20-Hydroxyeicosatetraenoic acid, also known as 20-HETE or 20-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid, is an eicosanoid metabolite of arachidonic acid that has a wide range of effects on the vascular system including the regulation of vascular tone, blood flow to specific organs, sodium and fluid transport in the kidney, and vascular pathway remodeling. These vascular and kidney effects of 20-HETE have been shown to be responsible for regulating blood pressure and blood flow to specific organs in rodents; genetic and preclinical studies suggest that 20-HETE may similarly regulate blood pressure and contribute to the development of stroke and heart attacks. Additionally the loss of its production appears to be one cause of the human neurological disease, hereditary spastic paraplegia. Preclinical studies also suggest that the overproduction of 20-HETE may contribute to the progression of certain human cancers, particularly those of the breast.
Biosynthesis Production in humans A subset of cytochrome P450 (CYP450) microsome-bound ω-hydroxylases, the cytochrome P450 omega hydroxylases, metabolize arachidonic acid to 20-HETE by an omega oxidation reaction. CYP450 enzymes belong to a superfamily which in humans is composed of at least 57 members and in mice at least 120 members. Among this superfamily, certain members of the CYP4A and CYP4F subfamilies in the CYP4 family are considered predominant cytochrome P450 enzymes that are responsible in most tissues for forming 20-HETE and, concurrently, smaller amounts of 19-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid (19-HETE). However, CYP2U1 may also contribute to the production of these two HETEs and CYP4F8 can metabolize arachidonic acid to 19-HETE while forming little or no 20-HETE.
The production of 19-HETE with 20-HETE may be significant since 19(R)-HETE, although not its stereoisomer, 19(S)-HETE, inhibits the action of 20-HETE on vascular endothelial cells. Based on studies analyzing the production of other HETEs by CYP enzymes, the production of 19-HETE by these enzymes may include both its R and S stereoisomers.
In humans, the CYP4 ω-hydroxylases include CYP4A11, CYP4F2, and CYP4F3 with the predominant 20-HETE-synthesizing enzymes being CYP4F2, which is the major 20-HETE producing enzyme in the human kidney, followed by CYP4A11. CYP4F3 is expressed as two distinct enzymes, CYP4F3A and CYP4F3B, due to alternative splicing of a single pre-mRNA precursor molecule; CYP4F3A is mostly expressed in leukocytes, CYP4F3B mostly in the liver. Human CYP4Z1, which is expressed in a limited range of tissues such as human breast and ovary, may also metabolize arachidonic acid to 20-HETE while human CYP4A22, once considered as contributing to 20-HETE production, is now regarded as being metabolically inactive. Finally, CYP2U1, the only member of the human CYP2U subfamily, is highly expressed in brain and thymus and to lesser extents in numerous other tissues such as kidney, lung and heart. CYP2U1 protein is also highly expressed, compared to several other cytochrome P450 enzymes, in malignant breast tissue; the MCF-7 human breast cancer cell line express messenger RNA for this cytochrome.
Production by rodents and other animals In mice, the only 20-HETE- and 19-HETE-producing enzymes of the Cyp4a subfamily are two extensively homologous ones, Cyp4a12a and Cyp4a12b; Cyp4a12a is expressed in the male kidney in an androgen hormone-dependent manner. In rats, Cyp4a1, Cyp4a2, Cyp4a3, and Cyp4a8 make 20-HETE. The tissue distribution of these enzymes differs from those of humans making extrapolations from rodent studies to humans somewhat complicated.
Mouse CYP2J9, rat CYP2J3, and sheep CYP2J metabolize arachidonic acid primarily to 19-HETE but also to smaller amounts of 20-HETE, and, in the case of the sheep enzyme, 18-HETE; human CYP2J2, however, is an epoxygenase, metabolizing arachidonic acid to epoxide products.
Factors regulating 20-HETE production Many agents stimulate cells and tissues to produce 20-HETE in vitro and in vivo. Androgens are particularly potent stimulators of this production. Other stimulators include the powerful vasoconstriction-inducing agents, angiotensin II, endothelins, and alpha adrenergic compounds (e.g. norepinephrine).
Nitric oxide, carbon monoxide, and superoxide inhibit 20-HETE production; these non-pharmacological agents do so by binding to the Heme binding site of the 20-HETE producing cytochrome p450 enzymes. Drugs that are substrates for the UDP-glucuronosyltransferase (UGT) enzymes which metabolize 20-HETE such as non-steroidal anti-inflammatory agents, opioids, gemfibrozil, Lasix, propanol, and various COX-2 inhibitors may act as perhaps unwanted side effects to increase the levels of 20-HETE. There are a variety of pharmacological agents which inhibit the synthesis of 20-HETE including various fatty acid analogs that compete reversibly with arachidonic acid for the substrate binding site in the CYP enzymes and benzene-based drugs.
Proviso on 20-HETE production The cytochrome ω-oxidases including those belonging to the CYP4A and CYP4F sub-families and CYPU21 hydroxylate not only arachidonic acid but also various shorter chain (e.g. lauric acid) and/or longer chain (e.g. docosahexaenoic acid) fatty acids. They can also ω-hydroxylate and thereby reduce the activity of various fatty acid metabolites (e.g. LTB4, 5-HETE, 5-oxo-eicosatetraenoic acid, 12-HETE, and several prostaglandins) that regulate inflammation, vascular responses, and other reactions. This metabolism-induced inactivation may underlie the proposed roles of the cytochromes in dampening inflammatory responses and the reported associations of certain CYP4F2 and CYP4F3 single nucleotide variants with human Krohn's disease and Coeliac disease, respectively. While many of the effects and diseases associated with the over- or under-expression, pharmacological inhibition, and single nucleotide or mutant variants of the cytochrome ω-hydroxylases have been attributed to their impact on 20-HETE production, the influence of these alternate metabolic actions have frequently not been defined.
