treated cells. DCU also caused a 4-fold increase in release of 14,15-EET when the cells were stimulated with a calcium ionophore. Furthermore, DCU decreased the conversion of [3H]11,12-EET to 11,12-DHET, increased 11,12-EET retention in PCEC lipids, and produced an accumulation of the partial beta-oxidation product 7,8-epoxy-16:2 in the medium. These findings suggest that in addition to being metabolized by sEH, EETs are substrates for beta-oxidation and chain elongation in endothelial cells and that there is considerable interaction among the three pathways. The modulation of EET metabolism by DCU provides novel insight into the mechanisms by which pharmacological or molecular inhibition of sEH effectively treats hypertension.

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. 12(S)-HETE release was markedly increased by pretreatment with 13(S)-hydroperoxyoctadecadienoic acid but not by the reduced congener 13(S)-hydroxyoctadecadienoic acid, suggesting oxidative upregulation of 12(S)-HETE output. 12(S)-HETE produced potent relaxation and hyperpolarization of PC microvessels (EC(50), expressed as -log[M] = 13.5 +/- 0.5). Moreover, 12(S)-HETE potently activated large-conductance Ca(2+)-activated K(+) currents in PC microvascular smooth muscle cells. In contrast, 12(S)-HETE was not a major product of conduit PC endothelial AA metabolism and did not exhibit potent bioactivity in conduit PC arteries. We suggest that, in the coronary microcirculation, 12(S)-HETE can function as a potent hyperpolarizing vasodilator that may contribute to endothelium-dependent relaxation, particularly in the setting of oxidative stress.

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. These metabolites accumulated primarily in the medium, but small amounts also were incorporated into the cell lipids. The most abundant 11, 12-EET product was 7,8-epoxyhexadecadienoic acid (7,8-epoxy-16:2), and two of the others that were identified are 9, 10-epoxyoctadecadienoic acid (9,10-epoxy-18:2) and 5, 6-epoxytetradecaenoic acid (5,6-epoxy-14:1). The main epoxy-fatty acid produced from 14,15-EET was 10,11-epoxyhexadecadienoic acid (10, 11-epoxy-16:2). [(3)H]8,9-EET was converted to a single metabolite with the chromatographic properties of a 16-carbon epoxy-fatty acid, but we were not able to identify this compound. Large amounts of the chain-shortened 11,12-EET metabolites were produced by long-chain acyl CoA dehydrogenase-deficient fibroblasts but not by Zellweger syndrome and acyl CoA oxidase-deficient fibroblasts. We conclude that the chain-shortened epoxy-fatty acids are produced primarily by peroxisomal beta-oxidation. This may serve as an alternate mechanism for EET inactivation and removal from the tissues. However, it is possible that the epoxy-fatty acid products may have metabolic or functional effects and that the purpose of the beta-oxidation pathway is to generate these products.

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. Porcine coronary artery endothelial cells were treated with an epoxide hydrolase inhibitor, 4-phenylchalcone oxide (4-PCO, 20 micromol/l), before being incubated with (3)H-labeled 14,15-EET (14,15-[(3)H]EET). 4-PCO blocked conversion of 14,15-[(3)H]EET to 14,15-[(3)H]DHET and doubled the amount of radiolabeled products incorporated into cell lipids, with >80% contained in phospholipids. Moreover, pretreatment with 4-PCO before incubation with 14,15-[(3)H]EET enhanced A-23187-induced release of radiolabeled products into the medium. In contrast, 4-PCO did not alter uptake, distribution, or release of [(3)H]arachidonic acid. In porcine coronary arteries, 4-PCO augmented 14,15-EET-induced potentiation of endothelium-dependent relaxation to bradykinin. These data suggest that epoxide hydrolases may play a role in regulating EET incorporation into phospholipids, thereby modulating endothelial function in the coronary vasculature.

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. However, Zellweger fibroblasts that are deficient in peroxisomal fatty acid oxidation did not, indicating that the oxidative removal of the carboxyl group occurs in the peroxisomes. [3H]Hexadecatrienoic acid (16:3) was the main product that accumulated when [3H]20:4 was incubated with normal, LCAD deficient, and very long-chain acyl coenzyme A dehydrogenase (VLCAD) deficient fibroblasts, but Zellweger fibroblasts did not form this product. Normal fibroblasts converted [3H]16:3 to radiolabeled 20:3 and arachidonic acid. These findings suggest that some of the 16:3 produced from arachidonic acid by peroxisomal beta-oxidation can be recycled and that this recycling process constitutes a novel pathway for the conversion of arachidonic acid to 20:3 in human fibroblasts.

