VEGF-induced relaxation of pulmonary arteries is mediated by endothelial cytochrome P-450 hydroxylase

The cytochrome P-450 metabolite 20-HETE induces calcium-, endothelial-, and nitric oxide (NO)-dependent relaxation of bovine pulmonary arteries (PA). VEGF is an NO-dependent dilator of systemic arteries and plays a key role in maintaining the integrity of the pulmonary vasculature. We tested the effect of VEGF on PA diameter and tone and the contribution of cytochrome P-450 family 4 (CYP4) to vasoactive effects of VEGF. Bovine PA rings (1 mm in diameter) relaxed with VEGF (0.1-10 nM) in an endothelial- and eNOS-dependent manner. This response was blunted by pretreatment with the CYP4 inhibitor dibromododecynyl methyl sulfonamide (DDMS) as well as a mechanistically different CYP4 inhibitor N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine. PAs also increased in diameter by 6-12% in the presence of VEGF (10 nM), and this increase was attenuated by DDMS. In contrast to that shown in PAs, 20-HETE constricted bovine renal arteries and did not increase intracellular Ca(2+) in renal artery endothelial cells as observed in bovine pulmonary artery endothelial cells (BPAECs). VEGF-evoked increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) in BPAECs were blunted by treatment with DDMS. Both VEGF (10 nM) and 20-HETE (1-5 microM) stimulated NO release from cultured BPAECs, and once again VEGF-induced increases were attenuated by pretreating the cells with DDMS. We conclude that CYP4/20-HETE contributes to VEGF-stimulated NO release and vasodilation in bovine PAs. Given the unique expression of 20-HETE-forming CYP4 in BPAECs vs. systemic arterial endothelial cells, CYP4 may be an important mediator of endothelial-dependent vasoreactivity in PAs.     

Prostaglandin and fatty acid omega- and (omega-1)-oxidation in rabbit lung. Acetylenic fatty acid mechanism-based inactivators as specific inhibitors

Terminal acetylenic fatty acid mechanism-based inhibitors (Ortiz de Montellano, P. R., and Reich, N. O. (1984) J. Biol. Chem. 259, 4136-4141) were used as probes in determining the substrate specificity of rabbit lung cytochrome P-450 isozymes of pregnant animals in both microsomes and reconstituted systems. Lung microsomal and reconstituted P-450 form 5-catalyzed lauric acid omega- and (omega-1)-hydroxylase activities were inhibited by a 12-carbon terminal acetylenic fatty acid, 11-dodecynoic acid (11-DDYA), and an 18-carbon terminal acetylenic fatty acid, 17-octadecynoic acid (17-ODYA). Rabbit lung microsomal lauric acid omega-hydroxylase activity was more sensitive to inhibition by 11-DDYA than was (omega-1)-hydroxylase activity. In reconstituted systems containing purified P-450 form 5, both omega- and (omega-1)-hydroxylation of lauric acid were inhibited in parallel when either 11-DDYA or 17-ODYA was used. These data suggest the presence of at least two P-450 isozymes in rabbit lung microsomes capable of lauric acid omega-hydroxylation. This is the first report indicating the multiplicity of lauric acid hydroxylases in lung microsomes. Lung microsomal prostaglandin omega-hydroxylation, mediated by the pregnancy-inducible P-450PG-omega (Williams, D. E., Hale, S. E., Okita, R. T., and Masters, B. S. S. (1984) J. Biol. Chem. 259, 14600-14608) was subject to inhibition by 17-ODYA only, whereas 11-DDYA acid was not an effective inhibitor of this hydroxylase. We have recently developed a new terminal acetylenic fatty acid, 12-hydroxy-16-heptadecynoic acid (12-HHDYA), that contains a hydroxyl group at the omega-6 position. We show that 12-HHDYA possesses a high degree of selectivity for the inactivation of rabbit lung microsomal prostaglandin omega-hydroxylase activity which cannot be obtained with the long chain acetylenic inhibitor, 17-ODYA. In addition, 12-HHDYA has no effect on lauric acid omega- or omega-1-hydroxylation or on benzphetamine N-demethylation. The development of this new terminal acetylenic fatty acid inhibitor provides us with a useful tool with which to study the physiological role of prostaglandin omega-hydroxylation in the rabbit lung during pregnancy.     

