Inhibition of cytochrome P450omega-hydroxylase: a novel endogenous cardioprotective pathway

Cytochrome P450s (CYP) and their arachidonic acid (AA) metabolites have important roles in regulating vascular tone, but their function and specific pathways involved in modulating myocardial ischemia-reperfusion injury have not been clearly established. Thus, we characterized the effects of several selective CYPomega-hydroxylase inhibitors and a CYPomega-hydroxylase metabolite of AA, 20-hydroxyeicosatetraenoic acid (20-HETE), on the extent of ischemia-reperfusion injury in canine hearts. During 60 minutes of ischemia and particularly after 3 hours of reperfusion, 20-HETE was produced at high concentrations. A nonspecific CYP inhibitor, miconazole, and 2 specific CYPomega-hydroxylase inhibitors, 17-octadecanoic acid (17-ODYA) and N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), markedly inhibited 20-HETE production during ischemia-reperfusion and produced a profound reduction in myocardial infarct size (expressed as a percent of the area at risk) (19.6+/-1.7% [control], 8.4+/-2.5% [0.96 mg/kg miconazole], 5.9+/-2.2% [0.28 mg/kg 17-ODYA], and 10.8+/-1.8% [0.40 mg/kg DDMS], P<0.05, respectively). Conversely, exogenous 20-HETE administration significantly increased infarct size (26.9+/-1.9%, P<0.05). Several CYPomega-hydroxylase isoforms, which are known to produce 20-HETE such as CYP4A1, CYP4A2, and CYP4F, were demonstrated to be present in canine heart tissue and their activity was markedly inhibited by incubation with 17-ODYA. These results indicate an important endogenous role for CYPomega-hydroxylases and in particular their product, 20-HETE, in exacerbating myocardial injury in canine myocardium. The full text of this article is available online at http://circres.ahajournals.org.     

花生四烯酸细胞色素P450代谢途径在高血压中作用的研究进展

花生四烯酸是人体必须脂肪酸之一，其代谢产物具有较强生物学活性并在众多生理病理过程中发挥着重要调节作用。其在人体内主要通过环氧化酶、脂氧化酶、细胞色素P450（CYP）三大途径进行代谢。其中，越来越多的研究表明，CYP代谢途径中其关键基因CYP通过调控经ω-羟化酶、表氧化酶作用的下游产物20羟-二十烷四烯酸及表氧一二十碳三烯酸的质／量，从而影响高血压的发生发展，并且对其分子生物学机制及遗传学的研究亦成为当前的研究焦点。本文主要对CYP途径在高血压中的作用简要做一综述。     

20-羟二十烷四烯酸在血压及血管舒缩功能中的调节作用

20-羟二十烷四烯酸(20-hydroxyeicosatetraenoic acid,20-HETE)是由细胞色素P450(cytochrome P450,CYP450)催化花生四烯酸(arachidonic acid,AA)的ω-羟基所生成的产物之一。随着研究进展,人们发现20-HETE在调节机体血压、脑血流量、心肌收缩力、肾功能等过程中发挥了重要作用,且它与肿瘤、炎症反应等疾病的发生和发展有着密切关系。现主要就20-HETE在机体血压及血管舒缩功能中的作用作一综述。     

Pulmonary intravascular macrophages metabolize arachidonic acid in vitro. Comparison with alveolar macrophages

