I. INTRODUCTION

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It has long been recognized that cyclooxygenase (COX) and lipoxygenase enzymes metabolize arachidonic acid (AA) to 5-, 12- and 15-hydroxyeicosatetraenoic acid (HETE), prostaglandins, prostacyclin, thromboxane, and leukotrienes (Fig. 1). These products modulate renal and pulmonary function, vascular tone, and inflammatory responses (155, 293, 303, 393). However, 20 years ago a third pathway for the metabolism of AA emerged. At this time, Capdevila et al. (51), Morrison and Pascoe (317), and Oliw et al. (340) reported that AA is also metabolized via cytochrome P-450 (CYP) enzymes in the liver and kidney to epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatetraenoic acids (DiHETEs). Subsequent work revealed that CYP enzymes in other extrahepatic tissues also catalyze the formation of 19- and 20-hydroxyeicosatetraenoic acids (19- and 20-HETE) and 7-, 10-, 12-, 13-, 15-, 16-, 17-, and 18-HETEs from AA (Fig. 1). These observations were largely ignored for many years due to the focus on the COX and lipoxygenase pathways, the limited availability of CYP metabolites of AA, and the lack of selective inhibitors of CYP enzymes. However, a decade ago, three key observations rekindled interest in the role of CYP metabolites of AA in the control of renal function and vascular tone. First, Iwai and Ingami (210) identified CYP4A2 as a gene that is regulated by salt intake and is overexpressed in the kidney of spontaneously hypertensive rats (SHR). Second, Sacerdoti et al. (406) and Omata and co-workers (342, 343) reported that the production of 20-HETE was elevated in the kidneys of the SHR. Finally, several investigators demonstrated that agents that induce heme oxygenase and reduce the renal formation of 20-HETE prevent the development hypertension in the SHR (104, 268, 409). These observations led to the hypothesis that an elevation in the renal production of 20-HETE may play a role in the development of hypertension (303). This proposal compelled a number of investigators to examine the role of CYP metabolites of AA in the regulation of renal function, vascular tone, and the long-term control of arterial pressure.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Pathways for the metabolism of arachidonic acid. Arachidonic acid is metabolized via the cyclooxygenase, lipoxygenase, and cytochrome P-450 (CYP) enzymes to prostaglandins (PG), prostacyclin (PGI2), thromboxane A2 (TxA2), or a series of hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (EETs), and dihydroxyeicosatrienoic acids (DiHETEs).

Remarkable progress has been made in the last 5 years. The work has been facilitated by the discovery of selective inhibitors of the formation of EETs and 20-HETE (9, 11, 39, 243, 300, 332, 427, 429, 475, 529) and by Dr. Camille Falck (University of Texas, Dallas, TX) who synthesized CYP metabolites of AA and freely distributed these compounds to investigators around the world. This area of research has entered an exponential growth phase similar to that seen in the nitric oxide (NO) field around 1990. More than 300 papers on biologic actions of CYP metabolites of AA have been published in the last 5 years. It now is now apparent that AA is primarily metabolized by CYP enzymes to EETs, DiHETEs, and 20-HETE in small blood vessels (155, 156, 203) and in the kidney (196, 205, 207, 342, 343), liver (512), lung (34, 211, 301, 513, 520), intestines (510), heart (68, 416, 502, 503), pancreas (416, 509), and white blood cells (29, 172, 402). These metabolites play an important role as paracrine factors and second messengers in the regulation of pulmonary, cardiac, renal, and vascular function (48, 212, 303, 378, 381, 393, 513) and modulate inflammatory (335) and growth responses (69, 70, 162, 272, 320, 321, 464). The focus of this review is to summarize the pathways for the formation of EETs, DiHETEs, and 20-HETE and the evidence that these products contribute to the regulation of renal and cardiovascular function.

	II. METABOLISM OF ARACHIDONIC ACID BY CYTOCHROME P-450 ENZYMES

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CYP genes encode for membrane-bound, heme-containing enzymes that catalyze NADPH-dependent oxidation of drugs, chemicals, and carcinogens (331, 416). CYP enzymes require NADPH reductase and CYPb₅ (NADH/NADPH oxidase) as cofactors (53). Because CYPb₅ oxidase is a major source of superoxide ions, there is considerable interest in determining whether the metabolism of AA by CYP enzymes contributes to oxidative stress. Indeed, there is evidence that the metabolism of various substrates by CYP enzymes leads to the formation of O radicals in the liver (37, 373). More recently, inhibitors of CYP2C9 were found to block the formation of O radicals by cultured porcine coronary artery endothelial cells (123). These studies suggest the metabolism of AA by CYP enzymes is a significant source of oxygen radicals in the vasculature.

More than 500 CYP genes have been identified and categorized by sequence homology (331). These genes are subdivided into 78 families. Fourteen of these families (29 subfamilies) are expressed in mammalian tissue. Up to 50 different CYP enzymes are expressed in a given species. Most of these enzymes are expressed in the liver and are involved in the metabolism of drugs and foreign chemicals. As such, CYP enzymes have been largely the province of biochemists and toxicologists. However, there has been a growing appreciation that many CYP enzymes are expressed in extrahepatic tissues and that they metabolize endogenous substrates like vitamins, steroids, and fatty acids, including AA. This review focuses on the formation of and cardiovascular actions of CYP metabolites of AA because most of the published work has focused on these metabolites. It is important to recognize that CYP enzymes metabolize other fatty acids (linolenic, linoleic, palmitic acid) as well or better than AA. The biologic actions of CYP metabolites of other fatty acids are unknown. However, it is likely that they are just as important as CYP metabolites of AA in the control of renal and cardiovascular function and will become a major focus of this field in the future.

Enzymes of the CYP4A, 4B, and 4F families catalyze the -hydroxylation of fatty acids. Several isoforms in these families produce 20-HETE when incubated with AA (144, 160, 240, 241, 370, 441, 431). These closely related gene families arose from ancient gene duplication, since the CYP4A and 4B gene families map to a conserved gene cluster on a single 450-kb YAC clone on chromosomes 4 in the mouse (169) and the homologous regions on chromosome 5 in the rat and chromosome 1 in humans. The 4F family appears to be a more ancient gene family that maps to a different region on chromosome 19 in humans (31).

cDNAs encoding for 15 different isoforms of the CYP4A family have been identified in various species (331). A summary of the isoforms responsible for the -hydroxylation of fatty acids in various species is presented in Table 1. CYP4A11, 4F2 (239, 264, 370), 4F11 (84), and 4F12 (44) are expressed in human liver and kidney. The substrate specificity of the CYP4F11 enzyme is unknown. CYP4F12 catalyzes the conversion of AA to 18-HETE (44). CYP4F2 appears to be the enzyme primarily responsible for the formation of 20-HETE in human kidney (370). This conclusion is based on the finding that a CYP4F2 antibody lowered the formation 20-HETE by microsomes prepared from human kidney by 67%, whereas a CYP4A11 antibody only reduced the formation of 20-HETE by 32% (370). Another member of the CYP4F family, CYP4F3, is highly expressed in human polymorphonuclear leukocytes (PMNs) (237). It catalyzes the -hydroxylation of leukotrienes. However, it remains to be determined if CYP4F3 is the source of 20-HETE produced by human PMNs (29).