20-HETE contributes to the acute fall in cerebral blood flow after subarachnoid hemorrhage in the rat

This study examined the effects of blocking the formation of 20-hydroxyeicosatetraenoic acid (20-HETE) on the acute fall in cerebral blood flow after subarachnoid hemorrhage (SAH) in the rat. In vehicle-treated rats, regional cerebral blood flow (rCBF) measured with laser-Doppler flowmetry fell by 30% 10 min after the injection of 0.3 ml of arterial blood into the cisterna magna, and it remained at this level for 2 h. Pretreatment with inhibitors of the formation of 20-HETE, 17-octadecynoic acid (17-ODYA; 1.5 nmol intrathecally) and N-hydroxy-N'-(4-butyl-2-methylphenyl)formamidine (HET0016; 10 mg/kg iv), reduced the initial fall in rCBF by 40%, and rCBF fully recovered 1 h after induction of SAH. The concentration of 20-HETE in the cerebrospinal fluid rose from 12 +/- 2 to 199 +/- 17 ng/ml after SAH in vehicle-treated rats. 20-HETE levels averaged only 15 +/- 11 and 39 +/- 13 ng/ml in rats pretreated with 17-ODYA or HET0016, respectively. HET0016 selectively inhibited the formation of 20-HETE in rat renal microsomes with an IC(50) of <15 nM and human recombinant CYP4A11, CYP4F2, and CYP4F3 enzymes with an IC(50) of 42, 125, and 100 nM, respectively. These results indicate that 20-HETE contributes to the acute fall in rCBF after SAH in rats.     

High dietary salt reduces the contribution of 20-HETE to arteriolar oxygen responsiveness in skeletal muscle

The coupling of tissue blood flow to cellular metabolic demand involves oxygen-dependent adjustments in arteriolar tone, and arteriolar responses to oxygen can be mediated, in part, by changes in local production of 20-HETE. In this study, we examined the long-term effect of dietary salt on arteriolar oxygen responsiveness in the exteriorized, superfused rat spinotrapezius muscle and the role of 20-HETE in this responsiveness. Rats were fed either a normal-salt (NS, 0.45%) or high-salt (HS, 4%) diet for 4-5 wk. There was no difference in steady-state tissue Po(2) between NS and HS rats, and elevation of superfusate oxygen content from 0% to 10% caused tissue Po(2) to increase by the same amount in both groups. However, the resulting reductions in arteriolar diameter and blood flow were less in HS rats than NS rats. Inhibition of 20-HETE formation with N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) or 17-octadecynoic acid (17-ODYA) attenuated oxygen-induced constriction in NS rats but not HS rats. Exogenous 20-HETE elicited arteriolar constriction that was greatly reduced by the large-conductance Ca(2+)-activated potassium (K(Ca)) channel inhibitors tetraethylammonium chloride (TEA) and iberiotoxin (IbTx) in NS rats and a smaller constriction that was less sensitive to TEA or IbTx in HS rats. Arteriolar responses to exogenous angiotensin II were similar in both groups but more sensitive to inhibition with DDMS in NS rats. Norepinephrine-induced arteriolar constriction was similar and insensitive to DDMS in both groups. We conclude that 20-HETE contributes to oxygen-induced constriction of skeletal muscle arterioles via inhibition of K(Ca) channels and that a high-salt diet impairs arteriolar responses to increased oxygen availability due to a reduction in vascular smooth muscle responsiveness to 20-HETE.     