Pulmonary intravascular macrophages are a recently identified component of the pulmonary mononuclear phagocyte system. It has been shown that alveolar macrophages are capable of metabolizing arachidonic acid (AA) to its biologically active inflammatory metabolites via the lipoxygenase and cyclooxygenase pathways. In this study, we have compared the ability of swine intravascular macrophages and alveolar macrophages to metabolize AA in vitro. Alveolar macrophages attached to a plastic substrate produced at least five identified AA metabolites including prostaglandin (PG)F2 alpha, hydroxyheptadecatrienoic acid (HHT), 5-hydroxyeicosatetraenoic acid (HETE), 12-HETE, and 15-HETE. In contrast, adherent intravascular macrophages produced eight identified metabolites including thromboxane (TX)B2, PGF2 alpha, PGD2, PGE2, HHT, 5-HETE, 12-HETE, and 15-HETE. The major lipoxygenase metabolite produced by both macrophage types was 5-HETE. The major cyclooxygenase metabolite produced by alveolar macrophages was PGF2 alpha, whereas the major metabolite produced by intravascular macrophages was HHT. Both macrophage populations treated with calcium ionophore (A23187) exhibited increased production of PGs, TXB2, leukotriene (LT)B4, 5-HETE, 12-HETE, and 15-HETE, but the most striking increase occurred in metabolism through the lipoxygenase pathway. The major lipoxygenase metabolite generated by ionophore-stimulated macrophages was 5-HETE, and in intravascular macrophages 12-HETE was also produced. Preincubation of macrophages with indomethacin and nordihydroguaiaretic acid attenuated the yield of cyclooxygenase metabolites and lipoxygenase metabolites, respectively. Studies of leukotriene formation demonstrated that both macrophage types produce LTC4 and LTB4 from the leukotriene precursor LTA4. Thus, we show that the pulmonary intravascular macrophage is capable of metabolizing AA and LTA4 to their inflammatory and vasoactive metabolites by the cyclooxygenase and lipoxygenase pathways.(ABSTRACT TRUNCATED AT 250 WORDS)     

20-Hydroxyeicosatetraenoic acid mediates calcium/calmodulin-dependent protein kinase II-induced mitogen-activated protein kinase activation in vascular smooth muscle cells

Norepinephrine (NE) and angiotensin II (Ang II), by promoting extracellular Ca²⁺ influx, increase Ca²⁺/calmodulin-dependent kinase II (CaMKII) activity, leading to activation of mitogen-activated protein kinase (MAPK) and cytosolic phospholipase A₂ (cPLA₂), resulting in release of arachidonic acid (AA) for prostacyclin synthesis in rabbit vascular smooth muscle cells. However, the mechanism by which CaMKII activates MAPK is unclear. The present study was conducted to determine the contribution of AA and its metabolites as possible mediators of CaMKII-induced MAPK activation by NE, Ang II, and epidermal growth factor (EGF) in vascular smooth muscle cells. NE-, Ang II-, and EGF-stimulated MAPK and cPLA₂ were reduced by inhibitors of cytochrome P450 (CYP450) and lipoxygenase but not by cyclooxygenase. NE-, Ang II-, and EGF-induced increases in Ras activity, measured by its translocation to plasma membrane, were abolished by CYP450, lipoxygenase, and farnesyltransferase inhibitors. An AA metabolite of CYP450, 20-hydroxyeicosatetraenoic acid (20-HETE), increased the activities of MAPK and cPLA₂ and caused translocation of Ras. These data suggest that activation of MAPK by NE, Ang II, and EGF is mediated by a signaling mechanism involving 20-HETE, which is generated by stimulation of cPLA₂ by CaMKII. Activation of Ras/MAPK by 20-HETE amplifies cPLA₂ activity and releases additional AA by a positive feedback mechanism. This mechanism of Ras/MAPK activation by 20-HETE may play a central role in the regulation of other cellular signaling molecules involved in cell proliferation and growth.     

Fenofibrate Attenuates Hypertension in Goldblatt Hypertensive Rats: Role of 20-Hydroxyeicosatetraenoic Acid in the Nonclipped Kidney

Background

There is vast evidence that the renin-angiotensin system is not the sole determinant of blood pressure (BP) elevation in human renovascular hypertension or the relevant experimental models. This study tested the hypothesis that kidney deficiency of 20-hydroxyeicosatetraenoic acid (20-HETE), a product of cytochrome P450 (CYP)-dependent ω-hydroxylase pathway of arachidonic acid metabolism, is important in the pathophysiology of the maintenance phase of 2-kidney, 1-clip (2K1C) Goldblatt hypertension.