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. The three major metabolites were identified by gas chromatography combined with mass spectrometry as 11-hydroxyhexadecadienoic acid (11-OH-16:2), 9-hydroxytetradecadienoic acid (9-OH-14:2), and 7-hydroxydodecadienoic acid (7-OH-12:2). Most of the radioactivity contained in the cell lipids remained as 13-HODE. However, some 11-OH-16:2 and several unidentified products with longer RT than 13-HODE were detected in the cell lipids. Normal human skin fibroblasts also converted 13-HODE to the three major chain-shortened metabolites, but Zellweger syndrome fibroblasts produced only a very small amount of 11-OH-16:2. Therefore, the chain-shortened products probably are formed primarily by peroxisomal beta-oxidation. These findings suggest that peroxisomal beta-oxidation may constitute a mechanism for the inactivation and removal of 13-HODE from the vascular wall. Because this is a gradual process, some 13-HODE that is initially incorporated remains in endothelial phospholipids, especially phosphatidylcholine. This may be the cause of some of the functional perturbations produced by 13-HODE in the vascular wall.

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. The inhibition occurred when substrate amounts of AA were used and was not accompanied by enhanced production of other PGs, suggesting an effect on PGH synthase; however, in murine cells, 14, 15-EET did not reduce PGH synthase mRNA or protein. Moreover, the 14, 15-EET-induced decrease in PGE2 production was overcome by increasing the concentration of AA, but not oleic acid (which is not a substrate for PGH synthase). These findings suggest that 14,15-EET competitively inhibits PGH synthase activity in vascular smooth muscle cells. The 14,15-EET-induced inhibition of PGE2 production resulted in potentiation of platelet-derived growth factor-induced smooth muscle cell proliferation, suggesting that the competitive inhibition of PGH synthase by 14,15-EET can affect growth responses in smooth muscle cells.

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). These metabolites, which were not formed from [1-14C]EPA and were not detected in the cells, were identified as tetradecatrienoic acid (14:3n-3), hexadecatetraenoic acid (16:4n-3), and octadecatetraenoic acid (18:4n-3). The most abundant product under all of the conditions tested was 16:4n-3. [3H]EPA was converted to 16:4n-3 and 14:3n-3 by fibroblasts deficient in mitochondrial long-chain acyl CoA dehydrogenase, but not by Zellweger syndrome or acyl CoA oxidase mutants that are deficient in peroxisomal beta-oxidation. Competition studies indicated that 16:4n-3 formation from 5 microM [3H]EPA was reduced by 60% when 10 microM arachidonic acid was added, but the conversion of [3H]arachidonic acid to its chain-shortened products was not decreased by the addition of 10 microM EPA. These findings demonstrate that as in the case of arachidonic acid, chain-shortened polyunsaturated fatty acid products accumulate when EPA undergoes peroxisomal beta-oxidation. While EPA does not reduce arachidonic acid utilization by this pathway, it is possible that some biological actions of EPA may be mediated by the formation of the corresponding EPA products, 16:4n-3 and 14:3n-3.

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. 14,15-EET and 11,12-EET (5 mumol/L) produced 75 +/- 9% and 52 +/- 4% relaxation, respectively, of U46619-contracted rings, whereas 8,9-EET and 5,6-EET did not produce significant relaxation. The corresponding DHET regioisomers produced comparable relaxation responses. Preincubation with 14,15-EET, 11,12-EET, 14,15-DHET, and 11,12-DHET augmented the magnitude and duration of bradykinin-induced relaxation, whereas endothelium-independent relaxations to aprikalim and sodium nitroprusside were not potentiated. Pretreatment with 2 mumol/L triacsin C (an inhibitor of acyl coenzyme A synthases) inhibited [3H]14,15-EET incorporation into endothelial phospholipids and blocked 11,12-EET- and 14,15-DHET-induced potentiation of relaxation to bradykinin. Exposure of [3H]14,15-EET-labeled endothelial cells to the Ca2+ ionophore A23187 (2 mumol/L) resulted in a 4-fold increased release of EET and DHET into the medium. We conclude that incorporation of EETs and DHETs into cell lipids results in potentiation of bradykinin-induced relaxation in porcine coronary arteries, providing the first evidence that incorporated EETs and DHETs are capable of modulating vascular function.