View this table: [in this window] [in a new window]   Table 1. -Hydroxylases

Four isoforms of the CYP4A gene family, CYP 4A1, 4A2, 4A3, and 4A8, are expressed in the liver, kidney, and brain of rats (144, 160, 168, 240, 241, 431, 441, 452) (Table 1). Enzymes of the CYP4F family (CYP4F1, 4F4, 4F5, 4F6) have also been cloned from rat tissues (226, 227, 238). CYP4F4-6 enzymes are expressed in the kidney, but the ability of these isoforms to metabolize AA has not been investigated. Recombinant CYP 4A1, 4A2, and 4A3 enzymes, but not 4A8, have been reported to produce 20-HETE when incubated with AA (Table 1) (332, 443, 444, 477). In contrast, there is one recent report indicating that CYP4A8 also produces 20-HETE when incubated with AA (176). The catalytic activity of the CYP4A1 enzyme for AA is 10 times greater than that of CYP4A2 or 4A3 (332). However, the results of RT-PCR and Western blot experiments suggest that CYP4A2 is the isoform that is constitutively expressed in the liver, kidney, and vasculature of rats (205, 452). Message and protein levels for CYP4A1 are relatively low in these tissues unless the expression of this isoform has been induced by fasting (376), diabetes, or fibrates (140, 205, 207). Messages for the CYP4A2 and 4A3 isoforms are also expressed in small arterioles in the kidney (203, 207), brain (140, 156), and skeletal muscle beds (255) of rats. In contrast, CYP4A1 message levels are very low in vessels isolated from the kidney (203, 205), even though it is easy to detect this isoform by RT-PCR in vessels microdissected from the cremaster muscle of the rat (159) and cerebral arteries of rats (140) and cats (141). Thus the isoforms that underlie the formation of 20-HETE in the peripheral vasculature and kidney remain in question. This issue is further clouded since there are no antibodies that can distinguish between CYP4A isoforms. There are also posttranslational modifications of the CYP4A proteins that make definitive identification of the isoforms on Western blots difficult. For example, CYP4A proteins can be glycosylated (256), and there is considerable evidence that the activity of CYP enzymes is regulated by phosphorylation via cAMP-dependent protein kinases (337). Finally, the high degree of homology between the CYP4A isoforms limits the usefulness of cDNA probes in Northern blots for measuring changes in the expression of these isoforms. The only technique that can specifically distinguish between the different CYP4A isoforms is RT-PCR (205).

Unlike CYP4A1 that only catalyzes the -hydroxylation of fatty acids, CYP4A2 and 4A3 can also produce EETs when incubated with AA (332, 477). However, this conclusion remains controversial in that Helvig et al. (168) and Hoch et al. (176) failed to confirm that recombinant CYP4A2 and 4A3 enzymes produce EETs. This discrepancy may be due to polymorphisms of the CYP4A isoforms between strains of rats, since the sequence of the CYP4A cDNAs that have been cloned and expressed by these groups are slightly different.

In rabbits, CYP4A4, 4A5, 4A6, and 4A7 all catalyze the -hydroxylation of fatty acids (Table 1) (360, 391). CYP4A6 and 4A7 also metabolize AA to 20-HETE (391), but the rate of reaction is half that seen when the shorter chain unsaturated fatty acid lauric acid is utilized as the substrate. The ability of CYP4A7 to metabolize AA is greatly enhanced by addition of cytochrome b₅ (284). In the presences of cytochrome b₅, the catalytic activity of CYP4A7 for AA exceeds that for other fatty acids (284). CYP4A5 exhibits little activity for AA, but it readily metabolizes lauric and palmitic acids (185, 391). CYP4A4 exhibits little activity toward lauric acid. It favors AA as a substrate (391). However, the rate of this reaction is only 20% of that seen when prostaglandin E₁ is used as a substrate for the reaction (391).

The expression of CYP4A6 is induced in the liver and kidney of rabbits by fibrates in a manner similar to the effect of fibrates on the CYP4A1 isoform in rats. The expression of CYP4A7 is induced to a lesser extent by fibrates. In this way, CYP4A7 resembles the CYP4A2 and 4A3 isoforms in rats (391). The expression of the CYP4A4 isoform is constitutively expressed at low levels in the liver and lung of control animals, but it is markedly induced by 20-fold during pregnancy (318, 360). It is also induced by progesterone and dexamethasone (298). The functional significance of the remarkable induction in the expression of CYP4A protein and enzyme activity during pregnancy remains to be determined.

CYP4A10 and CYP4A12 are the enzymes primarily responsible for the formation of 20-HETE in the liver and kidney of mice (Table 1) (31). CYP4A14 is also expressed in the kidney (184). However, this isoform favors the metabolism of short-chained, unsaturated fatty acids and exhibits little activity toward AA (184). Kikuta et al. (236) and Cui et al. (83) recently cloned members of the CYP4F family (CYP4F14, 4F15 and 4F16) from the liver of mice that catalyze the -hydroxylation of leukotrienes, prostaglandins, and lipoxins. Two other members of this family, CYP4F15 and 4F16, are expressed in murine liver and kidney and are induced by NO and fibrates (83). CYP4B1 is also another potential fatty acid -hydroxylase that is expressed in the kidney of mice (192). However, the importance of enzymes of the CYP4F and 4B families to the generation of 20-HETE in the liver and kidney has not been determined. CYP2J9 has recently been cloned from the brain of mice (375). This enzyme catalyzes the formation of 19-HETE from AA, and it is highly expressed in Purkinje cells. Because 19-HETE inhibits Ca²⁺ channel activity in Purkinje cells, a role for this enzyme in neurotransmitter release has been postulated (375).

The expression of CYP4A10 and CYP4A14 is induced by fibrates in murine liver and kidney similar to the CYP4A1 and 4A3 in rats (31, 184). CYP4A12 is constitutively expressed in the liver and the kidney of mice. The expression of CYP4A12 is weakly induced by fibrates (31). It is also differentially expressed in the liver and kidney of male versus female mice (169). In this way, its expression resembles that of CYP4A2 in rats (205, 452).

The formation of EETs from AA is very promiscuous. CYP enzymes of the P-450 1A, 2B, 2C, 2D, 2E, 2J, and 4A families (Table 2) have all been reported to catalyze the formation of EETs in various tissues and species (228, 243, 259, 288, 293, 387). CYP2C8, 9, and 19 as well as CYP1A2, 2J2, and CYP2B6 are all expressed in human liver and produce EETs when incubated with AA (46, 86, 387). The CYP2C8 isoform produces 14,15- and 11,12-EETs in a ratio of 1.25:1 (86). CYP2C9 produces 14,15-, 11,12-, and 8,9-EETs in a ratio of 2:1:1, whereas the CYP2J2 isoform produces 5,6-, 8,9-, 11,12-, and 14,15-EETs with equal efficiency (416, 503). Studies using the CYP2C9 inhibitor sulfaphenazole and the CYP1A/3A inhibitor -naphthoflavone suggest that CYP2C9 is responsible for 50% of the epoxygenase activity in human liver and that enzymes of the CYP1A and 3A families contribute 25% to this activity (387). On the other hand, this view has been challenged by the results of a recent study suggesting that CYP2C8 is the primary epoxygenase expressed in human liver (512). In this experiment, immunoprecipitation of human liver microsomes with a CYP2C8 antibody reduced the formation of EETs by 85% (512).