Lipoxygenase products of arachidonic acid stimulate LHRH release from rat median eminence

Exogenous arachidonic acid (AA) incubated in presence of male rat hypothalamus, shows a low rate of conversion (less than 1%) of the substrate with a major product, identified as 12-hydroxyeicosatetraenoic acid (12-HETE) by reverse phase-high performance liquid chromatography (rpHPLC) and gas chromatography-mass spectrometry (GC-MS). Furthermore, immunoreactive 12-HETE estimated after purification on rpHPLC is produced by hypothalamus slices or median eminences (MEs) incubated in absence of any exogenous precursor. The effect of 12-HETE was tested on the release of LHRH from rat MEs after a 30-min incubation and was compared to the effect of another lipoxygenase product, 5-HETE, and to the well-known stimulatory effect of prostaglandin E2 (PGE2). The three AA metabolites stimulate LHRH release. A significant stimulatory effect on LHRH release is obtained with 10(-9) M of 12-HETE and only with 10(-8) M of 5-HETE or PGE2. Furthermore, the effect of higher concentrations is different according to the eicosanoid tested. The maximal response (176% of the control) is reached with 12-HETE at 10(-8) M. No significant change is observed at 10(-7) and 10(-6) M. The response with 5-HETE is also maximal (162% of the control) at 10(-8) M but decreases significantly (only 117% of the control) at 10(-6) M. The amplitude of the response to PGE2 is larger and higher, reaching a plateau (300% of the control) at 10(-6) M. 12-HETE has no effect on somatostatin (SRIF), release, as already known for PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitric oxide/cytochrome P450 interactions in cyclosporin A-induced effects in the rat

INTRODUCTION: The present study evaluated the contribution of 20-hydroxyeicosatetraenoic acid (20-HETE) and its interaction with nitric oxide (NO) in cyclosporin A-induced nephrotoxicity and hypertension. METHODS AND RESULTS: The treatment of rats with cyclosporin A (25 mg/kg) for 7 days increased the renal microsomal conversion of arachidonic acid (AA) to 20-HETE (93 +/- 6%, P < 0.05), increased systolic blood pressure (SBP), reduced the urinary excretion of nitrite (53 +/- 8%, P < 0.05), induced renal damage as indicated by a marked increase in protein excretion (163 +/- 14%, P < 0.05), increased renal vasoconstrictor responses to AA (82 +/- 5%, P < 0.05) but not endothelin-1 or phenylephrine, and decreased vasodilator responses to bradykinin (42 +/- 10%, P < 0.05) and sodium nitroprusside (SNP; 56 +/- 13%, P < 0.05) in the renal preglomerular vessel treated with indomethacin and NO synthase inhibitor. The pretreatment of rats with HET0016 (10 mg/kg) or 1-aminobenzotriazole (50 mg/kg), inhibitors of cytochrome P450 (CYP450) activity, attenuated or prevented cyclosporin A-induced increases in 20-HETE production, SBP, and protein excretion, as did L-arginine (4 g/l), a substrate for NO synthase. L-Arginine but not HET0016 or 1-aminobenzotriazole blunted the cyclosporin A-induced decrease in nitrite excretion. Similarly, L-arginine blunted the enhanced vasoconstriction by AA as did HET0016 or 1-aminobenzotriazole. However, cyclosporin A-blunted dilator responses to bradykinin and SNP were not affected by L-arginine, HET0016, or 1-aminobenzotriazole. CONCLUSIONS: These data suggest that cyclosporin A-induced nephrotoxicity can be accounted for by reduced NO production and a consequent increase in 20-HETE. The cyclosporin A-induced nephrotoxicity is thus an ideal model for evaluating NO/CYP450 interactions.     

