20-hydroxyeicosatetraenoic acid (20-HETE) metabolism in coronary endothelial cells

We have investigated the role of endothelial cells in the metabolism of 20-hydroxyeicosatetraenoic acid (20-HETE), a vasoactive mediator synthesized from arachidonic acid by cytochrome P450 omega-oxidases. Porcine coronary artery endothelial cells (PCEC) incorporated 20-[(3)H]HETE primarily into the sn-2 position of phospholipids through a coenzyme A-dependent process. The incorporation was reduced by equimolar amounts of arachidonic, eicosapentaenoic or 8,9-epoxyeicosatrienoic acids, but some uptake persisted even when a 10-fold excess of arachidonic acid was available. The retention of 20-[(3)H]HETE increased substantially when methyl arachidonoyl fluorophosphonate, but not bromoenol lactone, was added, suggesting that a Ca(2+)-dependent cytosolic phospholipase A(2) released the 20-HETE contained in PCEC phospholipids. Addition of calcium ionophore A23187 produced a rapid release of 20-[(3)H]HETE from the PCEC, a finding that also is consistent with a Ca(2+)-dependent mobilization process. PCEC also converted 20-[(3)H]HETE to 20-carboxy-arachidonic acid (20-COOH-AA) and 18-, 16-, and 14-carbon beta-oxidation products. 20-COOH-AA produced vasodilation in porcine coronary arterioles, but 20-HETE was inactive. These results suggest that the incorporation of 20-HETE and its subsequent conversion to 20-COOH-AA in the endothelium may be important in modulating coronary vascular function.     

Low shear stress induces vascular eNOS uncoupling via autophagy-mediated eNOS phosphorylation

Uncoupled endothelial nitric oxide synthase (eNOS) produces O₂^? instead of nitric oxide (NO). Earlier, we reported rapamycin, an autophagy inducer and inhibitor of cellular proliferation, attenuated low shear stress (SS) induced O₂^? production. Nevertheless, it is unclear whether autophagy plays a critical role in the regulation of eNOS uncoupling. Therefore, this study aimed to investigate the modulation of autophagy on eNOS uncoupling induced by low SS exposure. We found that low SS induced endothelial O₂^? burst, which was accompanied by reduced NO release. Furthermore, inhibition of eNOS by L-NAME conspicuously attenuated low SS-induced O₂^? releasing, indicating eNOS uncoupling. Autophagy markers such as LC3 II/I ratio, amount of Beclin1, as well as ULK1/Atg1 were increased during low SS exposure, whereas autophagic degradation of p62/SQSTM1 was markedly reduced, implying impaired autophagic flux. Interestingly, low SS-induced NO reduction could be reversed by rapamycin, WYE-354 or ATG5 overexpression vector via restoration of autophagic flux, but not by N-acetylcysteine or apocynin. eNOS uncoupling might be ascribed to autophagic flux blockade because phosphorylation of eNOS Thr495 by low SS or PMA stimulation was also regulated by autophagy. In contrast, eNOS acetylation was not found to be regulated by low SS and autophagy. Notably, although low SS had no influence on eNOS Ser1177 phosphorylation, whereas boosted eNOS Ser1177 phosphorylation by rapamycin were in favor of the eNOS recoupling through restoration of autophagic flux. Taken together, we reported a novel mechanism for regulation of eNOS uncoupling by low SS via autophagy-mediated eNOS phosphorylation, which is implicated in geometrical nature of atherogenesis.     