CYP2J2 has emerged as one of the major enzymes involved in the formation of EETs in extrahepatic tissues in humans (503, 510, 511, 513, 515). Wu et al. (503) first reported that this isoform is highly expressed in cardiac myocytes. CYP2J2 is also expressed in the proximal tubule and medullary collecting duct in the kidney (288); -, -, -, and polypeptide-containing cells in the pancreas (509); in the lung (511); in the small intestine, and in the colon (503). This enzyme serves as the primary enzyme that catalyzes the formation of EETs in the heart. EETs diminish cardiac contractility by inhibiting the opening of sodium channels (265) and L-type calcium channels (68). CYP2J2 is also the enzyme primarily responsible for the formation of EETs in the pancreas of humans (509). In this tissue, EETs influence the secretion of insulin and glucagon (106). In the kidney, EETs regulate renal hemodynamics and inhibit sodium transport (213, 291, 381, 399, 412); however, the relative importance of CYP2J2 versus enzymes of the 2C and 4A families in generating EETs in human kidney has yet to be determined. Finally, EETs decrease bronchiolar tone and vascular resistance in human lung (212) (see sect. VII); however, little is known to what extent CYP2J enzymes versus other isoforms contribute to the production of EETs in the lung (383, 519, 521).

Human endothelial cells express enzymes of the CYP2C8, 2C9 (120, 273), 2J2 (335), 3A (178), and 2B1 (119, 178) families that all produce EETs. EETs dilate human coronary arteries (309, 310). They act by hyperpolarizing VSM by opening K⁺ channels and are a prime candidate for endothelial-dependent hyperpolarizing factor (EDHF) (48, 315). The recent findings that the vasodilator responses to acetylcholine and bradykinin are potentiated by an inducer of CYP2C8/9 protein (-naphthoflavone) (369) and are inhibited by sulfaphenazole (295) and antisense CYP2C8/9 oligonucleotides (120) suggest that CYP2C8 and 2C9 are likely the major isoforms responsible for the formation of EETs in human endothelial cells.

Enzymes of the CYP2C11 (159, 180, 451), 2C13 (507), 2C23 (180, 194, 223, 294), 2C24 (180), 2J3 (503), 2J4 (513), 2J10 (507), 2B1 and 2B2 (259), 2D18 (458), 2E1 (258), and 1A1 and 1A2 (387) families all produce EETs in rats (Table 2). CYP2C11 and 2J3 probably serve as the primary epoxygenases in the liver of male rats (perhaps CYP2C12 in females). However, the expression of CYP1A, 2B, and 2E families in the liver can be induced by hydrocarbons, barbiturates, and ethanol, respectively. As a result, these isoforms can become the major source of EETs in the liver and other tissues after exposure of rats to drugs and xenobiotics.

CYP2C11, 2C12, 2C23, 2C24, 2J3, 2J4, and 2J10 are expressed in the kidney of rats (Table 2). Holla et al. (180) concluded that CYP2C23 is the major epoxygenase enzyme expressed in the kidney of rats. More recently, a CYP2J enzyme has been reported to be highly expressed in the proximal tubule and collecting duct of rats (288). It is likely to also contribute to the formation of EETs in the kidney. The recent report that the production of EETs is elevated in the kidney of SHR and that this is associated with an increased expression of a protein that cross-reacts with a CYP2J antibody supports this view (507).

CYP2C11 protein is highly expressed in astrocytes in the brain of rats (7). EETs are formed by this cell type (7, 18), and they have been implicated in mediating reactive hyperemia in the brain (155) and the cerebral vasodilator response to glutamate (6). Recently, Thompson et al. (458) reported that CYP2D18 is also expressed in the brain of rats. The CYP2C18 enzyme that normally converts dopamine to aminochrome also catalyzes the formation of EETs from AA. The functional significance of this observation to control of neural function, reactive hyperemia, and the destruction and death of dopaminergic neurons in Parkinson disease are likely to become important new areas of investigation.

CYP2J3 is expressed in the heart, pancreas, liver, lung, and kidney of the rat. It appears to be the functional homolog of CYP2J2 enzyme expressed in humans. However, CYP2J3 does exhibit some differences with regard to the stereospecificity of the EETs produced. CYP2J3 favors the production of 14(S),15(R)- and 11(R),12(S)-EETs (502), whereas the 2J2 isoform produces 14(R), 15(S)- and a racemic mixture of 11,12-EETs (503). CYP2J4 is also expressed in the small intestine and liver of rats, but the importance of this isoform to the formation of EETs in these organs remains to be established (515).

Despite the fact that much of the work on endothelial-derived vasodilators and EDHF has been performed in rats, little is known about the CYP isoforms that produce EETs in rat endothelial cells. Message for CYP2C11 can be detected in cerebral, renal, splachnic, and skeletal muscle arterioles in rats (personal observation). CYP2J3 protein is also expressed in rat vascular endothelial cells (335). However, no one has determined the relative contribution of the various CYP isoforms to the production of EETs, nor is it known if the expression of CYP isoforms in the endothelium is altered by hypertension, diabetes, or hyperlipidemia. This information is critical since CYP isoforms exhibit different regio- and stereospecificity with regard to the type of EETs formed and the vascular response to EETs in some beds is both regio- and stereospecific (143, 200, 526).

CYP2C29, 2C38, 2C39, and 2C40 enzymes are expressed in the liver, kidney, and brain of mice (Table 2) (287). CYP2C38 is the most active epoxygenase. It preferentially metabolizes AA to 11,12-EET. CYP2C29, 2C39, and 2C40 are less active and produce 14,15-EET. CYP2B19 is expressed in the skin of mice. This enzyme produces EETs and 11-, 12-, and 15-HETE (228). CYP2J5 is highly expressed in the proximal tubule and collecting ducts in the kidney of mice (288). It produces 14,15-, 11,12-, and 8,9-EETs in a ratio of 2:1:1 when incubated with AA. More recently, cDNAs for CYP2J6, 7, 8, and 9 have also been cloned from the mouse. However, little is known about the expression or function of these isoforms (416).

CYP2C1, 2C4, and 2CAA are epoxygenases expressed in the liver of rabbits (Table 2) (85, 260). CYP2B, 2E, 2J, 1A, 2C, and 4A proteins are expressed in the pulmonary artery and peripheral lung tissue of rabbits (519). All of these isoforms produce EETs when incubated with AA. However, the observations that CYP2B4 inhibitors and antibodies completely inhibit the formation of EETs in rabbit lung (244, 513) suggest that CYP2B4 is probably the primary epoxygenase expressed in this tissue.

AA is also metabolized to 7-, 8-, 9-, 10-, 11-, 12-, 13-, 15-, 16-, 17-, and 18-HETE by CYP enzymes (Table 3). Brash et al. (40) reported that 7-, 10-, and 13-HETE are major CYP metabolites of AA produced in liver microsomes. Carroll and co-workers (57, 59) reported that 16-, 17-, and 18-HETE are produced in the kidney. They are incorporated into tissue phospholipid pools in the kidney, and high concentrations (ng/ml) of these compounds are excreted in the urine. Moreover, the release of 16-, 17-, and 18-HETEs from isolated perfused rabbit kidneys is stimulated by ANG II and other vasoconstrictors that stimulate phospholipases (59). Finally, Bednar and co-workers (29, 30) identified 16-HETE as one of the major metabolites of AA produced by human PMNs. The other major metabolite formed is 20-HETE (29, 172). The potential biologic importance of the myriad of HETEs formed by CYP isoforms has been highlighted by the recent discovery that structural analogs of 20-HETE, like 5-, 15-, and 19-HETE (likely 16-, 17-, and 18-HETE as well), serve as competitive antagonists of the vasoconstrictor actions of 20-HETE (9). Because 15-, 16-, and 19-HETE are produced in vivo, they could serve as endogenous inhibitors of the actions of 20-HETE.

