Murine cerebral microvascular endothelium incorporate and metabolize 12‐hydroxyeicosatetraenoic acid

Abstract

Cultured murine cerebromicrovascular endothelial cells were employed to study the metabolism of 12-hydroxyeicosatetraenoic acid (12-HETE) in an in vitro model of the blood-brain barrier. These endothelial cells convert 12-HETE to at least four, more polar compounds. Analysis of the least polar and predominant metabolite by gas chromatography combined with chemical ionization and electron impact mass spectrometry of reduced and nonreduced derivatives indicate that the compound is 8-hydroxyhexadecatrienoic acid (8-HHDTrE). The uptake of 12-HETE into cell phospholipids peaks at 2 hr, and is not saturable up to the highest concentration tested, 5 microM. Seventy-five to 92% of this 12-HETE is incorporated into phosphatidylcholine, while the remainder is divided between the inositol and ethanolamine phospholipids. Incorporation into neutral lipids is slower, with radioactivity gradually accumulating in triglycerides over 24 hr. Saponification of cell lipids demonstrated that not only 12-HETE, but also its major metabolite, 8-HHDTrE, is incorporated into the cell lipids. Prostacyclin and prostaglandin E2 production by the cerebral endothelial cells is inhibited by up to 56% with 1 microM and 90% with 5 microM 12-HETE. These data demonstrate that 12-HETE is actively metabolized by cerebral endothelium and suggest at least two mechanisms through which 12-HETE may alter cerebromicrovascular function: 1) incorporation into cerebral endothelial membranes and 2) inhibition of cerebral endothelial prostaglandin production. Conversion of 12-HETE to more polar compounds, particularly 8-HHDTrE, may be interpreted as either the inactivation of 12-HETE or the production of additional, biological mediators.

Hydroxyeicosatetraenoic acid metabolism in cultured human skin fibroblasts. Evidence for peroxisomal beta-oxidation.

“…These diHETEs are formed by lipoxygenase and cytochrome P450 pathways, respectively. In the human fibroblast, however, 12-HETE oxidation occurs primarily by the removal of 4 carbons from the carboxyl terminus, presumably through two successive ,3-oxidations, as has been observed in the smooth muscle and endothelial cells (15,16). Although the 15-HETE metabolite formed by the normal skin fibroblasts was not identified, its HPLC properties are identical to the product formed from 1 5-HETE by endothelial cells, 16:3(11-OH) (12).…”

“…This 16-carbon oxidative metabolite also is produced by human vascular smooth muscle cells (15) and murine cerebral microvascular endothelial cells (16). Conversion to is not the only oxidative pathway for 12-HETE.…”

“…action of stereospecific cytosolic lipoxygenase enzymes 1. Abbreviations used in this paper: HETE, hydroxyeicosatetraenoic acid; MDCK, Madin Darby canine kidney; MUO, 4-methylumbelliferyl oleate; DPBS, Dulbecco's phosphate-buffered saline; 16 that insert a single oxygen molecule into arachidonic acid at carbons 5, 12, or 15 (1). The initial product, an unstable hydroperoxide, is either spontaneously or peroxidatively reduced to the corresponding monohydroxylated product, a HETE, which is the stable end product.…”

Gammaherpesvirus 68 Infection of Endothelial Cells Requires both Host Autophagy Genes and Viral Oncogenes for Optimal Survival and Persistence

“…The endothelial cell line MB114 (50) was cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 5% fetal bovine serum (HyClone, Logan, UT), 2 mM L-glutamine, 10 U/ml penicillin, and 10 g/ml streptomycin sulfate (complete DMEM). Where indicated, medium was supplemented with 100 mM trehalose (Sigma Chemical, Saint Louis, MO) to induce autophagy, for a minimum of 48 h. 3-Methyladenine (3-MA) is a drug which inhibits the formation of autophagosomes at concentrations of 1 to 10 mM (59).…”

20-Hydroxyeicosatetraenoic Acid (20-HETE) Metabolism in Coronary Endothelial Cells

“…As opposed to 20-COOH-AA, these products appeared sequentially in the medium and then were converted to chain-shortened derivatives. Endothelial cells synthesize partial ␤-oxidation products from other eicosanoids, including intra-chain HETEs and EETs (27,39,50,51), and studies with mutant human skin fibroblasts indicate that this occurs through peroxisomal ␤-oxidation (52,53). While it seems likely that the ␤-oxidation pathway functions to degrade 20-HETE, the possibility that one or more of the chain-shortened metabolites may have functional activity cannot be excluded.…”