Mthfr deficiency induces endothelial progenitor cell senescence via uncoupling of eNOS and downregulation of SIRT1

Hyperhomocysteinemia (HHcy) has been shown to induce endothelial dysfunction in part as a result of enhanced oxidative stress. Function and survival of endothelial progenitor cells (EPCs, defined as sca1(+) c-kit(+) flk-1(+) bone marrow-derived cells), which significantly contribute to neovascularization and endothelial regeneration, depend on controlled production of reactive oxygen species (ROS). Mice heterozygous for the gene deletion of methylenetetrahydrofolate reductase (Mthfr(+/-)) have a 1.5- to 2-fold elevation in plasma homocysteine. This mild HHcy significantly reduced the number of circulating EPCs as well as their differentiation. Mthfr deficiency was also associated with increased ROS production and reduced nitric oxide (NO) generation in Mthfr(+/-) EPCs. Treatment of EPCs with sepiapterin, a precursor of tetrahydrobiopterin (BH(4)), a cofactor of endothelial nitric oxide synthase (eNOS), significantly reduced ROS and improved NO production. mRNA and protein expression of eNOS and the relative amount of eNOS dimer compared with monomer were decreased by Mthfr deficiency. Impaired differentiation of EPCs induced by Mthfr deficiency correlated with increased senescence, decreased telomere length, and reduced expression of SIRT1. Addition of sepiapterin maintained cell senescence and SIRT1 expression at levels comparable to the wild type. Taken together, these results demonstrate that Mthfr deficiency impairs EPC formation and increases EPC senescence by eNOS uncoupling and downregulation of SIRT1.     

Regulation of MAP kinase-mediated endothelial dysfunction in hyperglycemia via arginase I and eNOS dysregulation

Emerging evidence suggests that arginase contributes to endothelial dysfunction in diabetes. Intracellular signaling pathways, which interplay between arginase and eNOS enzyme activity leading to the development of endothelial dysfunction in hyperglycemia are not fully understood. Here, we analyzed the possible involvement of hyperglycemia (HG) induced arginase expression in eNOS protein regulation and activity and also the impact of arginase inhibition on eNOS activity. Furthermore, the roles of p38 MAPK and Erk1/2 phosphorylation in upregulation of arginase expression and eNOS dysregulation in endothelial cells (ECs) under hyperglycemia were evaluated. Protein analysis showed a concurrent increase in arginase I expression and decrease in eNOS expression and phosphorylation at Ser¹¹⁷⁷ under HG conditions. There was no simultaneous change in phosphorylation of eNOS at Thr⁴⁹⁵ in HG. Arginase inhibition prevented increased arginase activity, restored impaired NO bioavailability and reduced superoxide anion generation. Inhibition of MAP-kinases demonstrated that, unlike Erk1/2, p38 MAPK is an upstream activator in a signaling cascade leading to increased arginase I in HG conditions. P38 MAPK protein expression and phosphorylation were increased in response to HG. In the presence of a p38 MAPK inhibitor, HG-induced arginase expression was blunted. Although Erk1/2 was activated in HG, increased arginase expression was not blocked by co-treatment with an Erk1/2 inhibitor. Activation of both, p38 MAPK and Erk1/2 in HG, induced a downregulation in eNOS activity. Hence, applying MAPK inhibitors increased eNOS phosphorylation in HG.In conclusion, these findings demonstrate contributions of arginase I in the development of endothelial cell dysfunction under HG conditions via impaired eNOS regulation, which maybe mediated by p38 MAPK.     

Short-term passive smoking causes endothelial dysfunction via oxidative stress in nonsmokers

