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The studies of the P450 AA monooxygenase have uncovered new and important roles for P450 in the metabolism of endogenous substrates, and added P450 to the list of enzymes that participate in the metabolism of AA, a fatty acid that serves as the precursor for the biosynthesis of several physiologically important lipid mediators. The functional relevance of this metabolic pathway is suggested by the many important biological activities attributed to its products. These studies, as well as the documented endogenous roles of P450s in cholesterol, steroid, and vitamin metabolism are contributing to establish this enzyme system as a major participant in the regulation of cell, organ, and body physiology. Among these, the phenotypic analysis of mice carrying disrupted copies of the CYP4a14 gene unveiled new and important roles for the P450 enzymes in cardiovascular physiology and the control of systemic blood pressures, and suggested the human homologs of the rodent CYP 2C and 4A AA epoxygenases and ω-hydroxylases as candidate genes for the study of their role in the pathophysiology of hypertension, and cardiovascular disease.

During P450-dependent metabolism, arachidonic acid (AA) is: a) hydroxylated at its ω or ω-1 carbons to 19- and 20-hydroxyeicosatetraenoic acids (HETEs)(AA ω/ω-1 hydroxylase), or b) epoxidized at any one of its four olefins to 5,6-, 8,9-, 11,12-, or 14,15-epoxyeicosatrienoic acids (EETs)(AA epoxygenase). The demonstration that many of these P450- derived eicosanoids, were formed endogenously by several rat, rabbit, and human tissues, and that they were present in human and rat plasma and/or urine, established the P450 AA monooxygenase as a formal metabolic pathway, and stimulated an extensive functional characterization of its metabolites [1, 2, 3, 4, 5, 6, 7]. During the last 15 years, the list of biological activities attributed to the P450-eicosanoids has increased at a rapid pace, and led to an extensive in vitro characterization of their vasoactive properties, roles in cell and organ ion transport, and of their participation in hormone signaling cascades [1, 2, 3, 4, 5, 6, 7]. In male Sprague-Dawley rats, AA ω/ω-1 hydroxylation and epoxidation account for 70–80, and 20–30% of the total kidney microsomal monooxygenase activity, respectively, and 20-HETE and 11(R), 12(S)-EET (88% optical purity) are recovered as the major ω-hydroxylase and epoxygenase metabolites, respectively [[3]]. The high degree of structural homology displayed by the members of a P450 gene family [8] has complicated the identification of the P450 isoforms responsible for the metabolism of endogenous AA pools [[3],[5],[9]–[11]]. Nevertheless, extensive enzymatic, biochemical, immunological, and functional evidence indicates that CYPs of the 2 and 4 gene families are responsible for AA metabolism, and that, in rat kidney, CYPs 2C and 4A are the predominant and functionally significant AA epoxygenases and ω/ω-1 hydroxylases, respectively [[3],[9]–[19]]. For example, CYPs, 2C23, 2C24, and 2C11 are expressed in the rat kidney in decreasing order of abundance [[17]] but, of these, only CYP 2C23 metabolizes AA with a regio- and enantioselectivity that matches that of the microsomal enzymes [[16],[17]]. Furthermore, as shown in Figure 1,antibodies raised against CYP 2C23 inhibited better than 95% of the microsomal epoxygenase activity present in rat kidney microsomes, and had no effect on microsomal AA ω/ω-1 hydroxylation. The recognition of CYPs 2C and 4A as endogenous AA epoxygenases and ω/ hydroxylases, as well the early proposal of a role for these isoforms in the pathophysiology of experimental hypertension [1,[4]–[7]], open the way to the introduction of molecular approaches for the study of the functional roles of the AA monooxygenases, and the characterization of P450-isoform/gene-dependent phenotypes of renal dysfunction.

Arachidonic acid oxygenation by several cytochrome P450 monooxygenases yields epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs). Epoxide hydrolases can hydrolyze each of the EETs to the corresponding dihydroxyeicosatrienoic acids (DHETs). EETs and DHETs exhibit numerous biological actions and occur in most mammalian tissues, blood and urine (Node et al., 1999). However, unlike the cyclooxygenase and lipoxygenase metabolites of arachidonic acid, for which convenient, commercial immunoassays are available, analyses of EETs and DHETs presently require a tedious and costly analytical methodology.