Metabolism Glucuronidation of 20-HETE by UDP-glucuronosyltransferases (UGTs) is thought to be a primary pathway of 20-HETE elimination and thereby inactivation in humans.
There are several other pathways that metabolize 20-HETE. Human platelets and other tissues metabolize it via cyclooxygenase(s) to form the 20-hydroxy analogs of prostaglandin G2, thromboxane A2, thromboxane B2 and to 11(R)-hydroperoxy-20-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid which is rapidly reduced to 11,20-dihydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid; they also metabolize it through 12-lipoxygenase to form 12(S)-hydroperoxy-20-hydroxy-5Z,8Z,101E,14Z-eicosatetraenoic acid which is also rapidly reduced to 12,20-dihydroxy-5Z,8Z,101E,14Z-eicosatetraenoic acid. (The chirality of the hydroperoxy and hydroxyl residues at positions 11 and 12 in the eicosatetraenoic acids are predicted based on studies defining the chirality of the arachidonic metabolites made by these enzymes.) Since the prostaglandin and thromboxane metabolites of 20-HETE lack the platelet-stimulating activities to their prostaglandin and thromboxane precursors and since the 12-hydroxy and 11-hydroxy metabolites of 20-HETE may also be inactive, these metabolic pathways appear to function in inactivating 20-HETE with respect to the platelet system. However, the 20-hydroxy prostaglandin metabolites are able to contract rat aorta rings and thereby could contribute to the hypertensive actions of 20-HETE.
Cultured smooth muscle and endothelial cells from mouse brain microvasculature oxidize 20-HETE to its 20-carboxy analog, 20-carboxy- 5Z,8Z,11Z,14Z-eicosatetraenoic acid, then to 18-carboxy-5Z,8Z,10Z,14Z-octadecatetraenoic acid, and then to the further chain-shortened dicarboxylic acid, 16-carboxy-5Z,8Z,10E-hexadecatrrenoic acid, in a series of Beta oxidation reactions. These shortening pathways also are likely to serve in inactivating 20-HETE, although the initial product of this shortening pathway, 20-carboxy-HETE, dilates coronary microvessels in the pig heart and thereby could serve to antagonize the vasoconstrictor actions of 20-HETE, at least in this organ and species. Coronary artery endothelial cells isolated from pigs incorporate 20-HETE primarily into the sn-2 position of phospholipids through a coenzyme A-dependent process. It is likely, although not yet shown, that these mouse and pig 20-HETE metabolizing pathways also occur in humans.
Tissue distribution of 20-HETE-producing enzymes and/or activity
20-HETE-synthesizing enzymes are widely distributed to liver, kidney, brain, lung, intestine and blood vessels. In most vascular systems, 20-HETE synthesizing activity is limited to vascular smooth muscle of small blood vessels with little or no such activity in the vessel's endothelial cells or in large blood vessels. However, both the smooth muscle and endothelial cells obtained from mouse brain microvasculature, produce 20-HETE in culture.
20-HETE is produced by human neutrophils and platelets and by the ascending tubule cells in the medulla as well the pre-glomerular arterioles and certain other localized areas of the rabbit kidney.
Vascular-related activities
Rodent studies Blood vessel contraction In various rodent models, 20-HETE, at low concentrations (<50 nanomolar), acts to constrict arteries by sensitizing (i.e. increasing) the contraction responses of these artery's smooth muscle cells to other contracting agents such as alpha adrenergic agonists, vasopressin, endothelin, and a product of renin angiotensin system, angiotensin II. 20-HETE has a particularly complex interaction with the renin angiotensin system: angiotensin II stimulates the preglomerular microvessels of the rat kidney to produce 20-HETE; this production is required for angiotensin II to exert its full constrictor effects; and 20-HETE induces transcription of the enzyme which converts angiotensin I to angiotensin II, i.e. angiotensin-converting enzyme. Other agents such as Androgens and alpha adrenergic compounds such as norepinephrine. likewise stimulate 20-HETE production and have vasoconstrictive actions which are enhanced by 20-HETE. These circular or positive feedback interactions may serve to perpetuate vasoconstrictor responses.
Again in rodent models, 20-HETE acts to block Calcium-activated potassium channels to promote the entry of ionic calcium into vascular smooth muscle cells through the L-type calcium channel; the attendant rise in intracellular calcium triggers these muscles to contract.
Studies in rats also indicate that in vascular endothelial cells 20-HETE inhibits the association of the nitric oxide-producing enzyme, endothelial nitric oxide synthase (eNOS) with heat shock protein 90; this inhibits the ability of eNOS to become activated. The endothelial cells become dysfunctional in exhibiting decreased ability to produce the vasodilating agent, nitric oxide, and in containing elevated levels of a potentially injurious oxygen radical, superoxide anion; the blood vessels to which these dysfunctional endothelial cells belong are less able to dilate in response to the vasodilator, acetylcholine.
20-HETE can also constrict rodent (and human) artery preparations by directly activating the receptor for thromboxane A2. While significantly less potent than thromboxane A2 in activating this receptor, studies on rat and human cerebral artery preparations indicate that increased blood flow through these arteries triggers production of 20-HETE which in turn binds to thromboxane receptors to constrict these vessels and thereby reduce their blood blow. Acting in the latter capacity, 20-HETE, it is proposed, functions as a mediator regulating blood flow to the brain.