Hypoxic preconditioning protects rat hearts against ischemia–reperfusion injury via the arachidonate12-lipoxygenase/transient receptor potential vanilloid 1 pathway

Hypoxic preconditioning (HPC) protects rat hearts against ischemia–reperfusion (IR) injury. However, the role of transient receptor potential vanilloid 1 (TRPV1) in HPC-mediated cardioprotection remains unknown. TRPV1 is activated by endovanilloid 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], which is synthesized by arachidonate 12-lipoxygenase (ALOX12). Therefore, we examined whether HPC protects the myocardium against IR via the ALOX12/TRPV1 pathway. Compared to hearts of rats kept in room air, the hearts of rats kept in air with 10 % oxygen for 4 weeks had better post-ischemic recovery and less tissue damage when subjected to 30-min global ischemia and 4-h reflow in a Langendorff apparatus. Capsazepine, a specific TRPV1 blocker, administered 5 min before reperfusion markedly attenuated the effects of HPC, confirming that TRPV1 is a downstream effector in HPC-mediated cardioprotection. HPC resulted in the upregulation of ALOX12 and myocardial 12(S)-HETE, and prevented IR-induced 12(S)-HETE reduction. In addition, sarcolemmal ALOX12 expression in HPC hearts mainly co-localized with TRPV1 expression. Blockade of ALOX12 by cinnamyl-3,4-dihydroxy-α-cyanocinnamate or baicalein abrogated the effects of HPC, baicalein also decreased 12(S)-HETE expression. Mimicking HPC by given 12(S)-HETE or capsaicin to baicalien-treated hearts enhanced cardiac recovery during reperfusion. The cardiac protein kinase C (PKC) isoforms α, δ, ε, and ζ were preferentially expressed in the sarcolemmal membrane of HPC-treated hearts, indicating their high intrinsic activation state. Capsazepine or co-treatment with baicalein attenuated translocation of PKCα, PKCδ and PKCε, but not that of PKCζ. We conclude that HPC reduces heart susceptibly to IR via ALOX12/TRPV1/PKC pathway, as shown by increased 12(S)-HETE expression in HPC hearts.     

花生四烯酸的CYP代谢途径与心肌缺血再灌注损伤

花生四烯酸（AA）主要由环氧化酶（COX）、脂加氧酶（LOX）及细胞色素P450（CYP）途径代谢。近年来发现，AA的CYP代谢途径与心肌缺血再灌注（MIR）损伤的关系密切。本文就AA的CYP代谢酶在心脏中的表达、CYP代谢途径对MIR的影响及作用机制做一综述。     

Generation of lipoxygenase metabolites of arachidonic acid by monolayer cultures of tracheal epithelial cells and intact tracheal segments from rabbits

We compared the profile of lipoxygenase metabolites of arachidonic acid (AA) generated by cultured rabbit tracheal epithelial (TE) cells with that produced by intact rabbit tracheal segments at baseline and following addition of exogenous AA or calcium ionophore A23187. Lipoxygenase metabolites in effluent media were resolved by high-pressure liquid chromatography and quantitated by radioimmunoassay for monohydroxyeicosanoid (HETE) and leukotriene (LT) metabolites [5-, 12-, and 15-HETE; LTB4, LTC4, LTD4]. Following incubation with exogenous AA (10 micrograms/ml), cultured TE cells generated immunoreactive products that coeluted with authentic 5-, 12-, and 15-HETE standards. 12-HETE was the predominant metabolite. Whereas the generation of HETEs by TE monolayers was dependent on addition of exogenous AA, intact tracheal segments demonstrated a baseline production of 12-HETE and lesser amounts of 5- and 15-HETE as well as unidentified metabolites with UV absorbance at 280 nm. Incubation of tracheal segments with AA resulted in augmented metabolite production. In cultured TE cells, small quantities of HETEs were present intracellularly esterified to membrane phospholipids or free in the cytosol, and significant increases in free cytosolic 12- and 15-HETE were detected postincubation with AA. Calcium ionophore (5 microM) did not induce significant increases in HETE production in either cultured TE cells or tracheal segments. Minimal or no immunoreactive LTs B4, C4, and D4 were produced by TE monolayers or tracheal segments at baseline or following addition of AA or ionophore. Production of HETEs by cultured TE cells was not associated with decreased viability, release of intracellular lactic dehydrogenase, or loss of cells from the monolayers. Preincubation of monolayer cultures or tracheal segments with 5,8,11,14-eicosatetraynoic acid prior to addition of exogenous AA inhibition metabolite production. Our observations provide further documentation for the generation of lipoxygenase metabolites by TE cells and suggest that the array of metabolites generated by cultured TE cells may not be representative of the entire spectrum of AA metabolites produced by intact native epithelium.     