HO catalyzes the conversion of heme to bilirubin, free iron and CO. As the key enzyme in heme degradation, HO activity governs cellular heme concentration. Heme is ubiquitous and of vital importance in eukaryotes, functioning as the prosthetic moiety of various heme proteins including CYP, COX, thromboxane and prostacyclin synthases, NOS, catalase, peroxidase, and hemoglobin. Excess release of heme from myoglobin and hemoglobin has been shown to be highly toxic to several organs including the kidney. In view of the widespread utilization of heme and its potential toxicity,1 it is not surprising that tissues that normally process relatively large amounts of heme possess the biochemical means to efficiently and safely degrade the heme ring and regulate heme availability for essential functions.

Endothelial cells synthesize and release vasoactive mediators in response to various neurohumoral substances (e.g., acetylcholine, ATP, bradykinin, thrombin) and physical stimuli (e.g., shear stress exerted by the flowing blood) (Furchgott and Vanhoutte, 1989). Nitric oxide (NO) produced by the L-arginine—NO synthase pathway and prostacyclin produced from arachidonic acid by cyclooxygenase have been identified as potent endothelium-derived vasodilators (Moncada et al., 1976; Moncada and Vane, 1979; Furchgott and Zawadzki, 1980; Palmer et al., 1987, Palmer 1988). However, not all endothelium-dependent relaxations can be fully explained by the release of either NO or prostacyclin. Indeed, another unidentified substance(s) which hyperpolarizes the underlying vascular smooth muscle cells, termed endothelium-derived hyperpolarizing factor (EDHF), may contribute to endothelium-dependent relaxations (Furchgott and Vanhoutte, 1989; Komori and Vanhoutte, 1990; Feletou and Vanhoutte, 1996a; Mombouli and Vanhoutte, 1997).

There can be no doubt from what is now a considerable volume of literature that a host of endogenous extracellular messengers activate or inhibit vascular K+ channels directly or via intracellular or intercellular coupling mechanisms. It is also true, however, that some investigators have reported that K+ channel blockers have no effect on the actions of vasodilators and vasoconstrictors that have been suggested by other authors to involve K+ channels. Such apparent contradictions may be explained in some cases by the vasculature’s heterogeneity—between species and vascular beds, and the location, size, and type of blood vessel in question. However, this is only one possible explanation. Blood vessels also seem to have a wealth of parallel or backup mechanisms. As an example, for illustration purposes, a vasodilatory extracellular messenger may simultaneously activate K+ channels and suppress voltage-gated Ca2+ channels in vascular smooth muscle cells. Either effect may be sufficient for full vasodilation such that if one effect is prevented experimentally, there is no change in the size of the vasodilation. This is a parallel system. Alternatively, a backup system may exist whereby a mechanism that is not normally operative becomes essential in a certain condition, perhaps during or after ischemia. Developing this further, it is plausible (and there is supporting evidence) that vasoconstrictor and vasodilator mechanisms have commonality and thus interact in such a way that a vasodilatory mechanism is negated by one vasoconstrictor mechanism but not another. The combination of biological complexity, experimental variability, and the different experimental conditions used by investigators has, perhaps not surprisingly, produced a spectrum of conclusions from an essential functional role of K+ channels to no role at all.

Eicosanoid is a collective term for all biologically active, oxygenated metabolites of arachidonic acid and two other C20 polyunsaturated fatty acids. There are three major groups of eicosanoids. The prostaglandins (PGs), so named because they were first discovered in seminal fluids originating in the prostate gland, are products of the cyclooxygenase pathways. The cytochrome P450 “epoxygenase pathways” yield the epoxyeicosatrienoic acids. Various lipoxygenase pathways are responsible for biosynthesizing a wide range of compounds, including leukotrienes and hydroxyeicosatetraenoic acids. The biosynthesis and structures of representatives of the major groups of eicosanoids are outlined in Figure 1, and described in detail elsewhere (Stanley, 1999). One or more members of the major eicosanoid groups have been detected in virtually all mammalian tissues and body fluids, where they serve as lipid mediators of cellular events. Among their important actions, eicosanoids modulate ion transport physiology, mediate contraction or relaxation of smooth muscles and exert tremendous influence in host defense mechanisms. It would be difficult to exaggerate the importance of eicosanoids in the vertebrate immune system. Sir John Vane shared in the 1982 Nobel Prize in Medicine or Physiology, which recognized his discovery that the analgesic effects of aspirin are due to inhibition of prostaglandin biosynthesis. This discovery launched a very large pharmaceutical research enterprise aimed at discovery of new non-steriodal anti-inflammatory drugs, all of which act through their inhibitory influence on prostaglandin biosynthesis.