Recent studies have shown that passive smoking impairs vascular endothelial function and induces oxidative stress in humans. However, in most of the previous human data regarding tobacco-induced pathophysiology, vascular endothelial dysfunction and oxidative stress have been separately assessed. This study was designed to determine the association between the acute effect of passive smoking on vascular endothelial function and in-vivo oxidative stress status. We studied 30 healthy male Japanese volunteers (32 +/- 7 years) including 15 habitual smokers and 15 nonsmokers. After baseline echocardiographic, hemodynamic recording, and blood sampling, subjects were exposed to passive smoking for 30 min. Endothelium-dependent vasodilation was measured by using % flow-mediated vasodilation (%FMD) of the brachial artery and plasma levels of 8-isoprostane was measured by enzyme immunoassay before and after the passive smoking exposure. Baseline %FMD was lower (4.3% +/- 1.2% vs. 10.9% +/- 3.1%, p < 0.001) and baseline plasma 8-isoprostane level was higher (41.5 +/- 5.8 pg/mL vs. 26.9 +/- 5.4 pg/mL, p < 0.001) in smokers than those in nonsmokers. The %FMD and 8-isoprostane level did not change after passive smoking in smokers. In nonsmokers, however, the %FMD decreased (to 5.0% +/- 1.9%, p < 0.001) and the 8-isoprostane level increased (to 37.8 +/- 9.6 pg/mL, p < 0.001) significantly after 30 min passive smoking exposure, equivalently to the levels of smokers. Sixty corrected samples before and after passive smoking exposure in all patients showed a significant negative correlation between the % FMD and the plasma 8-isoprostane levels (n = 60, r = -0.69, p < 0.001). Even 30 min of passive smoking rapidly impairs vascular endothelial function, which is associated with oxidative stress. Our data provide the pathophysiological insight for the recent epidemiological evidence about the increased risk of coronary heart disease among nonsmokers exposed to passive smoking.     

Localization of 12-hydroxyeicosatetraenoic acid in endothelial cells

Bovine aortic endothelial cells take up 12-hydroxyeicosatetraenoic acid (12-HETE), a lipoxygenase product formed from arachidonic acid. The uptake of [3H]12-HETE reached a maximum in 2 to 4 h. At this time, from 75 to 80% of the incorporated radioactivity was contained in phospholipids, about 85% of the esterified radioactivity remained in the form of 12-HETE, and at least 90% of the phospholipid radioactivity was present in the sn-2-position. Subcellular fractionation on Percoll and sucrose gradients demonstrated that 65 to 74% of the radioactivity was present in membranes enriched in NADPH-cytochrome c reductase and UDP-galactosyl transferase. The specific radioactivity relative to protein of these intracellular membranes was 2.9-times higher than in a plasma membrane fraction enriched in 5'-nucleotidase. A similar intracellular localization was observed when [3H]5-HETE or [3H]arachidonic acid were taken up. The 12-HETE was contained primarily in the choline glycerophospholipids of the microsomal membranes. After incorporation, [3H]12-HETE was removed from the cell lipids much more rapidly than [3H]arachidonic acid, and 80% of the radioactivity released into the medium during the first hour remained as 12-HETE. Because it accumulates in microsomal membranes, 12-HETE uptake may perturb certain intracellular processes and thereby lead to endothelial dysfunction. The relatively rapid removal of the newly incorporated 12-HETE may be an important protective mechanism that prevents excessive accumulation and more extensive endothelial damage.     

IGF-I alleviates diabetes-induced RhoA activation, eNOS uncoupling, and myocardial dysfunction

IGF-I rescues diabetic heart defects and oxidative stress, although the underlying mechanism of action remains poorly understood. This study was designed to delineate the beneficial effects of IGF-I with a focus on RhoA, Akt, and eNOS coupling. Echocardiography was performed in normal or diabetic Friend Virus-B type (FVB) and IGF-I transgenic mice. Cardiomyocyte contractile properties were evaluated using peak shortening (PS), time-to-90% relengthening (TR90), and intracellular Ca2+ rise and decay. Diabetes reduced fraction shortening, PS, and intracellular Ca2+; it increased chamber size, prolonged TR90, and intracellular Ca2+ decay. Levels of RhoA mRNA, active RhoA, and O2(-) were elevated, whereas nitric oxide (NO) levels were reduced in diabetes. Diabetes-induced O2(-) accumulation was ablated by the NO synthase (NOS) inhibitor nitro-L-arginine methyl ester (L-NAME), indicating endothelial NOS (eNOS) uncoupling, all of which except heart size were negated by IGF-I. The IGF-I-elicited beneficial effects were mimicked by the Rho kinase inhibitor Y27632 and BH4. Diabetes depressed expression of Kv1.2 and dihydrofolate reductase (DHFR), increased beta-myosin heavy-chain expression, stimulated p38 MAPK, and reduced levels of total Akt and phosphorylated Akt/eNOS, all of which with the exception of myosin heavy chain were attenuated by IGF-I. In addition, Y27632 and the eNOS coupler folate abrogated glucose toxicity-induced PS decline, TR90 prolongation, while it increased O2(-) and decreased NO and Kv1.2 levels. The DHFR inhibitor methotrexate impaired myocyte function, NO/O2(-) balance, and rescued Y27632-induced cardiac protection. These results revealed that IGF-I benefits diabetic hearts via Rho inhibition and antagonism of diabetes-induced decrease in pAkt, eNOS uncoupling, and K+ channel expression.     