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). Fibroblasts deficient in mitochondrial long-chain acyl coenzyme A dehydrogenase produce increased amounts of 14:2 from arachidonic acid. By contrast, Zellweger fibroblasts which are deficient in peroxisomal beta-oxidation do not convert arachidonic acid to either 14:2 or 16:3. These results demonstrate that 14:2 can be synthesized from arachidonic acid, that this oxidative process occurs in the peroxisomes, and that the pathway does not function in Zellweger's syndrome and similar diseases where there is a genetic deficiency in peroxisomal beta-oxidation.

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). 13-HODE formation was reduced when intestinal FABP (I-FABP). liver FABP (L-FABP) or albumin was added. The relative ability of these proteins to reduce 15-LO induced formation of 13-HODE and 15-HETE was BSA > L-FABP > I-FABP. Smaller reductions in activity were observed with 20:4 as compared to 18:2. The IC50-values of I-FABP and L-FABP, using either 18:2 (3.4 microM) or 20:4 (3.4 microM), were 4.6 +/- 0.6 and 1.9 +/- 0.2 microM, respectively, for reduction of 13-HODE and 6.8 +/- 0.3 and 3.1 +/- 0.2 microM, respectively, for reduction of 15-HETE formation. The smaller 15-HETE reduction correlated with decreased binding of 20:4 to the FABP. Titration calorimetry also showed that the I-FABP IC50 for 18:2, 0.25 microM, was lower then for 20:4, 0.6 microM. Thus the reduction in fatty acid lipid peroxidation relates to the binding capacity of each FABP. We also demonstrated that 18:2 rapidly diffuses (flip-flops) across the phospholipid bilayer of small unilamellar vesicles (SUV) and measured partitioning of 18:2 between proteins and SUV by the pyranin fluorescence method [Kamp, F. and Hamilton, J.A. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 11367-11370]. Addition of proteins to SUV in buffer resulted in a complete desorption of 18:2 from SUV with a relative effect of BSA > L-FABP > I-FABP. This suggests that the relative effects of these proteins on 18:2 peroxidation will not be altered by the presence of membranes. Our results indicate that FAPBs protect intracellular polyunsaturated fatty acids against peroxidation and, through differential binding of 18:2 and 20:4, they may modulate the availability of these polyunsaturated fatty acids to intracellular oxidative pathways.

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. The relaxation slowly increases in magnitude, resulting in a response which is sustained for more than 10 min. Cultured porcine aortic smooth muscle cells rapidly take up [3H]14,15-EET. After 3 min, radioactivity is present in neutral lipids, phosphatidylcholine, and phosphatidylinositol. The cells also convert 14,15-EET to 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), and some DHET is detected in the medium after only 1 min of incubation. Like 14,15-EET, 14,15-DHET produces relaxation of the contracted coronary artery rings. These findings suggest that the incorporation into phospholipids and conversion to 14,15-DHET can occur at a rate that is fast enough to modulate the vasorelaxation produced by 14,15-EET.

Functional implications of a newly characterized pathway of 11,12-epoxyeicosatrienoic acid metabolism in arterial smooth muscle.

Epoxyeicosatrienoic acids (EETs) are potent vasodilators derived from cytochrome P-450 metabolism of arachidonic acid. The rapid conversion of EETs to their corresponding dihydroxyeicosatrienoic acids (DHETs) has been proposed as a process whereby EETs are rendered biologically inactive. However, the vascular metabolism of EETs and the vasoactivities of EET metabolites have not been extensively studied. Accordingly, 11,12-EET metabolism was characterized in porcine aortic smooth muscle cells. The cells converted [3H]11,12-EET to 11,12-DHET and to a newly identified metabolite, 7,8-dihydroxy-hexadecadienoic acid (DHHD). 11,12-DHET accumulation in the medium reached a maximum in 2 to 4 hours and then declined, whereas 7,8-DHHD accumulation increased continuously and exceeded the amount of 11,12-DHET by 8 hours. [3H]11,12-EET conversion to radiolabeled 7,8-DHHD was reduced in the presence of unlabeled 11,12-DHET, indicating that 11,12-DHET is an intermediate in the conversion of 11,12-EET to 7,8-DHHD. This is consistent with a pathway whereby 11,12-EET is converted by an epoxide hydrolase to 11,12-DHET, which then undergoes two beta-oxidations to form 7,8-DHHD. In porcine coronary artery rings contracted with a thromboxane mimetic, 11,12-DHET produced relaxation similar in magnitude to that produced by 11,12-EET (77% versus 64% relaxation at 5 mumol/L, respectively). 7,8-DHHD also produced vasorelaxation. Thus, the vasoactivity of 11,12-EET is not eliminated by conversion to 11,12-DHET and 7,8-DHHD. These results suggest that 11,12-DHET and its metabolite, 7,8-DHHD, may contribute to the regulation of vascular tone in the porcine coronary artery and possibly other vascular tissues.