A summary of the isoforms responsible for the formation of the bisallylic and subterminal hydroxyl metabolites of AA is presented in Table 3. CYP1A2 and 3A4 enzymes catalyze the formation of 7-, 10-, and 13-HETE from AA in human liver (40, 46). In addition to catalyzing the formation of EETs, CYP2C8 also catalyzes the formation of 11-, 13-, and 15-HETE (46), whereas CYP2C9 catalyzes the formation of 12- and 13-HETE (46). CYP4F8 (45) and CYP4F12 (44) catalyze the formation of 18-HETE in humans. CYP4F12 is expressed in the liver, kidney, small intestine, and heart (44). Finally, CYP2C37 and CYP2C40 have recently been cloned from the liver of the mouse (287). CYP2C37 produces 12-HETE (287), whereas CYP2C40 catalyzes the formation of 16-HETE (462).

A summary of the metabolic fates of EETs, DiHETEs, and 19- and 20-HETEs in various tissues is presented in Figure 2. EETs are converted by soluble or microsomal epoxide hydroxylases (sEH or mEH) to the corresponding DiHETEs. In renal arterioles, EETs are potent vasodilators, whereas the corresponding DiHETEs are much less active (200). Thus the conversion of EETs to DiHETEs is thought to limit the actions of EETs. The exception to this may be in the coronary microcirculation, where DiHETEs and EETs are equipotent as dilators of porcine coronary arteries (109, 341). There are strain differences in the expression of epoxide hydrolase enzymes that affect the ratio of EETs to DiHETEs produced in various tissues. For example, microsomes prepared from the kidneys of SHR produce DiHETEs, whereas those prepared from the kidneys of Wistar-Kyoto (WKY) rats produce EETs (142, 196). More recently, Yu et al. (507) have reported that the expression of sEH is elevated in the kidneys of SHR. They also found that administration of an inhibitor of sEH increases the renal formation of EETs and lowers blood pressure in SHR. Further evidence for a role for sEH and EETs in the control of blood pressure has been provided by Sinal et al. (432). They found that knockout of the sEH gene increased the renal formation of EETs and lowers blood pressure by 13 mmHg in male mice (432).

View larger version (15K): [in this window] [in a new window]   Fig. 2. Metabolic fate of cytochrome P-450 metabolites of arachidonic acid (AA). Once formed, epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (HETE) are metabolized by -oxidation to 18- and 16-carbon derivatives, which are less biologically active. 5,6-EET, 8,9-EET, and 20-HETE can also be metabolized by cyclooxygenase (COX) to vasoconstrictor endoperoxides or to vasodilator prostaglandin or prostacyclin-like derivatives. EETs, DiHETEs, and 20-HETE are reincorporated into membrane phospholipid pools. They can be released again through the actions of agents that activate phospholipases. All of these fatty acids are also avidly bound by plasma proteins, which limits their biologic activity in the circulation and restricts their distribution to plasma when given via a blood-borne route of administration.

EETs, DiHETEs, and 19- and 20-HETE predominately act as autocrine factors. There are several arguments that support this view. First, these compounds are very lipophilic and are found in higher concentrations within tissue than in plasma, urine, or interstitial fluid. Second, EETs and 20-HETE avidly bind to proteins (492). The binding of these compounds to albumin likely restricts the distribution of these compounds to the vascular space when they are given systemically. In this regard, EETs and 20-HETE readily alter vascular tone and epithelial transport when studied in vitro. However, it is extremely difficult to demonstrate that they have any effect on renal function or vascular tone when given systemically because of protein binding. Finally, EETs, HETEs, and 20-HETE are rapidly incorporated into membrane phospholipids in many cell types (468, 469, 486). Karara and co-workers (221, 222) reported that the concentration of EETs incorporated in lipids extracted from human kidney is in the micromolar range. Similar values have also been reported in kidneys of rabbits (107) and rats (453). High concentrations of EETs have also been reported in phospholipids extracted from human platelets (523), VSM cells (108), vascular endothelial cells (468, 469), human and rat hearts (265, 502, 503), and the brain (100). Carroll et al. (57) demonstrated that 16-, 17-, 18-, 19-, and 20-HETE are incorporated in phospholipids extracted from the kidney of rabbits. EETs and 20-HETE are released from membrane phospholipids in response to vasoactive hormones and other stimuli that activate phospholipases. Weintrab et al. (486) reported that bradykinin releases EETs and DiHETEs from endothelial cells, and they contribute to the vasodilator response to bradykinin in porcine coronary arteries. Similarly, Carroll et al. (57) reported that ANG II increases the release of 16-, 17-, 18-, 19-, and 20-HETE into the urine and renal venous effluent of isolated perfused rabbit kidneys even after blockade of the synthesis of 20-HETE with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS). Thus it may take considerable time to fully deplete tissue pools of EETs and 20-HETE after administration of CYP inhibitors. This concept has been largely ignored by most investigators studying the effects of CYP inhibitors on renal function and vascular tone and will likely have major impact on the interpretation of future studies.

A further demonstration that the release of EETs from membrane phospholipids contributes to the vasodilator response to bradykinin in canine coronary arteries has recently been provided by Mombouli et al. (314). They found that the epoxygenase inhibitor miconazole prevented the vasodilator response to AA in canine coronary arteries, indicating the dose of this agent was sufficient to completely block the synthesis of EETs from AA. Miconazole had no effect on the vasodilator response to bradykinin in this same study. The response to bradykinin was blocked by an inhibitor of phospholipase A₂. Moreover, the vasodilator response to repeated challenges with bradykinin gradually fell as the tissue pool of EETs became depleted in vessels treated with miconazole. Mombouli et al. (314) suggested that the failure of miconazole to acutely block the vasodilator response to bradykinin was due to the sustained release of EETs from membrane phospholipid pools. The results of this study are extremely important because they illustrate that finding that a CYP inhibitor has no effect on a vascular response is not sufficient to conclude that CYP metabolites of AA play no role in mediating that response.

EETs and HETEs are rapidly metabolized by -oxidation to 18- and 16-carbon products (108, 110, 112) that are generally less active than the parent compounds. They are also excellent substrates for chain elongation (110). In many tissues, EETs and HETEs can be further metabolized by CYP, lipoxygenase, and cyclooxygenase enzymes. For example, we reported that 11,12- and 14,15-EETs are metabolized by CYP4A enzymes in the kidney of Dahl rats to 20-hydroxy-11,12- and 14,15-EET (290). Others have described the formation of trihydroxy metabolites of AA that can arise from the metabolism of EETs by lipoxygenase or cyclooxygenase enzymes (317, 340).

The metabolism of EETs and 20-HETE by COX also plays a role in modifying the biologic actions of these metabolites. For example, 5,6-EETs can be metabolized by COX to both vasodilator and vasoconstrictor compounds. 5,6-EET constricts the renal circulation of rats both in vivo (453) and in vitro (60, 200). This effect is blocked by COX inhibitors and thromboxane receptor antagonists and is mediated by the formation of an endoperoxide intermediate. Similarly, Schwartzman et al. (421) and Escalante and co-workers (103, 105) have reported that the vasoconstrictor response to 20-HETE in peripheral arteries of rats and rabbits is blocked by indomethacin and endoperoxide receptor antagonists, suggesting it is related to the formation of endoperoxides.