Detection of 20-hydroxyeicosatetraenoic acid in rat urine

20-Hydroxyeicosatetraenoic acid (20-HETE), an arachidonate metabolite of the cytochrome P450 omega hydroxylase, was detected in rat urine by gas chromatography-mass spectrometric techniques. The concentration of 20-HETE in urine from 7-week-old hypertensive and normotensive rats was 2.1 and 1.3 nM, respectively. This is the first demonstration of 20-HETE urinary excretion and thus calls attention to the possibility that 20-HETE participates in the regulation of renal function via its effect on vascular tone and ion transport processes.     

Conversion of 15-hydroxyeicosatetraenoic acid to 11-hydroxyhexadecatrienoic acid by endothelial cells

Cultured endothelial cells take up 15-hydroxyeicosatetraenoic acid (15-HETE), a lipoxygenase product formed from arachidonic acid, and incorporate it into cellular phospholipids and glycerides. Uptake can occur from either the apical or basolateral surface. A substantial amount of the 15-HETE incorporated into phospholipids is present in the inositol phosphoglycerides. 15-HETE is converted into several metabolic products that accumulate in teh extracellular fluid; this conversion does not require stimulation by agonists. The main product has been identified as 11-hydroxyhexadecatrienoic acid [16:3(11-OH)], a metabolite of 15-HETE that has not been described previously. Formation of 16:3(11-OH) decreases when 4-pentenoic acid is present, suggesting that it is produced by beta-oxidation. The endothelial cells can take up 16:3(11-OH) only 25% as effectively as 15-HETE, and 16:3(11-OH) is almost entirely excluded from the inositol phosphoglycerides. These results suggest that the endothelial cells can incorporate 15-HETE when it is released into their environment. Through partial oxidation, the endothelium can process 15-HETE to a novel metabolite that is less effectively taken up and, in particular, is excluded from the inositol phosphoglycerides.     

20-hydroxyeicosatetraenoic acid (20-HETE): structural determinants for renal vasoconstriction

The effects of natural and synthetic eicosanoids on the diameter of rat interlobular arteries studied in vitro were compared to that of the potent, endogenous vasoconstrictor 20-HETE. Vasoconstrictor activity was optimum for chain lengths of 20-22 carbons with at least one olefin or epoxide between located between C(13)-C(15) and an oxygen substituent at C(20)-C(22). The presence of delta (Zou et al. Am. J. Physiol. 1996, 270, R228; Gebremedhin, D. et al. Am. J. Physiol. 1998, 507, 771)-, delta (Carroll et al. Am. J. Physiol. 1996, 271, R863; Vazquez et al. Life Sci. 1995, 56, 1455)-, or delta (Imig et al. Hypertension 2000, 35, 307; Lopez et al. Amer. J. Physiol. 2001, 281, F420)-olefins had no influence on the vasoconstrictor response whereas the introduction of a C(7)-thiomethylene enhanced potency. A sulfonamide or alcohol, but not a lactone, could replace the C(1)-carboxylate. These data were used to construct a putative binding domain map of the 20-HETE receptor consisting of: (i) a comparatively open, hydrophilic binding site accommodating the C(1)-functionality; (ii) a hydrophobic trough spanning the olefins; (iii) a shallow pocket containing a critical pi-pi binding site in the vicinity of the pi (Ito et al. Am. J. Physiol. 1998, 274, F395; Quigley, R.; Baum, M.; Reddy, K. M.; Griener, J. C.; Falck, J. R. Am. J. Physiol. 2000, 278, F949)-olefin; and (iv) an oxyphilic binding site proximate to the omega-terminus.     