Cytochromes P450 from family 4 are the main omega hydroxylating enzymes in humans: CYP4F3B is the prominent player in PUFA metabolism

Human CYP450 omega-hydroxylases of the CYP4 family are known to convert arachidonic acid (AA) to its metabolite 20-hydroxyeicosatetraenoic acid (20-HETE). This study deals with hydroxylations of four PUFAs, eicosatrienoic acid (ETA), AA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) by either human recombinant CYP4s enzymes or human liver microsomal preparations. CYP4F3A and CYP4F3B were the most efficient omega-hydroxylases of these PUFAs. Moreover, the differences in the number of unsaturations of ETA, AA, and EPA allowed us to demonstrate a rise in the metabolic rate of hydroxylation when the double bond in 14-15 or 17-18 was missing. With the CYP4F enzymes, the main pathway was always the omega-hydroxylation of PUFAs, whereas it was the (omega-1)-hydroxylation with CYP1A1, CYP2C19, and CYP2E1. Finally, we demonstrated that the omega9 and omega3 PUFAs (ETA, EPA, and DHA) could all be used as alternative substrates in AA metabolism by human CYP4F2 and -4F3B. Thus, they decreased the ability of these enzymes to convert AA to 20-HETE. However, although ETA was the most hydroxylated substrate, EPA and DHA were the most potent inhibitors of the conversion of AA to 20-HETE. These findings suggest that some physiological effects of omega3 FAs could partly result from a shift in the generation of active hydroxylated metabolites of AA through a CYP-mediated catalysis.     

Src tyrosine kinase is the trigger but not the mediator of ischemic preconditioning

The present study determined the role of 20-hydroxyeicosatetraenoic acid [20-HETE; produced by omega-hydroxylation of arachidonic acid via cytochrome P-450 (CP450) 4A enzymes] in regulating myogenic activation of skeletal muscle resistance arteries from normotensive (NT) and hypertensive (HT) Dahl salt-sensitive (SS) rats. Gracilis arteries (GA) were isolated from each rat and viewed via television microscopy, and changes in vessel diameter with altered transmural pressure were measured with a video micrometer. Under control conditions, GA from both groups exhibited strong, endothelium-independent myogenic activation. Treatment of GA with 17-octadecynoic acid (17-ODYA; inhibitor of CP450 4A enzymes) did not alter myogenic activation in NT rats, but impaired this response in HT animals. Treatment of GA from HT rats with dibromo-dodecynyl-methylsulfimide (DDMS; inhibitor of 20-HETE production) impaired myogenic activation, as did application of 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid, an antagonist for 20-HETE receptors. Application of iberiotoxin, a Ca(2+)-activated potassium (K(Ca)) channel inhibitor, restored myogenic activation from HT rats treated with DDMS. These results suggest that myogenic activation of skeletal muscle resistance arteries from NT Dahl-SS rats does not depend on CP450, whereas myogenic activation of these vessels in HT Dahl-SS rats is partly a function of 20-HETE production, inhibiting K(Ca) channels through a receptor-mediated process.     

Effects of high-salt diet on CYP450-4A omega-hydroxylase expression and active tone in mesenteric resistance arteries

This study investigated the role of changes in the expression of the cytochrome P-450 4A (CYP450-4A) enzymes that produce 20-hydroxyeicosatetraenoic acid (20-HETE) in modulating the responses of rat mesenteric resistance arteries to norepinephrine (NE) and reduced Po(2) after short-term (3-day) changes in dietary salt intake. The CYP450-4A2, -4A3, and -4A8 isoforms were all detected by RT-PCR in arteries obtained from rats fed a high-salt (HS, 4% NaCl) diet, whereas only the CYP450-4A3 isoform was detected in vessels from rats fed a low-salt (LS, 0.4% NaCl) diet. Expression of the 51-kDa CYP450-4A protein was significantly increased by a HS diet. Inhibiting 20-HETE synthesis with 30 muM N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) reduced the vasoconstrictor response to NE in arteries obtained from rats fed either a LS or HS diet, but NE sensitivity after DDMS treatment was significantly lower in vessels from rats on a HS diet. DDMS treatment also restored the vasodilator response to reduced Po(2) that was impaired in arteries from rats on a HS diet. These findings suggest that 1) a HS diet increases the expression of CYP450-4A enzymes in the mesenteric vasculature, 2) 20-HETE contributes to the vasoconstrictor response to NE in mesenteric resistance arteries, 3) the contribution of 20-HETE to the vasoconstrictor response to NE is greater in rats fed a HS diet than in rats fed a LS diet, and 4) upregulation of the production of 20-HETE contributes to the impaired dilation of mesenteric resistance arteries in response to hypoxia in rats fed a HS diet.     