Production of arachidonic acid metabolites by macrophages exposed in vitro to asbestos, carbonyl iron particles, or calcium ionophore

Consequent to asbestos deposition, alveolar macrophages (AM) accumulate at alveolar duct bifurcations where they phagocytize fibers. Because phagocytosis can stimulate the release of arachidonic acid (AA) metabolites, the possibility that secretion of these powerful mediators of inflammation might be induced by chrysotile asbestos was investigated in vitro. Rat AM were treated in vitro with chrysotile asbestos, and the cyclooxygenase products--prostaglandins, thromboxane B2 (TXB2), 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT)--and lipoxygenase products--leukotrienes (LT), hydroxyeicosatetraenoic acids (HETE)--secreted in the medium were isolated by high-performance liquid chromatography. Composition of the AA metabolites released was compared with that from those stimulated by the calcium ionophore A 23187 (20 microM) and by another particulate phagocytic stimulus, i.e., carbonyl iron beads. Calcium ionophore stimulation induced a marked release of various AA metabolites in the medium from both the cyclooxygenase pathway (HHT, TXB2, and PGE2, in decreasing quantities, respectively) and the lipoxygenase pathway (LTB4, 5-HETE, 12-HETE, and LTC4). The major product was LTB4. Treatment of the macrophages with asbestos fibers induced the release of a similar array of AA metabolites, although there were smaller amounts of LTC4 and 12-HETE, but increased quantities of PGF2 alpha. A time course study showed a steady increase in metabolite production for 1 h, followed by a plateau. In addition, the amount of metabolites released was dependent on asbestos concentrations. Phagocytosis of iron beads induced the secretion of the same metabolites as asbestos stimulation, but in larger quantities, probably reflecting the lack of cytotoxicity of the particle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Smooth muscle--specific expression of CYP4A1 induces endothelial sprouting in renal arterial microvessels

Cytochrome P450 (CYP) 4A1 has been characterized as the most efficient arachidonic acid omega-hydroxylase catalyzing the formation of 20-hydroxyeicosatetraenoic acid (20-HETE), a potent constrictor of the renal and cerebral microcirculation and a mitogen for smooth muscle cells. We constructed adenoviruses expressing the CYP4A1 cDNA or LacZ under the control of the smooth muscle cell-specific promoter SM22alpha (Ad-SM22-4A1 and Ad-SM22-nLacZ, respectively). Beta-galactosidase expression was detected in Ad-SM22-nLacZ-transduced vascular smooth muscle A7r5 and PAC1 cells, but not in Ad-SM22-nLacZ-transduced 3T3 fibroblasts or vascular endothelial cells. Likewise, CYP4A1 mRNA and protein were detected in Ad-SM22-4A1-transduced A7r5 and PAC1 cells. Ad-SM22-4A1-transduced A7r5 cells metabolized lauric acid to 12-hydroxy-lauric acid at a rate 5 times greater than that of cells transduced with Ad-SM22-nLacZ (4.79+/-1.77 versus 0.97+/-0.57 nmol 12-hydroxy lauric acid/10(6) cells per h). Smooth muscle-specific LacZ expression was also detected in microdissected renal interlobar arteries transduced with Ad-SM22-nLacZ. Arteries transduced with Ad-SM22-4A1 produced higher levels of 20-HETE (4.04+/-0.29 and 13.43+/-2.84 ng/mg protein in Ad-SM22-nLacZ-transduced and Ad-SM22-4A1-transduced arteries, respectively) and demonstrated a marked angiogenic activity measured as the total length of sprouting neovessels (12.63+/-3.66 mm in Ad-SM22-4A1-transduced vessels versus 1.79+/-0.89 mm in Ad-SM22-nLacZ-transduced vessels). This angiogenic activity represented endothelial cell sprouting and was fully blocked by treatment with HET0016, a selective inhibitor of CYP4A-catalyzed reactions. The inhibitory effect of HET0016 was reversed by addition of a 20-HETE agonist. We conclude that Ad-SM22-4A1 drives a smooth muscle-specific functional expression of CYP4A1 and demonstrates increased angiogenesis, presumably via increased production of 20-HETE.     