Peroxynitrite causes endothelial cell monolayer barrier dysfunction

Nitric oxide (·NO) attenuates hydrogen peroxide(H₂O₂)-mediated barrier dysfunction in culturedporcine pulmonary artery endothelial cells (PAEC) (Gupta MP, Ober MD,Patterson C, Al-Hassani M, Natarajan V, and Hart, CM. Am JPhysiol Lung Cell Mol Physiol 280: L116-L126, 2001). However,·NO rapidly combines with superoxide (O) to formthe powerful oxidant peroxynitrite (ONOO), which wehypothesized would cause PAEC monolayer barrier dysfunction. To testthis hypothesis, we treated PAEC with ONOO (500 µM) or3-morpholinosydnonimine hydrochloride (SIN-1; 1-500 µM).SIN-1-mediated ONOO formation was confirmed by monitoringthe oxidation of dihydrorhodamine 123 to rhodamine. BothONOO and SIN-1 increased albumin clearance(P < 0.05) in the absence of cytotoxicity and alteredthe architecture of the cytoskeletal proteins actin and -catenin asdetected by immunofluorescent confocal imaging.ONOO-induced barrier dysfunction was partially reversibleand was attenuated by cysteine. Both ONOO and SIN-1nitrated tyrosine residues, including those on -catenin and actin,and oxidized proteins in PAEC. The introduction of actin treated withONOO into PAEC monolayers via liposomes alsoresulted in barrier dysfunction. These results indicate thatONOO directly alters endothelial cytoskeletal proteins,leading to barrier dysfunction.

     

eNOS function is developmentally regulated: uncoupling of eNOS occurs postnatally

At birth, the transition to gas breathing requires the function of endothelial vasoactive agents. We investigated the function of endothelial nitric oxide synthase (eNOS) in pulmonary artery (PA) vessels and endothelial cells isolated from fetal and young (4-wk) sheep. We found greater relaxations to the NOS activator A-23187 in 4-wk-old compared with fetal vessels and that the NOS inhibitor nitro-L-arginine blocked relaxations in both groups. Relaxations in 4-wk vessels were not blocked by an inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, but were partially blocked by catalase. We therefore hypothesized that activation of eNOS produced reactive oxygen species in 4-wk but not fetal PA. To address this question, we studied NO and superoxide production by endothelial cells at baseline and following NOS stimulation with A-23187, VEGF, and laminar shear stress. Stimulation of NOS induced phosphorylation at serine 1177, and this event correlated with an increase in NO production in both ages. Upon stimulation of eNOS, fetal PA endothelial cells (PAEC) produced only NO. In contrast 4-wk-old PAEC produced superoxide in addition to NO. Superoxide production was blocked by L-NAME but not by apocynin (an NADPH oxidase inhibitor). L-Arginine increased NO production in both cell types but did not block superoxide production. Heat shock protein 90/eNOS association increased upon stimulation and did not change with developmental age. Cellular levels of total and reduced biopterin were higher in fetal vs. 4-wk cells. Sepiapterin [a tetrahydrobiopterin (BH4) precursor] increased basal and stimulated NO levels and completely blocked superoxide production. We conclude that the normal function of eNOS becomes uncoupled after birth, leading to a developmental adaptation of the pulmonary vascular system to produce oxygen species other than NO. We speculate this may be related to cellular production and/or maintenance of BH4 levels.     