Nitric oxide contributes to 20-HETE-induced relaxation of pulmonary arteries.

In contrast to its constrictor effects on peripheral arteries, 20-hydroxyeicosatetraenoic acid (20-HETE) is an endothelial-dependent dilator of pulmonary arteries (PAs). The present study examined the hypothesis that the vasodilator effects of 20-HETE in PAs are due to an elevation of intracellular calcium concentration ([Ca(2+)](i)) and the release of nitric oxide (NO) from bovine PA endothelial cells (BPAECs). BPAECs express cytochrome P-450 4A (CYP4A) protein and produce 20-HETE. 20-HETE dilated PAs preconstricted with U-46619 or norepinephrine and treated with the cytochrome P-450 inhibitor 17-octadecynoic acid and the cyclooxygenase inhibitor indomethacin. The dilator effect of 20-HETE was blocked by the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) or by removal of endothelium. 20-HETE significantly increased [Ca(2+)](i) and NO production in BPAECs. 20-HETE-induced NO release was blunted by removal of extracellular calcium, as well as NO synthase inhibitors (L-NAME). These results suggest that 20-HETE dilates PAs at least in part by increasing [Ca(2+)](i) and NO release in BPAECs.     

Evaluation of cytochrome P450 4 family as mediator of phospholipase D activation in aortic vascular smooth muscle cells

Norepinephrine (NE) stimulates phospholipase D (PLD) activity via phospholipase A2-dependent arachidonic acid release in rabbit aortic vascular smooth muscle cells (VSMC). We have previously shown that exogenous 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid generated through the cytochrome P450 (CYP) 4A pathway in vivo, stimulates PLD activity. Whether endogenous CYP4-derived arachidonic acid metabolites act as intracellular mediators of NE-induced PLD activation in VSMC is not known. In rabbit aortic VSMC, prototypical hepatic/renal CYP4A inducers such as fenofibrate and Wy 14643 inhibited both basal and NE-induced PLD activity after 48 h of exposure. The level of CYP4F, and to a lesser extent CYP4A, was also decreased by these agents. The expression levels of rabbit aortic VSMC CYP4A and CYP4F isoforms were reduced by antisense oligonucleotides treatment for 48 hours as measured by RTQ-PCR or Western blotting. This reduction in CYP4A or CYP4F levels did not change NE-induced PLD activation. The corresponding CYP4A scrambled and CYP4F sense oligonucleotides did not alter CYP levels. PLD activity was increased by ~70% after 15 min of stimulation with NE, whereas lauric acid omega-hydroxylase activity, a measure of fatty acid omega-hydroxylation, was unchanged. Inhibition of omega-hydroxylation with DDMS and HET0016, selective omega-hydroxylase inhibitors, and 20-HEDE, an antagonist of 20-HETE, increased PLD activity in a concentration-dependent manner and did not alter NE-induced PLD activation. These data suggest that PLD activation by NE is independent of the CYP4A/4F enzymes in rabbit aortic VSMC.     

