Review
Epoxyeicosatrienoic acids (EETs): metabolism and biochemical function

https://doi.org/10.1016/S0163-7827(03)00049-3Get rights and content

Abstract

Epoxyeicosatrienoic acids (EETs), which are synthesized from arachidonic acid by cytochrome P450 epoxygenases, function primarily as autocrine and paracrine effectors in the cardiovascular system and kidney. They modulate ion transport and gene expression, producing vasorelaxation as well as anti-inflammatory and pro-fibrinolytic effects. EETs are incorporated into the sn-2 position of phospholipids and are rapidly mobilized when a cell is treated with a Ca2+ ionophore, suggesting that they may play a role in phospholipid-mediated signal transduction processes. Soluble epoxide hydrolase (sEH) converts EETs to dihydroxyeicosatrienoic acids (DHETs), and inhibition of sEH is a potential approach for enhancing the biological activity of EETs. EETs also undergo chain-elongation and β-oxidation, and the accumulation of partial β-oxidation products increases when sEH is inhibited. Some functional effects of EETs occur through activation of either the guanine nucleotide binding protein Gαs or the Src signal transduction pathways, suggesting that EETs act by binding to membrane receptors. However, other evidence indicates that the modulation of gene expression occurs through an intracellular action of EETs. Because of the diversity of biochemical and functional responses produced by EETs, it is doubtful that a single mechanism or signal transduction pathway can account for all of their actions.

Introduction

Three classes of eicosanoid biomediators are synthesized from arachidonic acid; the cyclooxygenase, lipoxygenase and cytochrome P450 (CYP) products [1]. The CYP class is subdivided into two groups; the epoxyeicosatrienoic acids (EETs) formed by CYP epoxgenases, primarily the CYP2C and 2J isoforms in humans, and the arachidonic acid derivatives that are hydroxylated at or near the ω-terminus by CYP ω-oxidases, primarily the CYP 4A and 4F isoforms in humans [2], [3]. As shown in Fig. 1, CYP epoxygenases produce four EET regioisomers from arachidonic acid; 5,6-, 8,9-, 11,12-, and 14,15-EET.

Each CYP epoxygenase isozyme produces all four EET regioisomers, but one or two usually are the predominant products. For example, 14,15-EET accounts for 41% of the EETs produced by rat heart CYP 2J3 [4], and 11,12-EET accounts for 58% of the EETs produced by rat kidney CYP 2J23 [5]. These two regioisomers are the predominant EETs produced by many different cells and tissues: for example, they account for 67–80% of the total EETs produced by five purified and reconstituted rat CYP epoxygenases [2]. Each regioisomer consists of a mixture of R,S- and S,R-enantiomers. As a further complication, the enantiofacial selectivity is different for each CYP epoxygenase, and the same isozyme can have a different selectivity for epoxidation at each of the arachidonic acid double bonds [2].

The various types of functional effects produced by EETs are listed in Table 1. EETs are important modulators of cardiovascular function, and they produce these effects by acting primarily on the vasculature and in the kidney [6], [7], [8]. They are synthesized in blood vessels by CYP epoxygenases contained in endothelial cells [9], and they produce vasodilation in a number of vascular beds by activating the smooth muscle large conductance Ca2+-activated K+ channels (BKCa) [10]. This hyperpolarizes the smooth muscle and produces vasorelaxation, lowering blood pressure [6], [8], [11]. A substantial amount of evidence indicates that EETs may function as endothelium-derived hyperpolarizing factors (EDHF) [12], particularly in the coronary circulation [13]. Furthermore, effects of EETs on smooth muscle migration [14], prostaglandin (PG) E2 production [15], Ca2+ influx [16] and aromatase activity [17] have been observed. In the endothelium, EETs inhibit cytokine-induced inflammatory responses [18], [19], increase Ca2+ entry [20], [21], enhance fibrinolysis [22], and stimulate tube formation [23]. Functional effects also occur in other tissues [24], [25], [26], [27], [28], [29], [30], [31], [32]. In addition, EET binding to intact cell membranes and to recombinant fatty acid binding protein (FABP) has been observed [31], [32].

EETs are enzymatically hydrated to the corresponding dihydroxyeicosatrienoic acid (DHET) by epoxide hydrolases [33], [34]. This reaction is illustrated in Fig. 2, which shows the conversion of 14,15-EET to 14,15-DHET. DHETs were initially thought to be inactivation products of EETs, but several recent studies indicate that, like EETs, they produce vasodilation and activate smooth muscle BKCa channels [35], [36], [37], [38]. There are two major epoxide hydrolase isozymes in mammalian tissues; soluble epoxide hydrolase (sEH) contained primarily in the cytosol [33], [34], and microsomal epoxide hydrolase (mEH) bound to intracellular membranes [39]. sEH is the main isozyme that acts on the EETs.