The metabolism of CYP metabolites of AA by COX appears to be both tissue and species specific. For example, 5,6-EET dilates the isolated perfused rabbit kidney (58). This effect is COX dependent and thought to be related to the formation of 20-hydroxy-PGE₁ or PGI₂ by the endothelium. On the other hand, 20-HETE constricts in vitro perfused rabbit afferent arterioles preconstricted with phenylephrine (23). Only a small part of this response was blocked by indomethacin. Arima et al. (23) concluded that the vasoconstrictor response to 20-HETE in the rabbit afferent arteriole is due to a direct effect of 20-HETE on VSM and a minor component that is dependent on the metabolism of 20-HETE by COX to a thromboxane/endoperoxide metabolite.

It is also important to recognize that 5,6- and 8,9-EETs, 20-HETE, and subterminal HETEs (16-, 17-, 18-, and 19-HETE) are good substrates for COX. Thus the biologic responses to CYP metabolites of AA are a function of their direct effects on the target tissue as well as those of any COX metabolites formed that augment or oppose the actions of the parent compound. Moreover, experimental conditions alter the expression of COX in various tissues. This will change the types and amounts of COX metabolites of EETs and 20-HETE formed and modify the overall vascular response to these compounds.

In addition to being metabolized by COX and lipoxygenase enzymes, 20-HETE and EETs can affect the release of COX and/or lipoxygenase metabolites from tissues. For example, 20-HETE has been reported to dilate canine coronary arteries by stimulating the release of prostacyclin from the endothelium (371). Finally, it should be noted that most of the antibodies used to measure COX and lipoxygenase metabolites of AA cross-react with 20-hydroxy metabolites of the parent compounds (personal observations). Thus much of the previous work attributing the actions of COX inhibitors to blockade of the formation of prostaglandins, thromboxane, and prostacyclin needs to be reevaluated to determine if some of the actions of these agents are due to inhibition of the formation of COX metabolites of EETs and 20-HETE.

Many factors influence the expression of the CYP enzymes that metabolize AA. CYP4A1 and 4A3 mRNA and protein are highly expressed in the kidney of neonatal rats, but the levels decline in adulthood (254, 342). In contrast, CYP4A2 protein is not expressed in the kidney of neonates, but the levels increase with age until it becomes the dominant isoform expressed in the kidney of adult rats (342). Because 20-HETE is a potent mitogen (272, 321, 323), the upregulation of CYP4A1 expression (the most active isoform for the formation of 20-HETE) just after birth may contribute to the postnatal growth and development of the kidney.

In the proximal tubule, ANG II increases the formation of EETs (291, 381), whereas epidermal growth factor (272), dopamine (21, 336), and parathyroid hormone (PTH) (345, 386, 430) increase the formation of 20-HETE. In the thick ascending loop of Henle (TALH), a variety of peptide hormones increase the formation of 20-HETE (16, 145, 150, 286, 303, 481). The expression of CYP4A proteins in the kidney and vasculature falls in rats fed a high-salt diet (207, 210, 290, 439, 441), and this can be prevented if circulating ANG II levels are maintained by an intravenous infusion (11). In contrast, a high salt intake increases the expression of CYP2C23 and the formation of EETs in the kidney of rats (55, 294, 358). Because EETs inhibit sodium transport in the proximal tubule and collecting duct (291, 399, 412), upregulation of renal epoxygenase activity has been postulated to play an important role in the chronic adaptation of animals to a high-salt diet (293, 294) and in the development of salt-sensitive hypertension in humans (261) and rats (294). However, we have recently noted that there is considerable heterogeneity among inbred strains of rats regarding the effects of changes in salt intake on the expression of CYP enzymes in the kidney and the formation of EETs and 20-HETE (175). Thus it remains to be determined whether this reflects genetic mutations in CYP genes and whether these mutations are linked to differences in the salt sensitivity of blood pressure in different strains of rats.

The expression of CYP4A protein is increased by glucocorticoids, mineralocorticoids, and progesterone in the kidney and the lung (263, 298, 431). Antilipidemic agents, such as clofibrate, induce the expression of CYP4A1 and 4A3 and the synthesis of 20-HETE in the liver and kidney (20, 144, 160, 398, 431) by interacting with the peroxisome proliferator-activated receptor (PPAR)- receptor (149, 255). There is also evidence that chronic exposure to NO increases the expression of CYP4A mRNA and protein in the liver and kidney (208, 425, 426), even though NO itself inhibits the formation of EETs and 20-HETE (13, 8, 358, 446). In contrast, NO downregulates the formation of CYP2C isoforms that produce EETs (283, 425, 426). Other inducers of CYP enzymes, such as phenobarbital, 3-methylcholantherene, and 3,4-benzo(a)pyrene, do not alter renal CYP4A activity (431). Nearly all vasoconstrictor agents, i.e., ANG II, norepinephrine, vasopressin, and endothelin, activate phospholipases and increase the synthesis and release of 20-HETE in the kidney and VSM cells (57, 59, 82, 303, 350, 354, 470). The expression of CYP enzymes involved in the metabolism of AA is altered in diabetes (25, 193, 255), pregnancy (64, 144, 301, 298, 318), hepatorenal syndrome (407), cyclosporin- induced (43, 408, 506) and cisplatin-induced (325) nephrotoxicity, alcohol-induced liver disease (327), chronic renal disease (267), and various models of hypertension (104, 268, 290, 294, 343, 353, 381, 394, 398, 439, 441, 494). It is also altered by changes in dietary sodium (180, 207, 293, 294, 358) or potassium (62, 480, 514) intake and by fasting (255, 376). However, the role of 20-HETE in mediating the changes in renal function and/or vascular tone associated with these conditions has yet to be established. The patterns of expression of CYP isoforms of the 2C and 4A families differ in male versus female rats (205, 290, 451, 452); however, the functional significance of these observations remains to be determined.

F.  CYP Inhibitors and Inducers

1.  Nonselective inhibitors

The development of selective inhibitors and inducers of the formation of 20-HETE and EETs has been the catalyst driving the exponential growth of information regarding the roles of these substances in the control of cardiovascular function. A summary of the agents that have been used to manipulate the metabolism of AA is presented in Figure 3. The release of AA from membrane phospholipids can be blocked by the relatively nonspecific phospholipase inhibitors quinacrine or heparin or the more specific inhibitor of cytoplasm phospholipase A₂ arachidonyl-trifluoromethyl ketone (AACOF₃).

View larger version (24K): [in this window] [in a new window]   Fig. 3. Summary of agents available to induce or inhibit various pathways for the metabolism of arachidonic acid. The complete chemical names of the various drugs are presented in text. PLA2, phospholipase A2; EETs, epoxyeicosatrienoic acids; 20-HETE, 20-hydroxyeicosatetraenoic acid; DiHETEs, dihydroxyeicosatrienoic acids; NO, nitric oxide; CO, carbon monoxide.