Oxygenation of omega-3 fatty acids by human cytochrome P450 4F3B: effect on 20-hydroxyeicosatetraenoic acid production

Cytochrome P450 (CYP) omega-oxidases convert arachidonic acid (AA) to 20-hydroxyeicosatetraenoic acid (20-HETE), a lipid mediator that modulates vascular tone. We observed that a microsomal preparation containing recombinant human CYP4F3B, which converts AA to 20-HETE, converted eicosapentaenoic acid (EPA) to 20-OH-EPA. Likewise, docosahexaenoic acid (DHA) was converted to 22-OH-DHA, indicating that human CYP4F3B also can oxidize 22-carbon omega-3 fatty acids. Consistent with these findings, addition of 0.5-5 microM EPA, DHA or omega-3 docosapentaenoic acid (DPA) to incubations containing 0.5 microM [3H]AA inhibited [3H]20-HETE production by 15-65%. [3H]20-OH-EPA was rapidly taken up by COS-7 cells, and almost all of the incorporated radioactivity remained as unmodified 20-OH-EPA. The 20-OH-EPA stimulated luciferase activity in COS-7 cells that express peroxisome proliferator-activated receptor alpha, indicating that this EPA metabolite may function as a lipid mediator. These findings suggest that some functional effects of omega-3 fatty acid supplementation may be due to inhibition of 20-HETE formation or the conversion of EPA to the corresponding omega-oxidized product.     

Effects of selective inhibition of cytochrome P-450 omega-hydroxylases and ischemic preconditioning in myocardial protection

Cytochrome P-450 (CYP) omega-hydroxylases and their arachidonic acid (AA) metabolite, 20-hydroxyeicosatetraenoic acid (20-HETE), produce a detrimental effect on ischemia-reperfusion injury in canine hearts, and the inhibition of CYP omega-hydroxylases markedly reduces myocardial infarct size expressed as a percentage of the area at risk (IS/AAR, %). In this study, we demonstrated that a specific CYP omega-hydroxylase inhibitor, N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), markedly reduced 20-HETE production during ischemia-reperfusion and reduced myocardial infarct size compared with control [19.5 +/- 1.0% (control), 9.6 +/- 1.5% (0.40 mg/kg DDMS), 4.0 +/- 2.0% (0.81 mg/kg DDMS), P < 0.01]. In addition, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE, a putative 20-HETE antagonist) significantly reduced myocardial infarct size from control [10.3 +/- 1.3% (0.032 mg/kg 20-HEDE) and 5.9 +/- 1.9% (0.064 mg/kg 20-HEDE), P < 0.05]. We further demonstrated that one 5-min period of ischemic preconditioning (IPC) reduced infarct size to a similar extent as that observed with the high doses of DDMS and 20-HEDE, and the higher dose of DDMS given simultaneously with IPC augmented the infarct size reduction [9.9 +/- 2.8% (IPC) to 2.5 +/- 1.4% (0.81 mg/kg DDMS), P < 0.05] to a greater degree than that observed with either treatment alone. These results suggest an important negative role for endogenous CYP omega-hydroxylases and their product, 20-HETE, to exacerbate myocardial injury in canine myocardium. Furthermore, for the first time, this study demonstrates that the effect of IPC and the inhibition of CYP omega-hydroxylase synthesis (DDMS) or its actions (20-HEDE) may have additive effects in protecting the canine heart from ischemia-reperfusion injury.     

Inhibition of 20-HETE abolishes the myogenic response during NOS antagonism in the ovine fetal pulmonary circulation

Mechanisms that maintain high pulmonary vascular resistance (PVR) and oppose vasodilation in the fetal lung are poorly understood. In fetal lambs, increased pulmonary artery pressure evokes a potent vasoconstriction, suggesting that a myogenic response contributes to high PVR in the fetus. In adult systemic circulations, the arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) has been shown to modulate the myogenic response, but its role in the fetal lung is unknown. We hypothesized that acute increases in pulmonary artery pressure release 20-HETE, which causes vasoconstriction, or a myogenic response, in the fetal lung. To address this hypothesis, we studied the hemodynamic effects of N-methylsufonyl-12,12-dibromododec-11-enamide (DDMS), a specific inhibitor of 20-HETE production, on the pulmonary vasoconstriction caused by acute compression of the ductus arteriosus (DA) in chronically prepared fetal sheep. An inflatable vascular occluder around the DA was used to increase pulmonary artery pressure under three study conditions: control, after pretreatment with nitro-L-arginine (L-NA; to inhibit shear-stress vasodilation), and after combined treatment with both L-NA and a specific 20-HETE inhibitor, DDMS. We found that DA compression after L-NA treatment increased PVR by 44 +/- 12%. Although intrapulmonary DDMS infusion did not affect basal PVR, DDMS completely abolished the vasoconstrictor response to DA compression in the presence of L-NA (44 +/- 12% vs. 2 +/- 4% change in PVR, L-NA vs. L-NA + DDMS, P < 0.05). We conclude that 20-HETE mediates the myogenic response in the fetal pulmonary circulation and speculate that pharmacological inhibition of 20-HETE might have a therapeutic role in neonatal conditions characterized by pulmonary hypertension.     