Arachidonic acid diols produced by cytochrome P-450 monooxygenases are incorporated into phospholipids of vascular endothelial cells.

Epoxyeicosatrienoic acids (EETs) are synthesized by cytochrome P-450 monooxygenases and released into the blood. When taken up by vascular endothelial and smooth muscle cells, the EETs are primarily esterified to phospholipids or converted to dihydroxyeicosatetraenoic acids (DHETs) and released. In the present studies, radiolabeled 8,9-, 11,12-, and 14,15-DHETs released into the medium from vascular smooth muscle cells were isolated and incubated for 4-16 h with cultured bovine aortic endothelial cells. The uptake ranged from 2 to 50% for the three regioisomers. Hydrolysis of the endothelial lipids and gas chromatographic-mass spectral analyses of the products indicated that all three DHET regioisomers were incorporated intact into phosphatidylcholine and phosphatidylinositol. Similar incubations with EETs confirmed that small amounts of DHETs were also esterified to endothelial phospholipids. These studies indicate that DHETs are incorporated into phospholipids either at the time of EET conversion to DHET or upon release and re-uptake of DHETs. Beside demonstrating for the first time that fatty acid diols are incorporated intact into endothelial lipids, these studies raise the possibility that both EETs and DHETs remain long enough in the vascular wall to produce chronic vasoactive effects.

Epoxyeicosatrienoic acid metabolism in arterial smooth muscle cells.

Epoxyeicosatrienoic acids (EETs) are eicosanoids synthesized from arachidonic acid by the cytochrome P450 eposygenase pathway. The present studies demonstrate that 8,9-, 11,12-, and 14,15-EET are rapidly taken up by porcine aortic smooth muscle cells. About half of the uptake is incorporated into phospholipids, and saponification indicates that most of this remains in the form of EET. The EETs also are converted to the corresponding dihydroxyeicosatrienoic acids (DHETs) and during prolonged incubations, additional metabolites that do not retain the EET carboxyl group are formed. Most of these products are released into the medium. However, some DHET and metabolites less polar than EET are incorporated into the phospholipids, and a small amount of unesterified EET is also present in the cells. The incorporation of 14,15-EET and its conversion to DHET did not approach saturation until the concentration exceeded 10-20 microM, indicating that vascular smooth muscle has a large capacity to utilize this EET. These findings suggest that certain vasoactive effects of EETs may be due to their incorporation by smooth muscle cells. Furthermore, through conversion to DHET and other oxidized metabolites, smooth muscle apparently has the capacity to inactivate EETs that are either formed in or penetrate into the vascular wall.

Formation and release of a peroxisome-dependent arachidonic acid metabolite by human skin fibroblasts.

Human skin fibroblasts labeled with [5,6,8,9,11,12,-14,15-3H]arachidonic acid produce a radioactive metabolite that has a shorter retention time on reverse-phase high-performance liquid chromatography than arachidonic acid. This product is not retained in the cells; it is released entirely into the extracellular fluid in a time-dependent manner. The metabolite does not cochromatograph with any of the eicosanoid standards, and its formation is not prevented by the addition of cyclooxygenase, lipoxygenase, or cytochrome P-450 inhibitors. The compound is not produced by fibroblasts labeled with [1-14C]arachidonic acid, suggesting that it is formed through an oxidative process. Chemical analyses indicated that the metabolite is 4,7,10-hexadecatrienoic acid (16:3). Peroxisome-deficient human skin fibroblasts did not produce 16:3, indicating that it probably is formed through peroxisomal beta-oxidation. Human umbilical vein endothelial cells and porcine pulmonary artery smooth muscle cells also release radioactive 16:3 following labeling with [3H]arachidonic acid. Therefore, the production of this metabolite is not limited only to fibroblasts. The fact that 16:3 is released into the extra-cellular fluid suggests that it may be a new type of lipid mediator derived from arachidonic acid, formed through a peroxisome-dependent oxidative process.