Much of the information regarding CYP metabolites of AA in the control of renal and cardiovascular function has been deduced by comparing the effects of COX (indomethacin, meclofenamate) and lipoxygenase [baicalein, cinnamyl-3,4-dihydroxy--cyano-cinnamate (CDC)] inhibitors with those obtained after blockade of all three pathways for the metabolism of AA with 5,8,11,14-eicosatetraynoic acid (ETYA). Other approaches to reduce the formation of EETs and 20-HETE have been to use -diethyl-aminoethyldiphenylpropylacetate (SKF-525) that binds to heme in CYP enzymes (55) or heme arginate, SnCl₂, or CoCl₂ that induce heme oxygenase (104, 268, 350, 353, 354, 409) and reduce CYP activity by diminishing the availability of heme for incorporation into CYP enzymes. However, these agents are not very specific and reduce the activity of all CYP enzymes, including those involved in steroid metabolism. They also inhibit other heme-containing enzymes, like NO synthase. The interpretation of the results of studies using heme oxygenase inducers is further complicated since these agents stimulate the formation of carbon monoxide (CO), which inactivates both CYP and NOS enzymes and has direct effects on ion channels and vascular tone (479).

More specific suicide-substrate CYP inhibitors like 1-aminobenzotriazole (ABT) were developed (243, 300, 445) about 10 years ago. These agents have an advantage that they should only bind to and inactivate CYP isoforms that utilize these compounds as substrates. In this regard, ABT (50 mg/kg ip) has recently been reported to selectively inhibit enzymes of the CYP4A family and reduce the formation of 20-HETE in the kidney of rats (445). However, others have reported that ABT also blocks the activity of other CYP isoforms (CYP2B, 1A, 2C) that produce EETs in the liver and lung (243, 300). We have found that ABT (50 mg/kg ip) completely blocks the formation of EETs, DiHETEs, and 20-HETE in the kidney of rats within 2 h and reduces the 24-h excretion of 20-HETE by >50% (292). It also reduces the formation of 20-HETE and EETs in the liver and lung by ~50%. Even though ABT is nonselective in blocking the formation of EETs versus 20-HETE, it is still useful for evaluating the role of CYP metabolites of AA in mediating biologic responses in vivo, since it is inexpensive, readily available (Sigma), orally active, and well tolerated by rats for >2 wk (304). It also irreversibly inactivates CYP enzymes so that the organs can be collected and the tissue prepared, and one can assess the degree of inhibition of various CYP isoforms using in vitro assays.

Another useful agent is 17-octadecynoic acid (17-ODYA). This compound was synthesized by Dr. Ortiz de Montellano to be a specific suicide substrate inhibitor of the CYP4A enzymes that metabolize AA to 20-HETE (318, 348, 427). Subsequent studies demonstrated that 17-ODYA irreversibly blocks the formation of 20-HETE at concentrations >1 µM (529). Unfortunately, it is equally as effective in blocking the formation of EETs in various tissues (529).

The endogenous gaseous mediators NO and CO bind to heme and inactivate CYP enzymes. They completely block the formation of EETs and 20-HETE in renal and hepatic microsomes and renal and cerebral arteries at concentrations <100 nM (13, 446, 448). It is believed that they serve as modulators of the activity of CYP metabolism of AA in the kidney and peripheral vasculature in vivo. Indeed, many of the cGMP-independent actions of NO and CO on vascular tone and epithelial transport have been shown to be related to their ability to inhibit the endogenous formation of 20-HETE in various tissues (12, 13, 77, 276, 446, 448).

Dr. Falck synthesized 12,12-dibromododec-11-enamide (DBDD) and DDMS as a second generation of inhibitors to selectively block the formation of 20-HETE (8, 475). These compounds competitively inhibit the formation of 20-HETE by renal microsomes at concentrations <10 µM, whereas epoxygenase activity is only reduced by 10-20% (8). At higher concentrations (50 µM), however, they are equally effective at inhibiting the formation of EETs and 20-HETE. The main limitation in using these inhibitors for in vivo studies is that they are fatty acids that avidly bind to plasma proteins. This property limits their ability to diffuse into tissues and inhibit the formation of 20-HETE when given via a blood-borne route. Thus these compounds are only effective at inhibiting the formation of 20-HETE when added to protein-free solutions in vitro or when directly applied to tissues in vivo.

More recent studies have employed a new inhibitor of the formation of 20-HETE, N-hydroxy-N'-(4-butyl-2 methylphenyl)formamidine (HET-0016) (229, 311). This compound selectively inhibits the formation of 20-HETE at a concentration <10 nM. It has no effect on epoxygenase, COX, or lipoxygenase activity at concentrations up to 1 µM (311). At this concentration, it also has minimal effects on the activity of other CYP isoforms (2CYP2C9, 2D6, 3A4) involved in drug metabolism in humans (311). HET-0016 appears to be the most specific inhibitor of the synthesis of 20-HETE currently available. However, little is yet known about the biologic effects of HET-0016 in vivo or its potential side effects.

A number of analogs of 20-HETE, such as 5-, 15-, and 19-HETE, block the vasoconstrictor actions of 20-HETE in renal (9) and cerebral arteries (140). The most effective analog appears to be 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid [6(Z),15(Z)-20-HEDE or WIT-002]. Unlike 5-, 15-, and 19-HETE, 6(Z),15(Z)-20-HEDE lacks double bonds at the 8,9- and 11,12-positions, so it is not metabolized by COX. It completely blocks the vasoconstrictor response to 20-HETE in renal (9), cerebral (140), and skeletal muscle (128, 132) arterioles at a concentration of 1 µM. It has become an important adjunct to enzyme inhibitors to establish a role for 20-HETE in the myogenic response of renal, cerebral, and skeletal muscle arterioles and the vasoconstrictor response to elevated PO₂ in cerebral and skeletal muscle arterioles (131, 132, 140). However, 6(Z),15(Z)-20-HEDE also binds to plasma protein. This property limits its usefulness for in vivo experiments. Like most fatty acids, it also is a competitive inhibitor of the synthesis of 20-HETE and EETs at high concentrations.

The most recent approach to block the formation of 20-HETE has been the use of antisense cDNA oligonucleotides directed against CYP4A1. Wang and co-workers (476, 478) demonstrated that daily intravenous injections of a 20-mer antisense CYP4A1 oligonucleotide for 5 days reduced the expression of CYP4A1 and 4A2 protein and the production of 20-HETE in renal arterioles of Sprague-Dawley rats. Blockade of this pathway was associated with a modest fall in arterial blood pressure in Sprague-Dawley rats (476) and SHR (478). The advantage of antisense oligonucleotides to block the expression of CYP proteins is that they can be very specific provided there is enough difference in the sequence of the targeted region between CYP isoforms. The recent successes using antisense oligonucleotides suggest that it may be possible to achieve long-term knockdown of the expression of specific CYP isoforms by transfection of animals with viral constructs that produce antisense cRNAs.

A number of compounds have been identified that selectively inhibit epoxygenase activity. These include the antimycotic agents miconazole, econazole, ketoconazole, and clotrimazole (52, 529). Miconazole (1 µM) selectively reduces the formation of EETs by renal and hepatic microsomes by 70% and has much less effect on the formation of 20-HETE (529). At higher concentrations (10 µM), it completely inhibits epoxygenase activity while reducing formation of 20-HETE by 50% (52, 529). However, antimycotic agents are not specific epoxygenase inhibitors. They bind to heme and inhibit enzymes involved in drug metabolism, steroidogenesis (390, 429), and the synthesis of NO (151, 186). They also alter intracellular calcium concentration and ion channel activity in various types of cells (88, 94, 388, 389).