Ursolic acid prevents doxorubicin‐induced cardiac toxicity in mice through eNOS activation and inhibition of eNOS uncoupling

In addition to the known antitumour effects of ursolic acid (UA), increasing evidence indicates that this molecule plays a role in cardiac protection. In this study, the effects of ursolic acid on the heart in mice treated with doxorubicin (DOX) were assessed. The results showed that ursolic acid improved left ventrical fractional shortening (LVFS) and left ventrical ejection fraction (LVEF) of the heart, increased nitrogen oxide (NO) levels, inhibited reactive oxygen species (ROS) production and decreased cardiac apoptosis in mice treated with doxorubicin. Mechanistically, ursolic acid increased AKT and endothelial nitric‐oxide synthase (eNOS) phosphorylation levels, and enhanced eNOS expression, while inhibiting doxorubicin induced eNOS uncoupling through NADPH oxidase 4 (NOX4) down‐regulation. These effects of ursolic acid resulted in heart protection from doxorubicin‐induced injury. Therefore, ursolic acid may be considered a potential therapeutic agent for doxorubicin‐associated cardiac toxicity in clinical practice.     

Exposure to polychlorinated biphenyls causes endothelial cell dysfunction

Environmental chemicals, such as polychlorinated biphenyls (PCBs), may be atherogenic by disrupting normal functions of the vascular endothelium. To investigate this hypothesis, porcine pulmonary artery-derived endothelial cells were exposed to 3,3′,4,4′-tetrachlorobiphenyl (PCB 77), 2,3,4,4′,5-pentachlorobiphenyl (PCB 114), or 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB 153) for up to 24 hours. These PCBs were selected for their varying binding avidities with the aryl hydrocarbon (Ah) receptor and differences in their induction of cytochrome P450. PCB 77 and PCB 114 significantly disrupted, in a dose-dependent manner, endothelial barrier function by allowing an increase in albumin transfer across endothelial monolayers. These PCBs also contributed markedly to cellular oxidative stress, as measured by 2,7-dichlorofluorescin (DCF) fluorescence and lipid hydroperoxides, and caused a significant increase in intracellular calcium ([Ca²⁺]_i) levels. Enhanced oxidative stress and [Ca²⁺]_i in PCB 77- and PCB 114-treated cells were accompanied by increased activity and content of cytochrome P450 1A and by a decrease in the vitamin E content in the culture medium. In contrast to the effects of PCB 77 and PCB 114, cell exposure to PCB 153 had no effect on cellular oxidation, [Ca²⁺]_i, or endothelial barrier function. These results suggest that certain PCBs may play a role in the development of atherosclerosis by causing endothelial cell dysfunction and a decrease in the barrier function of the vascular endothelium. It is possible that interaction of PCBs with the Ah receptor and activation of the cytochrome P450 1A subfamily are involved in this pathology. © 1995 John Wiley & Sons, Inc.     