The CYP450 hydroxylase pathway contributes to P2X receptor-mediated afferent arteriolar vasoconstriction

This study was conducted to test the hypothesis that the cytochrome P-450 (CYP450) metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) contributes to the afferent arteriolar response to P2 receptor activation. Afferent arteriolar responses to ATP, the P2X agonist, alpha,beta-methylene ATP and the P2Y agonist UTP were determined before and after treatment with the selective CYP450 hydroxylase inhibitor, N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) or the 20-HETE antagonist, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (20-HEDE). Stimulation with 1.0 and 10 microM ATP elicited an initial preglomerular vasoconstriction of 12 +/- 1% and 45 +/- 4% and a sustained vasoconstriction of 11 +/- 1% and 11 +/- 2%, respectively. DDMS or 20-HEDE significantly attenuated the sustained afferent arteriolar constrictor response to ATP. alpha,beta-Methylene ATP (1 microM) induced a rapid initial afferent vasoconstriction of 64 +/- 3%, which partially recovered to a stable diameter 10 +/- 1% smaller than control. Both DDMS and 20-HEDE significantly attenuated the initial vasoconstriction and abolished the sustained vasoconstrictor response to alpha,beta-methylene ATP. UTP decreased afferent diameter by 50 +/- 5% and 20-HEDE did not change this response. In addition, the ATP-induced increase in the intracellular Ca2+ concentration in preglomerular microvascular smooth muscle cells was significantly attenuated by 20-HEDE. Taken together, these results are consistent with the hypothesis that the CYP450 metabolite 20-HETE participates in the afferent arteriolar response to activation of P2X receptors.     

Transfection of CYP4A1 cDNA decreases diameter and increases responsiveness of gracilis muscle arterioles to constrictor stimuli

Cytochrome P-450-4A1 (CYP4A1) is an omega-hydroxylase that catalyzes the metabolism of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE). The goal of this study was to determine the vasomotor consequences of vascular overexpression of CYP4A1. Isolated rat gracilis muscle arterioles transfected ex vivo with an expression plasmid containing CYP4A1 cDNA expressed more CYP4A protein than vessels transfected with the control plasmid. In arterioles pressurized to 80 mmHg, the internal diameter of vessels transfected with CYP4A1 cDNA (55 +/- 3 microm) was surpassed (P < 0.05) by that of vessels transfected with control plasmid (97 +/- 4 microm). Treatment with a CYP4A inhibitor (N-methylsulfonyl-12,12-dibromododec-11-enamide; DDMS) or with an antagonist of 20-HETE actions [20-hydroxyeicosa-6(Z),15(Z)-dienoic acid; 20-HEDE] elicited robust dilation of arterioles transfected with CYP4A1 cDNA, whereas the treatment had little or no effect in vessels transfected with control plasmid. Examination of the intraluminal pressure-internal diameter relationship revealed that pressure increments over the range of 40-100 mmHg elicited a more intense (P < 0.05) myogenic constrictor response in arterioles transfected with CYP4A1 cDNA than in those with control plasmid. Arterioles transfected with CYP4A1 cDNA also displayed enhanced sensitivity to the constrictor action of phenylephrine. Treatment with DDMS or 20-HEDE greatly attenuated the constrictor responsiveness to both constrictor stimuli in vessels overexpressing CYP4A1, whereas the treatment had much less effect in control vessels. These data suggest that CYP4A1 overexpression promotes constriction of gracilis muscle arterioles by intensifying the responsiveness of vascular smooth muscle to constrictor stimuli. This effect of CYP4A1 overexpression appears to be mediated by a CYP4A1 product.     