The synthesis of 20-HETE in small porcine coronary arteries antagonizes EDHF-mediated relaxation

OBJECTIVE: Exogenous application of 20-hydroxyeicosatetraenoic acid (20-HETE) to small (300-500 microm) porcine coronary arteries elicits contraction by activating the Rho kinase and increasing the sensitivity of contractile proteins to Ca2+. Here, we determined whether 20-HETE is involved in the regulation of coronary artery tone as well as its role in the modulation of endothelium-derived hyperpolarizing factor (EDHF)-mediated responses. METHODS AND RESULTS: Small porcine coronary arteries expressed cytochrome P450 (CYP) 4A, as demonstrated by Western blot analysis, and generated 20-HETE. Moreover, 20-HETE production was increased two- and threefold over basal levels in response to isometric stretch or the thromboxane analogue U46619, respectively, and was inhibited by the CYP 4A inhibitor N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS). In vascular reactivity studies, DDMS attenuated U46619-induced contractions and induced a concentration-dependent but endothelium-independent relaxation of precontracted arterial rings. Endogenously generated 20-HETE significantly inhibited the EDHF-mediated relaxation of coronary arteries, which was potentiated by the phospholipase A2 inhibitors AACOCF3 and ONO-RS-082, as well as by the omega-hydroxylase inhibitors 17-octadecynoic acid and DDMS. EDHF-mediated relaxation was not affected by either the nonselective epoxygenase inhibitors miconazole and clotrimazole or the CYP 2C inhibitor sulfaphenazole but was abolished by the Na-K-ATPase inhibitor, ouabain. Exogenous application of 20-HETE inhibited EDHF-mediated relaxations and caused a concomitant increase in the phosphorylation of protein kinase Calpha (PKCalpha). This effect was reversed by the PKC inhibitor Ro-318220 and mimicked by the PKC activator phorbol-12 myristate 13-acetate. CONCLUSIONS: These results indicate that vascular tone in small porcine coronary arteries is partly determined by the endogenous production of 20-HETE. In addition, 20-HETE functionally antagonizes EDHF-mediated relaxation via a PKCalpha-dependent mechanism, probably involving the inhibition of the Na-K-ATPase.     

5-Hydroxyeicosatetraenoic acid increases prolactin release from rat anterior pituitary cells