15-羟二十碳四烯酸下调肺动脉内皮型一氧化氮合酶的活性

15-羟二十碳四烯酸（15-hydroxyeicosatetraenoic acid,15-HETE）在低氧性肺血管收缩中起着重要作用,低氧肺动脉高压下调内皮型。氧化氮合酶（endothelial nitric oxide synthase,eNOS）,使一氧化氮（nitric oxide,NO）的产量下降,但目前尚无关于15-HETE与eNOS／NO相互作用研究的报道。我们通过Wistar大鼠肺动脉环张力、牛肺动脉内皮细胞NO产量、总eNOS表达及eNOS磷酸化测定等方法对15-HETE与eNOS／NO的相互作用进行研究。首先分离人鼠肺动脉,分为eNOS抑制剂L-NAME组（0．1mmol／L）、去缸管内皮组与内皮完整组,用15-HETE作用夫鼠离体肺动脉环,测定肺动脉张力。结果表明,L-NAME组、去除内皮组与内皮完整组分别比较,15-HETE对血管的收缩作用增强,且都有统计学意义（P〈0．05）。培养牛肺动脉内皮细胞,分别用15-HETE、15-脂氧酶（15-lipoxygenase,15-LO）抑制剂[（cinnamyl 3,4-dihydroxy-[alpha]-cyanocinnamate,CDC）和（nordihydroguiairetic acid,YDGA）]处理细胞,通过Greiss方法检测亚硝酸盐含量,间接测定NO产量,与对照组比较,1μmol／L 15-HETE明显降低肺动脉内皮细胞NO水平（P〈0．05）,10μmol／L CDC和0．1mmol／L NDGA显著增加NO水平（分别是P〈0．05,P〈0．01）;通过Western blot检测不同时间（5,10,15,20,30,60min）eNOS的表达情况,结果显示,15-HETE的不同作用时间,没有引起eNOS表达的明显不同;用苏氨酸495位点磷酸化eNOS（Thr495）抗体进行免疫沉淀,再用总eNOS抗体和15-LO抗体通过Western blot检测磷酸化型含量,问接测定eNOS活性,结果表明15-HETE增强Thr495磷酸化型eNOS含量。由于Thr495为eNOS抑制性磷酸化位点,因此15-HETE降低eNOS活性。这些数据表明：15-HETE的缩血管作用有eNOS／NO参与,15-HETE可以通过磷酸化Thr495位点降低eNOS活性,并且首次发现磷酸化eNOS（Thr495）和15-LO之间存在蛋白质相互作用。     

Quantification of 20-hydroxyeicosatetraenoic acid by colorimetric competitive enzyme linked immunosorbent assay

Analysis of 20-hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictor produced by the cytochrome P450 pathway, presently requires high-performance liquid chromatography (HPLC) and gas chromatography/ mass spectrometry (GC/MS). To simplify 20-HETE analysis, competitive ELISAs were developed using polyclonal anti-20-HETE coated ELISA plates to which free 20-HETE and 20-HETE conjugated to horseradish peroxidase (HRP) or alkaline phosphatase (AP) were added. Assays were developed with and without a pro prietary enhancer solution which allows for the extraction-free measurement of 20-HETE in urine samples. The bound 20-HETE-HRP or 20-HETE-AP was detected using 3,3 ,5,5, -tetramethylbenzidine and p-nitrophenyl phosphate, respectively. Sensitivities expressed as 80% B/B0, were 0.1 ng/ml for the HRP assay, and 0 5 ng/ml for the AP assay, with r2 = 0 99 for both formats. Of the 17 lipids tested for cross-reactivity, arachidonic acid showed the highest (0.32%) followed by racemic 5-HETE (0.07%) and 8,9-dihydroxyeicosatrienoic acid (DHET) (0.04%). Preliminary validation experiments examining serum and urine concentrations of 20-HETE yield values that fall within the ranges established by GC/MS in the literature. These ELISAs provide simple and inexpensive methods for the analysis of 20-HETE in biological samples.     

20-hydroxyeicosatetraenoic acid is a vasoconstrictor in the newborn piglet pulmonary microcirculation