Complicating about 10% of all pregnancies pregnancy-induced hypertensive diseases account for an enormous number of maternal deaths. At present, termination of pregnancy represents the only treatment of the cause. The important role of eicosanoids in pregnancy-induced hypertension is generally accepted1–2. As humoral vasoactive factors they are important for vascular tone regulation of the non-innervated uteroplacental vessels. In PIH and related diseases, these vessels fail to dilate normally, leading to a chronic preplacental decrease in circulation. A systemic lack of prostacyclin may also promote an increased vasoconstriction and coagulation disturbances in PIH leading to endothelial cell disorder, hemolysis and the vicious circle of disseminated intravasai coagulation3–4.

We have previously found that 5,6-EET (epoxyeicosatrienoic acid)(50 nM) significantly dilates the vascular bed(42%) of the isolated, constantly perfused rabbit lung, which has been constricted with U46619(5-8 pM). We studied the role of EDRF-NO and prostaglandins in the 5,6-EET-induced vascular relaxation. Dilation to 5,6-EET was evident only when the pulmonary vascular tone was increased. L-NNA (N omega-nitro-L-arginine, 10(-4) M), an inhibitor of NO synthase(NOS); U46619(5-10 pM), a thromboxane mimetic; and L-NNA + INDO(indomethacin, 10(-5) M), a cyclooxygenase inhibitor, all increased the pressure of pulmonary artery(PPa) from baseline, to a peak range of 28-38mmHg(32.75 [symbol: see text] 2.2), whereas INDO alone increased Ppa only by 10mmHg. L-NNA + INDO,L-NNA alone, and INDO + U46619 attenuated the 5,6-EET relaxing effect by 100%, 88% and 64.5%, respectively. In the presence of L-NNA and 5,6-EET, SNAP(S-nitroso-N-acetyl-D,L-penicillamine, 10(-6) M), a NO donor, reduced Ppa by 75%. We conclude that the mechanism of vasodilation to 5,6-EET in the rabbit pulmonary circulation is via both EDRF-NO and PG pathways and that the vasodilation is largely EDRF-NO dependent.

The present study examined the metabolism of ADP-ribose (ADPR) and cyclic ADP-ribose (cADPR) in small bovine coronary arterial homogenates and characterized the effects of these nucleotides on the activity of potassium (K+) channels in coronary smooth muscle cells. ADPR and cADPR were produced from NAD+ (lmM) by homogenates from small bovine coronary arteries. The conversion rate was 2.81 ± 0.19 nmol/min/100 µg protein for ADPR and 1.37 ± 0.03 nmol/min/100 µg protein for cADPR. In patch clamp experiments, ADPR produced a concentration-dependent increase in the activity of a calcium activated K (KCa) channel in inside-out membrane patches of coronary arterial smooth muscle cells at concentrations of 0.1, 1 and 10 µM. The open state probability (NPo) of KCa channel was maximally increased 5-fold at a concentration of 10 µM. cADPR reduced the activity of KCa channel at concentrations of 1 and 10 µM. The NPo was decreased by 45% and 75%, respectively. The results indicate that there is an enzymatic pathway in the coronary arterial smooth muscle to produce ADPR and cADPR. These nucleotides may play a role in the control of coronary vascular tone by altering the activity of the KCa channel in vascular smooth muscle cells.