The enzymatic breakdown of phospholipids to form arachidonic acid and its subsequent conversion to metabolites produced via the lipoxygenase pathway in anterior pituitary cells may contribute to the process of PRL release. The incubation of primary cultures of pituitary cells from female rats with the lipoxygenase product 5-hydroxyeicosatetraenoic acid (5-HETE; 5-100 microM) significantly increased PRL release in a concentration-dependent manner. The release of PRL induced by 45 microM 5-HETE was completely blocked by 1 microM dopamine. Penfluridol, an agent that binds to and inactivates several Ca+2-binding proteins, including calmodulin, decreased (P less than 0.01) basal and 5-HETE-stimulated PRL release. Similarly, 50 microM D-600, a Ca+2 channel antagonist, significantly (P less than 0.01) reduced basal and 5-HETE-induced PRL release. BW755c or RHC 80267, both of which reduce the production of arachidonic acid metabolites, including 5-HETE, significantly reduced basal PRL release. The inhibitory effects of BW755c and RHC 80267 on PRL release, however, could be overcome by the addition of 5-HETE. In conclusion, 5-HETE or similar lipoxygenase metabolites may be important cellular components in the process of PRL release, and the inhibitory action of dopamine on PRL would seem to be mediated at some step after stimulation by these metabolites.     

The effects of lipoxygenase products on progesterone and prostaglandin production by human corpora lutea

The present study was undertaken to assess the effects of the products of the lipoxygenase pathway on steroidogenesis and the production of prostaglandins (PGs) by human corpora lutea in the midluteal phase. In the first experiment luteal cells were cultured with 5-hydroxyeicosatetraenoic acid (5-HETE) at 10, 100, 500, or 1000 ng/mL in the presence or absence of hCG at 100 ng/mL for 10 days. The addition of 5-HETE dose-dependently inhibited progesterone (P) production by the cultural luteal cells. P production stimulated by exposure to hCG was also reduced significantly in response to 5-HETE. However, 5-HETE had no effect on the production of 6-keto-PGF1 alpha, PGF2 alpha, or PGE2 by cultured luteal cells at any point during the culture period. In the second experiment the reaction products of soybean lipoxidase of arachidonic acid (AA-LIP) were added to cultured luteal cells. Treatment with either AA or LIP alone had no effect on basal P production. The addition of AA-LIP at all concentrations tested reduced P production by cultured luteal cells in the presence or absence of hCG. AA-LIP significantly reduced basal 6-keto-PGF1 alpha secretion in cultured luteal cells on day 2. Although the stimulatory effect of AA on luteal PGE2 production was maintained throughout the entire culture period, the lipoxygenase products of AA did not affect AA-stimulated PGE2 production by cultured luteal cell. These results suggest that the products of the lipoxygenase pathway may be important in the involution of human corpora lutea.     

Cytochrome P450 omega-hydroxylase inhibition reduces infarct size during reperfusion via the sarcolemmal KATP channel

Inhibition of 20-hydroxyeicosatrienoic acid (20-HETE), by pretreatment with pharmacological inhibitors of cytochrome P450 (CYP) omega-hydroxylase, has been shown to reduce infarct size in canines when administered prior to ischemia. However, it is unknown whether these agents reduce infarct size when administered just prior to reperfusion and if the sarcolemmal and/or mitochondrial K(ATP) channels (sK(ATP) and mK(ATP)) contribute to cardioprotection. Therefore, we determined whether specific CYP inhibitors for epoxygenases and omega-hydroxylases are cardioprotective when given either prior to ischemia or prior to reperfusion and furthermore, if selective inhibition of the sK(ATP) by HMR-1098 or mK(ATP) by 5-hydroxydecanoic acid (5-HD) could abrogate this effect. Male Sprague-Dawley rats underwent 30 minutes of ischemia followed by 2 hours of reperfusion. Groups received either miconazole (MIC, non-selective CYP inhibitor, 3 mg/kg), 17-octadecynoic acid (17-ODYA, CYP omega-hydroxylase inhibitor, 0,3 or 3 mg/kg), N-methylsulfonyl-12, 12-dibromododec-11-enamide (DDMS, CYP omega-hydroxylase inhibitor, 0,4 or 4 mg/kg), N-methanesulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH, CYP epoxygenase inhibitor, 3 mg/kg), or vehicle either 10 minutes prior to ischemia or 5 minutes prior to reperfusion. Rats also received either HMR-1098 (6 mg/kg) or 5-HD (10 mg/kg) 10 minutes prior to reperfusion, with subsets of rats also receiving either MIC or 17-ODYA 5 minutes prior to reperfusion. DDMS and 17-ODYA dose dependently reduced infarct size. Rats treated with MIC, 17-ODYA and DDMS, but not MS-PPOH, produced comparable reductions in infarct size when administered prior to ischemia or reperfusion compared to vehicle. HMR-1098, but not 5-HD, also blocked the infarct size reduction afforded by MIC and 17-ODYA. These data suggest a novel cardioprotective pathway involving CYP omega-hydroxylase inhibition and subsequent activation of the sK(ATP) channel during reperfusion.     