20-Hydroxyeicosatetraenoic acid (20-HETE), a cytochrome p-450 metabolite of arachidonic acid, is a vasoconstrictor in the systemic circulation and a vasodilator in the adult pulmonary circulation. Little is known about the vasoactive properties of 20-HETE in the newborn pulmonary circulation. The objectives of this study were to determine the vascular effects of 20-HETE and to explore the signaling mechanism(s) that mediate these effects in newborn pulmonary resistance-level arteries (PRA). Our findings demonstrate that, in contrast to the adult pulmonary circulation where 20-HETE mediates vasodilation, it causes constriction in newborn PRA at resting tone. Furthermore, inhibition of cyclooxygenase (COX) with indomethacin augments 20-HETE-induced constriction. The enhanced constrictor response to 20-HETE under conditions of COX inhibition is abolished in endothelium-disrupted PRA, suggesting that 20-HETE either stimulates endothelium-derived COX to release a counteracting vasodilator or is rapidly metabolized by COX to a less potent vasoconstrictor. 20-HETE-induced constriction is significantly inhibited by blocking calcium-dependent K(+) (K(Ca)) channels and the thromboxane-PGH(2) receptor. Altogether, our data indicate that the vascular actions of 20-HETE are partially mediated via the activation of K(Ca) channels and are significantly modulated by interactions with the COX-prostaglandin pathway.     

The mitogenic effect of 15- and 12-hydroxyeicosatetraenoic acid on endothelial cells may be mediated via diacylglycerol kinase inhibition

15-Hydroxyeicosatetraenoic acid (15-HETE), a major lipoxygenase metabolite of arachidonic acid in fetal bovine aortic endothelial cells, was a mitogen for these cells, stimulating both cell proliferation and DNA synthesis in the presence of serum and serum-deprived cells. In [14C]arachidonic acid-labeled confluent endothelial cell monolayers, 15-HETE (30 microM) caused an elevation of [14C]diacylglycerol (DAG) with a concomitant decrease in cellular [14C]phosphatidylinositol (PI) in both unstimulated and stimulated cells. 1-Oleoyl-2-acetylglycerol, a synthetic DAG analog, stimulated endothelial cell DNA synthesis in a concentration-dependent manner. In [3H]inositol-labeled cells, 15-HETE also caused a decrease in cellular PI content under both basal and stimulated conditions. 15-HETE, however, had no effect on either isolated phospholipase C activity or phosphoinositide turnover in lithium chloride-treated cells. In intact cells, 15-HETE (30 microM) inhibited the synthesis of [3H]PI from [3H]inositol (80% inhibition, p less than 0.001). In human red cell membranes, the production of phosphatidic acid from endogenous DAG was inhibited by 15-HETE in a concentration-dependent manner with an IC50 of 41 microM. Although 12-HETE had effects similar to those of 15-HETE, the parent compound arachidonic acid did not affect DNA synthesis or DAG kinase activity. Our study thus demonstrates that the mitogenic activity of 15- and 12-HETE on endothelial cells may be mediated via DAG kinase inhibition with the concomitant accumulation of cellular DAG.     

Pathogenetic role of eNOS uncoupling in cardiopulmonary disorders

The homodimeric flavohemeprotein endothelial nitric oxide synthase (eNOS) oxidizes l-arginine to l-citrulline and nitric oxide (NO), which acutely vasodilates blood vessels and inhibits platelet aggregation. Chronically, eNOS has a major role in the regulation of blood pressure and prevention of atherosclerosis by decreasing leukocyte adhesion and smooth muscle proliferation. However, a disturbed vascular redox balance results in eNOS damage and uncoupling of oxygen activation from l-arginine conversion. Uncoupled eNOS monomerizes and generates reactive oxygen species (ROS) rather than NO. Indeed, eNOS uncoupling has been suggested as one of the main pathomechanisms in a broad range of cardiovascular and pulmonary disorders such as atherosclerosis, ventricular remodeling, and pulmonary hypertension. Therefore, modulating uncoupled eNOS, in particular eNOS-dependent ROS generation, is an attractive therapeutic approach to preventing and/or treating cardiopulmonary disorders, including protective effects during cardiothoracic surgery. This review provides a comprehensive overview of the pathogenetic role of uncoupled eNOS in both cardiovascular and pulmonary disorders. In addition, the related therapeutic possibilities such as supplementation with the eNOS substrate l-arginine, volatile NO, and direct NO donors as well as eNOS modulators such as the eNOS cofactor tetrahydrobiopterin and folic acid are discussed in detail.