Two recent findings in the cardiovascular system have heightened interest in EETs. One is that 11,12-EET appears to be a strong candidate for EDHF in the coronary circulation [13]. The other is that disruption of the sEH gene [40] or inhibition of sEH by N,N′-dicyclohexylurea (DCU), a selective sEH inhibitor [41], reduces blood pressure [42], suggesting that sEH is a potential pharmacological target for hypertension.

This review will focus on the metabolism and biochemical mechanisms of action of EETs. The reader is referred to other recent reviews for in-depth coverage of EET chemistry, physiology and pharmacology [2], [3], [6], [7], [8], [11], [43], [44].

Section snippets

EET content of tissue and plasma lipids

Animal and human tissue lipids contain EETs. Rat liver phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) contain 70, 85 and 106 μmol EET/mol phospholipid, respectively [45]. These phosphoglycerides contain 8,9-, 11,12- and 14,15-EET, and all of the EETs are esterified at the sn-2 position of the glycerol moiety. Rabbit kidney contains 8,9- and 14,15-EET [46], and human kidney cortex contains 8,9-, 11,12- and 14,15-EET [47]. The enantiomeric structures of the

Major pathways of EET metabolism

Most of the detailed information about EET metabolism has been obtained from incubations of cultured mammalian cells with chemically synthesized [1-14C]EETs or [5,6,8,9,11,12,14,15-3H]EETs [50], [51]. These studies have been done with murine mastocytoma cells [52], porcine and human endothelial cells [50], [53], [54], porcine arterial smooth muscle cells [15], [35], [55], [56], [57], human skin fibroblasts (HSF) [58] and rat cerebral astrocytes [59].

Additional pathways of EET metabolism

Besides the major pathways illustrated in Fig. 3, EETs can undergo a number of other metabolic conversions. These additional reactions, which include partial β-oxidation, chain-elongation, oxidation by the cyclooxygenase, lipoxygenase and CYP ω-oxidase enzymes, and conjugation with glutathione are illustrated schematically in Fig. 4.

Stimulated release of EET from cells

Much more EET is released into the culture medium when cells are incubated with a Ca2+ ionophore than under basal conditions. For example, mastocytoma cells loaded with radiolabeled 14,15-EET released 50% of the EET into the extracellular fluid in 15 min when they were incubated with A23187, a Ca2+ ionophore [52]. Likewise, PCEC incubated with A23187 released 10-times more [3H]14,15-EET than under basal conditions [35], [63]. Because most of the intracellular EET is present in phospholipids,

ω-3 Fatty acid analogues of EET

Eicosapentaenoic acid (EPA, 20:5ω-3), the ω-3 fatty acid analogue of arachidonic acid, can be converted to a 17,18-epoxy-derivative by vascular smooth muscle cells [87]. This apparently occurs through the action of either the CYP 4A1 or 4A3 that is expressed in these cells. The 17,18-epoxy-derivative of EPA produces greater activation of the rat cerebral artery BKCa channel than 11,12-EET. Likewise, epoxides synthesized chemically by incubation of EPA with m-chloroperoxybenzoic acid [88],

Soluble epoxide hydrolase (sEH)

Epoxide hydrolases (EC 3.3.2.3) are a group of enzymes that convert the epoxide group of chemical compounds to corresponding diols by the addition of water. The main epoxide hydrolases have been classified based on the structure of their substrates and their cellular localization. They are cholesterol 5,6-oxide hydrolase, hepoxilin A3 hydrolase, leukotriene A4 hydrolase, sEH (formerly referred to as cytosolic EH), and mEH [91], [92]. sEH is the primary enzyme responsible for the conversion of

Biochemical response to EETs: mechanisms of action

Considerable progress has been made during the last two decades in determining the functional effects and metabolism of EETs [2], [7], [8], [11], [44]. In contrast, much less is known about the biochemical mechanisms through which EETs produce their responses. Because the physiological actions vary widely, depending upon the EET regioisomer and the specific cell type (see Table 1), a single biochemical response mechanism probably does not exist. Prostaglandins and thromboxanes act through a

Acknowledgements

The work from our laboratory described in this article was supported by the National Institutes of Health grants HL72845, HL49264 and HL62984, and by American Heart Association grants 0060413Z and 0230096N.

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