15-HETE对缺氧兔肺动脉平滑肌钾离子通道的影响

用肺动脉环和全细胞膜片钳技术研究15-羟化二十烷四烯酸(15-HETE)对缺氧兔肺动脉平滑肌钾离子通道的影响。新出生的幼兔分两组,一组放入吸氧分数为0．12的低氧舱内;另一组保持正常氧环境。9d后,称重、取肺动脉进行细胞培养并制作肺动脉环。分别加入4-氨基吡啶(4-aminopyridione,4-AP)、四乙胺(tetraethylammonium,TEA)、glyburide(GLYB)三种特异性钾离子通道阻断剂,观察15-HETE对兔肺动脉平滑肌钾离子通道的作用变化,同时采用全细胞膜片钳测定钾电流。结果显示：5mmol／L 4-AP阻断Kv通道后可以抑制15-HETE诱导的缺氧兔肺动脉收缩;TEA和GLYB分别阻断大电导型钙激活钾通道(BKCa)和KATP通道后并不影响15-HETE诱导的缺氧兔肺动脉收缩;15-HETE可降低兔肺动脉平滑肌细胞钾电流幅度。上述结果提示：缺氧兔肺动脉中,15-HETE阻断电压依赖钾通道(Kv通道),引起膜去极化,可能是缺氧性肺血管收缩的机制之一。     

CYP4A mRNA, protein, and product in rat lungs: novel localization in vascular endothelium.

The vasodilatory effect of 20-hydroxyeicosatetraenoic acid (20-HETE) on lung arteries is opposite to the constrictor effect seen in cerebral and renal vessels. These observations raise questions about the cellular localization of 20-HETE-forming isoforms in pulmonary arteries and other tissues. Using in situ hybridization, we demonstrate for the first time CYP4A (a family of cytochrome P-450 enzymes catalyzing formation of 20-HETE from the substrate arachidonic acid) mRNA in pulmonary arterial endothelial and smooth muscle cells, bronchial smooth muscle and bronchial epithelial cells, type I epithelial cells, and macrophages in adult male rat lungs. Moreover, we detect CYP4A protein in rat pulmonary arteries and bronchi as well as cultured endothelial cells. Finally, we identify endogenously formed 20-HETE by using fluorescent HPLC techniques, as well as the capacity to convert arachidonic acid into 20-HETE in pulmonary arteries, bronchi, and endothelium. These data show that 20-HETE is an endogenous product of several pulmonary cell types and is localized to tissues that optimally position it to modulate physiological functions such as smooth muscle tone or electrolyte flux.     

Contribution of cytochrome P-450 omega-hydroxylase to altered arteriolar reactivity with high-salt diet and hypertension

The present study evaluated the contribution of cytochrome P-450 omega-hydroxylase in modulating the reactivity of cremaster muscle arterioles in normotensive rats on high-salt (HS) and low-salt (LS) diet and in rats with reduced renal mass hypertension (RRM-HT). Changes in arteriolar diameter in response to ACh, sodium nitroprusside (SNP), ANG II, and elevated O(2) were measured via television microscopy under control conditions and following cytochrome P-450 omega-hydroxylase inhibition with 17-octadecynoic acid (17-ODYA) or N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS). In normotensive rats on either LS or HS diet, resting tone was unaffected and arteriolar reactivity to ACh or SNP was minimally affected by cytochrome P-450 omega-hydroxylase inhibition. In RRM-HT rats, cytochrome P-450 omega-hydroxylase inhibition reduced resting tone and significantly enhanced arteriolar dilation to ACh and SNP. Treatment with 17-ODYA or DDMS inhibited arteriolar constriction to ANG II and O(2) in all the groups, although the degree of inhibition was greater in RRM-HT than in normotensive animals. These results suggest that metabolites of cytochrome P-450 omega-hydroxylase contribute to the altered reactivity of skeletal muscle arterioles to vasoconstrictor and vasodilator stimuli in RRM-HT.     