More recently, Wang et al. (475) and Nguyen et al. (332) have reported that 6-(2-propargyloxyphenyl)-hexanamide (PPOH) and methylsuphonyl-6-(2-propargyloxyphenyl)-hexanamide (PPOMS) block CYP4A2- and 4A3-catalyzed epoxidation of AA. They also block epoxygenase activity in renal tissue at concentrations of 1-10 µM and have little effect on the formation of 20-HETE. At higher concentrations (20 µM), however, we found that these compounds competitively reduce the synthesis of 20-HETE. There is also evidence that these compounds can be effective as epoxygenase inhibitors in vivo. In this regard, Brand-Schieber et al. (39) reported that an acute intravenous injection of MS-PPOH (5 mg/kg) in rats selectively lowered renal epoxygenase activity for up to 6 h. This was associated with an increase in urine flow and sodium excretion. These results suggest that endogenously formed EETs may have antidiuretic and antinatriuretic properties in the kidney.

Others have begun to use compounds that are metabolized by CYP isoforms that produce EETs as competitive inhibitors. These compounds include sulfaphenazole (CYP2C9) (295, 411), metyrapone (2B) (134, 244), N--methylbenzyl-1-aminobenzotriazole 7-ethoxyresorufin (CYP1A), 7-pentoxyresorufin (CYP2B1), -naphthoflavone (CYP1A1, 1A2, 2C8, 2C9) (66, 411), diazepam (CYP2C19), diethyldithiocarbamate (CYP2E1, 1A1, 1A2, 2A6, 2B6, 2C8, 3A3, 3A4) (66, 134, 411), quinidine (CYP2D6) (411), orpenadrine (CYP2B6) (411), triacetyloleandomycin (CYP3A3, 3A44, 3A5) (66), toleandomycin (134), methohexitone, or thiopentone (274). In theory, this approach of providing a competing substrate to a CYP isoform should reduce its ability to produce EETs depending on the relative affinity of the isoform for AA versus the inhibitor. However, the specificity of these agents has yet to be established, nor has anyone documented that these competitive substrate inhibitors actually reduce the concentration of EETs in the targeted tissues.

The conversion of EETs to DiHETEs can be inhibited using trichloropropylene oxide (TCPO) (73) or 4-phenylchalcone oxide (4-PCO) (341) that block mEH or N,N-dicyclohexylurea (DCU) (110, 508) that blocks sEH. Triacsin C (486) blocks the reincorporation of EETs, DiHETEs, and HETEs into membrane phospholipid pools by acyl CoA synthase. This augments the concentration of EETs released from tissues and potentiates the vascular responses to EETs and DiHetes (108, 486).

EETs receptor antagonists have yet to be identified. However, 11(R),12(S)-EET dilates rat renal arterioles and activates K⁺ channels in renal VSM cells, whereas, the S,R-isomer is inactive (200, 526). Given the recent findings that inactive 20-HETE analogs serve as competitive antagonists of the vasoconstrictor actions of 20-HETE, one might predict that S,R-isomers of EETs may block the vasodilator responses to the active R,S-isomers.

Recently, Fisslthaler and co-workers (119, 120) and others (36, 123) demonstrated the utility of using molecular approaches to obtain isoform-specific inhibition of the formation of EETs. They found that incubating porcine coronary arteries with antisense oligionucleotides to human CYP2C8/9 reduced the expression of an immunoreactive CYP2C8/9-like protein and the vasodilator response to AA and bradykinin. They concluded that a homolog of human CYP2C8/9 is likely responsible for the formation of EETs that serves as EDHF in these arteries.

	III. 20-HYDROXYEICOSATETRAENOIC ACID AND VASCULAR TONE

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The concept that small arteries throughout the body express CYP4A protein and produce 20-HETE and that this substance plays a central role in the regulation of vascular tone and growth emerged from work done 10 years ago (225). A summary of these experiments is presented in Figure 4. The effects of inhibitors of AA metabolism on the response of canine renal arcuate arteries to elevations in transmural pressure were examined. These vessels constrict in response to elevations in transmural pressure, and AA markedly enhanced the myogenic response of these arteries (225). The effects of AA were enhanced by indomethacin and were blocked by several mechanistically different CYP inhibitors, including SKF-525, miconazole, and 12-hydroxy-16-heptadecynoic acid (12-HHDYA). These studies were the first to suggest a role for endogenously formed CYP metabolites of AA in modulating the myogenic response of renal arterioles.

View larger version (18K): [in this window] [in a new window]   Fig. 4. Effects of arachidonic acid (AA; 50 µM) and cytochrome P-450 (CYP) inhibitors (ketoconazole, SKF 525, ABT, or 12-hydroxy-16-hepatadecynoic acid) on the pressure-diameter relation in dog renal arcuate arteries. [Redrawn from Kauser et al. (225).]

CYP inhibitors also impair the vasodilator response to acetylcholine (225, 289). This observation was ignored for many years because it was hard to understand how CYP inhibitors could block a NO-mediated response. However, it has since become apparent that a NO-induced fall in 20-HETE levels mediates the cGMP-independent effect of NO on K⁺ channel activity and vascular tone in renal and cerebral arteries (8, 12, 13, 351, 446, 447) and that endothelial-derived CYP metabolites of AA serve as EDHF at least in some vascular beds (48, 120, 139). These observations likely explain why inhibitors of CYP activity attenuate the response to endothelial-dependent vasodilators.

The metabolites of AA produced by canine renal interlobular arteries (289), rat renal (203, 527) and cerebral arterioles (140, 448), and cat cerebral arterioles (141, 156) were identified using HPLC and gas chromatography-mass spectroscopy (GC-MS). In one of these studies (Fig. 5), renal microvessels were isolated from the kidney of rats with a magnet after filling the vasculature with iron oxide (203, 527). These vessels produced 20-HETE (retention time 10 min) and lesser quantities of 14,15- and 11,12-EETs (17-20 min) and the corresponding DiHETEs (7-9 min) (203, 527) when incubated with AA. Similar results were obtained when cerebral arteries of rats (140, 448) and cats (156) and microsomes prepared from canine renal arteries (289) were incubated with AA.

View larger version (48K): [in this window] [in a new window]   Fig. 5. Metabolism of 14C-labeled arachidonic acid by rat renal microvessels. Left panel: appearance of renal microvessels isolated using a magnet after filling the kidney with iron oxide. Right panel: typical reverse-phase HPLC chromatogram depicting the metabolites formed when renal microvessels were incubated with 14C-labeled arachidonic acid (AA; 1 µCi/ml, 10 µM) for 30 min at 37°C in a 0.1 M KPO4 buffer containing 1 mM NADPH. The peaks eluting at 9.5 min and 25 min coelute with 20-HETE and AA standards, respectively. Those appearing at 8 and 9 min coelute with 11,12- and 8,9-dihydroxyeicosatrienoic acids, respectively. Peaks appearing between 12 and 17 min coelute with the hydroxyeicosatrienoic acids (18-, 16-, 15-, 12-, 5-HETEs). [Redrawn from Alonso-Galicia et al. (13) and Zou et al. (527).]