Arachidonate lipoxygenases as essential regulators of cell survival and apoptosis.

Arachidonic acid (AA) metabolites derived from both cyclooxygenase (COX) and lipoxygenase (LOX) pathways transduce a variety of signals related to cell growth. Here, we report that the AA LOX pathway also functions as a critical regulator of cell survival and apoptosis. Rat Walker 256 (W256) carcinosarcoma cells express 12-LOX and synthesize 12(S)- and 15(S)-hydroxyeicosatetraenoic acids as their major LOX metabolites. W256 cells transfected with 12-LOX-specific antisense oligonucleotide or antisense oligonucleotides directed to conserved regions of LOXs underwent time- and dose-dependent apoptosis. Likewise, treatment of W256 cells with various LOX but not COX inhibitors induced apoptotic cell death, which could be partially inhibited by exogenous 12(S)- or 15(S)-hydroxyeicosatetraenoic acids. The W256 cell apoptosis induced by antisense oligos and LOX inhibitors was followed by a rapid downregulation of bcl-2 protein, a dramatic decrease in the bcl-2/bax ratio, and could be suppressed by bcl-2 overexpression. In contrast, p53, which is wild type in W256 cells, did not undergo alterations during apoptosis induction. The results suggest that the LOX pathway plays an important physiological role in regulating apoptosis.     

A single nucleotide polymorphism in the CYP4F2 but not CYP4A11 gene is associated with increased 20-HETE excretion and blood pressure

Arachidonic acid is a major fatty acid that can be metabolized by the cytochrome P450 enzyme to a number of bioactive eicosanoids. A major metabolite of this oxidation is 20-hydroxyeicosatetraenoic acid, which acts as a potent vasoconstrictor. However, in the kidney, its vasoconstrictor actions can be offset by its natriuretic properties. A guanine-to-adenine polymorphism in the CYP4F2 gene was associated with a reduction in 20-hydroxyeicosatetraenoic acid production in vitro. A thymidine-to-cytosine polymorphism in the CYP4A11 gene reduced catalytic activity by >50% in vitro and was associated with hypertension. The aim was to determine whether these 2 mutations are associated with urinary 20-hydroxyeicosatetraenoic acid excretion and blood pressure in humans. For the CYP4F2, 51% were homozygous for the G allele, 40% were carriers, and 9% were homozygous for the A allele. For CYP4A11, 72% were homozygous for the T allele, 25% were carriers, and 3% were homozygous for the C allele. The CYP4F2 GA/AA genotype was significantly associated with an increase in both 20-hydroxyeicosatetraenoic acid excretion and systolic blood pressure. The CYP4A11 CC/TC genotype was significantly associated with a reduction in 20-hydroxyeicosatetraenoic acid excretion but was not associated with blood pressure. We have demonstrated for the first time in humans that polymorphisms of the CYP4F2 and CYP4A11 genes have opposite effects on 20-hydroxyeicosatetraenoic acid excretion. The positive association between the CYP4F2 GA/AA genotype and both systolic blood pressure and 20-hydroxyeicosatetraenoic acid excretion strengthens a role for 20-hydroxyeicosatetraenoic acid in the modulation of blood pressure.