Enalapril reverses high-fat diet-induced alterations in cytochrome P450-mediated eicosanoid metabolism

Metabolism of arachidonic acid by cytochrome P450 (CYP) to biologically active eicosanoids has been recognized increasingly as an integral mediator in the pathogenesis of cardiovascular and metabolic disease. CYP epoxygenase-derived epoxyeicosatrienoic and dihydroxyeicosatrienoic acids (EET + DHET) and CYP ω-hydroxylase-derived 20-hydroxyeicosatetraenoic acid (20-HETE) exhibit divergent effects in the regulation of vascular tone and inflammation; thus, alterations in the functional balance between these parallel pathways in liver and kidney may contribute to the pathogenesis and progression of metabolic syndrome. However, the impact of metabolic dysfunction on CYP-mediated formation of endogenous eicosanoids has not been well characterized. Therefore, we evaluated CYP epoxygenase (EET + DHET) and ω-hydroxylase (20-HETE) metabolic activity in liver and kidney in apoE(-/-) and wild-type mice fed a high-fat diet, which promoted weight gain and increased plasma insulin levels significantly. Hepatic CYP epoxygenase metabolic activity was significantly suppressed, whereas renal CYP ω-hydroxylase metabolic activity was induced significantly in high-fat diet-fed mice regardless of genotype, resulting in a significantly higher 20-HETE/EET + DHET formation rate ratio in both tissues. Treatment with enalapril, but not metformin or losartan, reversed the suppression of hepatic CYP epoxygenase metabolic activity and induction of renal CYP ω-hydroxylase metabolic activity, thereby restoring the functional balance between the pathways. Collectively, these findings suggest that the kinin-kallikrein system and angiotensin II type 2 receptor are key regulators of hepatic and renal CYP-mediated eicosanoid metabolism in the presence of metabolic syndrome. Future studies delineating the underlying mechanisms and evaluating the therapeutic potential of modulating CYP-derived EETs and 20-HETE in metabolic diseases are warranted.     

Postmenopausal hypertension: role of 20-HETE

Blood pressure (BP) increases after menopause. However, the mechanisms responsible have not been elucidated. In this study we tested the hypothesis that 20-hydroxyeicosatetraenoic acids (20-HETE), produced by cytochrome P-450 (CYP450) ω-hydroxylase, contributes to the hypertension in a model of postmenopausal hypertension, aged female spontaneously hypertensive rats (PMR). 1-Aminobenzotriazole, a nonselective inhibitor of arachidonic acid metabolism, for 7 days, reduced BP in PMR but had no effect in young females. Acute intravenous infusion of HET-0016, a specific inhibitor of 20-HETE, over 3 h, also reduced BP in PMR. CYP4A isoform mRNA expression showed no difference in renal CYP4A1 or CYP4A3 but increases in CYP4A2 and decreases in CYP4A8. CYP4A protein expression was decreased in kidney of PMR compared with young females. Endogenous 20-HETE was significantly higher in cerebral vessels of PMR than young females (YF) but was significantly lower in renal vessels of PMR. Omega-hydroxylase activity in cerebral vessels was also higher in PMR but was similar in kidney vessels in both groups. In renal microsomal preparations, endogenous 20-HETE was not different in PMR and young females, but ω-hydroxylase activity was significantly lower in PMR than YF. The data with blockers suggest that 20-HETE contributes to postmenopausal hypertension in SHR. The data also suggest that cerebral production of 20-HETE may be increased and renal tubular production may be decreased in PMR, thus both contributing to their elevated BP.