Eukaryotic cells contain substantial amounts of arachidonic acid (AA; 5,8,11,14-eicosatetraenoic acid) esterified predominantly to the sn-2 position of cellular glycerophospholipids. As with many lipid-derived mediators, e.g., cholesterol, phosphoinositides, diglycerides, AA serves a structural role, as a component of cellular membranes, and an important functional role, as a participant in a variety of receptor/agonist-mediated signaling cascades.1–4 In the absence of stimuli, the intracellular levels of nonesterified AA are nearly undetectable. However, most organ cells possess an elaborate enzymatic machinery that, in response to a variety of stimuli, catalyzes: (1) the hydrolytic cleavage of the AA molecule form selected, hormonally sensitive phospholipid pools, (2) the transduction of chemical information into the fatty acid molecular template by means of regio- and stereospecific oxygenation reactions, and (3) the decoding of that chemical information either by receptor-mediated processes or, alternatively, by the direct effects of these oxygenated metabolites on metabolic pathways1–4 (Fig. 1). As a net result, these processes provide cells with a rapid and versatile on/off molecular switch for the intra- or intercellular transduction and/or amplification of functionally meaningful information.

The ′epoxygenase pathway′ involves the metabolism of arachidonic acid by the cytochrome P-450 monooxygenase system first elucidated by Bate’s group in the early 1980’ s (1-5). The four regioisomeric products are the result of enzymatic addition of a highly reactive epoxide functionality across one of four double bonds present in the arachidonate molecule (Figure 1 ). Two regioisomers are of particular biological interest. 14,15-epoxyeicosatrienoic acid (14,15-EET) has the ability to inhibit platelet aggregation via its inhibition of platelet cyclooxygenase (6,7) as well as the ability to stimulate thymidine incorporation in cultured rat glomerular mesangial cells (8), and secretion of growth hormones from somatotrophs via modulation of cAMP and Ca2+(9). 5,6-epoxyeicosatrienoic acid is a substrate for

Cyclooxygenase and lipoxygenase metabolites of arachidonic acid (AA) are known to affect neurotransmitter function and inflammatory processes. Cyclooxygenase metabolism of arachidonic acid has been shown to be associated with oxygen free radical formation 1. These cyclooxygenase- dependent free radicals are produced after experimental neural trauma or acute hypertension and contribute to endothelial lesions in the cerebral microcirculation 2. Recently a third pathway of arachidonic acid metabolism has been examined, the so-called third pathway, or epoxygenase pathway 3. It is known that this pathway produces epoxides of arachidonic acid as well as 19- and 20-hydroxylated derivatives of arachidonic acid. One of the products of the epoxygenase pathway, 5,6-epoxyeicosatrienoic acid (5,6-EET) is known to be metabolized by cyclooxygenase enzyme 4, therefore offering the possibility that third pathway metabolites may also produce oxygen radicals. The goal of the current study was to determine if brain makes epoxyeicosatrienoic acids (EETs) from AA and if so whether they affect cerebral vascular resistance or in vivo platelet aggregation.

The renal epoxygenase has been demonstrated to be an active pathway for the conversion of PGI2 to a new, previously unreported, metabolite. This metabolite was isolated and identified by radiogas-chromatography-mass spectrometry as 5-hydroxy-6-keto PGF1α. Its structure was further confirmed by comparison of the mass-spectra to that of the synthetic standard. The formation of 5-hydroxy-6-keto PGF1α in the kidney suggested epoxidation of prostacyclin via the renal epoxygenase as an alternative pathway of PGI2 metabolism.

The renal epoxygenase has been demonstrated to be an active pathway for the conversion of arachidonic acid (AA) to 11,12-epoxide, 19-hydroxy and 20-hydroxy-eicosatetraenoic acids. The epoxygenase activity was found to be located predominantly in the renal cortex. This renal epoxygenase pathway also transformed PGI2 to a new, previously unreported, metabolite. This metabolite was isolated and identified by radio gas chromatography and gas chromatography-mass spectrometry as 5-hydroxy-6-keto-PGFlα. Its structure was further confirmed by comparison of the mass-spectra to that of the synthetic standard. The formation of 5-hydroxy-6-keto-PGF1α in the kidney suggested epoxidation of prostacyclin via the renal epoxygenase as an alternative pathway of PGI2 metabolism.