EETs are produced by CYP enzymes of the 2C and 2J families in vascular endothelial cells (119, 120, 335, 416). They are potent vasodilators that hyperpolarize VSM by increasing the activity of large-conductance, Ca²⁺-activated K⁺ channels (47, 49, 95, 97, 139, 143, 164, 187, 270, 526) and have been proposed to be an EDHF (48, 120, 165, 314). In contrast, VSM cells in renal, cerebral, pulmonary, and skeletal muscle arterioles (34, 140, 156, 203, 205, 256) express CYP4A mRNA and protein and appear to be the source of the 20-HETE produced in these vessels.

C.  Actions

1.  Vascular tone

20-HETE constricts renal (9, 203, 289, 446, 447), cerebral (140, 155, 156), mesenteric (478), and skeletal muscle arterioles (158, 257). An illustration of the effects of 20-HETE on renal afferent arterioles of the rat (203) is presented in Figure 6. In this experiment, indomethacin, baicalein, and 17-ODYA were added to the bath to block the endogenous formation of metabolites of AA and the metabolism of 20-HETE via the COX, lipoxygenase, and CYP pathways. 20-HETE at a concentration of 10⁸ M significantly reduces the diameter of rat afferent arterioles. This concentration of 20-HETE is similar to those reported to constrict cerebral arteries of cats (156), skeletal muscle arterioles of the rat (158), and the afferent arteriole of rabbits (23). It should be noted that the endogenous level of 20-HETE in microvessels is ~100 nM (140, 446). This probably explains why most investigators find that high concentrations of 20-HETE (1-10 µM) are required to constrict renal and cerebral arterioles when the endogenous formation of this substance is not blocked.

View larger version (13K): [in this window] [in a new window]   Fig. 6. Effects of 20-hydroxyeicosatetraenoic acid (20-HETE) on the diameter of rat afferent arterioles studied using the in vitro perfused juxtamedullary nephron preparation. *Significant difference from the control diameter at 90 mmHg. [Redrawn from Imig et al. (203).]

Although 20-HETE is a potent constrictor of small arteries and arterioles (<100 µm), it has little or no effect on larger arteries or the aorta. The reason for the differential response to 20-HETE in large arteries versus small arterioles remains to be determined. 20-HETE seems to have equivalent mitogenic effects in VSM cells isolated from the aorta and small vessels of the rat (321, 323), so the lack of effect of 20-HETE on vascular tone in large vessels is surprising. It may have something to do with differences in the expression of K⁺ channel types or the signal transduction pathways expressed in large versus small blood vessels.

The initial studies on the mechanism of action of 20-HETE in canine renal arteries revealed that it is associated with a 10-mV depolarization of membrane potential and a rise in intracellular Ca²⁺ concentration in VSM cells (289). Because membrane potential is dependent on K⁺ conductance in VSM cells (154), our lab in collaboration with Drs. Harder and Gebremedhin studied the effects of 20-HETE on K⁺ channel activity in VSM cells isolated from renal and cerebral arterioles (141, 156, 525). The results of a typical experiment in VSM cells isolated from cat cerebral arteries (156) are presented in Figure 7. Inhibition of the formation of 20-HETE with 17-ODYA activates a large-conductance, Ca²⁺-activated K⁺ (K_Ca) channel recorded from VSM cells isolated from cat cerebral arteries (156). This effect is completely reversed by the addition of 1 nM 20-HETE to the bath. Similar results were obtained using VSM cells isolated from rat renal arterioles (525). Further studies indicated that the vasoconstrictor response to 20-HETE in renal arterioles is mimicked by blocking K_Ca channels with TEA or iberiotoxin and that 20-HETE has no effect on vascular tone after administration of these agents (203). These findings indicate that 20-HETE is normally produced by VSM cells, where it serves as an endogenous inhibitor of the K_Ca channel.

View larger version (29K): [in this window] [in a new window]   Fig. 7. Effects of blockade of the endogenous formation of 20-HETE on K+ channel activity in vascular smooth muscle cells isolated from a middle cerebral artery of a cat. Top panel: tracing of signal channel activity recorded from a cell-attached patch from a cell bathed in normal physiological salt solution at resting membrane potential. Middle panel: marked increase in K+ activity after blocking the endogenous formation of 20-HETE with 17-octadecynoic acid (17-ODYA; 10 µM). Bottom panel: ability of 1 nM 20-HETE to reverse the effects of 17-ODYA on K+ channel activity in this cell. [Data from Harder et al. (156).]

A summary of the role of 20-HETE in the regulation of vascular tone is presented in Figure 8. Membrane stretch and a number of vasoactive hormones and mitogens activate phospholipase C that increases the synthesis of inositol trisphosphate (IP₃) and triggers the release of intracellular Ca²⁺. The rise in intracellular Ca²⁺ concentration activates K_Ca channels, leading to membrane hyperpolarization, which oppose vascular contraction by limiting Ca²⁺ influx through voltage-sensitive Ca²⁺ channels. Therefore, some intrinsic mechanism must exist that buffers against activation of K_Ca channels after elevations in intracellular Ca²⁺ concentration. This is where 20-HETE likely plays a critical role, since elevations in intracellular Ca²⁺ concentration are known to activate Ca²⁺-sensitive phospholipase A and diacylglycerol (DAG) lipase to release AA. AA is then converted to 20-HETE, which blocks K_Ca channels. Blockade of this channel depolarizes VSM and enhances Ca²⁺ entry and vascular contraction by enhancing the opening of voltage-sensitive Ca²⁺ channels. There is also evidence that 20-HETE has an additional effect to enhance the opening of L-type Ca²⁺ channels (141).

View larger version (36K): [in this window] [in a new window]   Fig. 8. Summary of the role of cytochrome P-450 metabolites of arachidonic acid (AA) in the control of vascular tone. Membrane stretch and vasoactive agents [angiotensin II (ANG II), norepinephrine (NE)] activate phospholipase C (PLC) to release inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 triggers the release of intracellular Ca2+ from the endoplasmic reticulum. Elevations in intracellular Ca2+ concentration activate Ca2+-sensitive phospholipase A2 (PLA) and diacylglycerol (DAG) lipase to release AA and stimulate the formation of 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE blocks the large-conductance, calcium-activated potassium (KCa) channel in vascular smooth muscle (VSM) cells, leading to a fall in membrane potential (Em), which enhances Ca2+ influx via L-type, voltage-sensitive Ca2+ channels. Epoxyeicosatrienoic acids (EETs) are produced by the endothelium and are potent vasodilators that hyperpolarize VSM cells by increasing the activity of the KCa channel. Acetylcholine and bradykinin stimulate the release of EETs from the endothelium, and they serve as endothelial-derived hyperpolarizing factor (EDHF) in some vascular beds.

The mechanism by which 20-HETE blocks the K_Ca channel remains to be determined. No one has yet identified a 20-HETE receptor or demonstrated that it alters the production of second messengers (DAG, IP₃, cAMP, or cGMP). However, the finding (9) that structural analogs of 20-HETE block the vasoconstrictor response to 20-HETE suggests that there is some type of receptor in VSM cells. In addition, the observations that 20-HETE blocks K⁺ channel activity in cell-attached, but not in excised membrane patches, suggest that the inhibitory effect of 20-HETE on the K_Ca channel requires an intact intracellular signal transduction pathway (262, 447).

20-HETE activates protein kinase C (PKC) in VSM cells isolated from cat cerebral arteries (262) and in proximal tubule cells (336). Inhibitors of PKC block th