Mediation of H2O2-induced vascular relaxation by endothelium-derived relaxing factor

We investigated the effects of H₂O₂ generated by glucose (G) and glucose oxidase (GO) on the isolated rabbit aorta suspended in Krebs-Ringer solution. H₂O₂ produced contraction in small concentration and relaxation followed by contraction in large concentration. Contraction produced by large concentration was smaller than that produced by small concentration of H₂O₂. Relaxation was prevented by deendothelialization or N^G-monomethyl-L-arginine, an inhibitor of nitric oxide synthesis. These results suggest that H₂O₂ in large concentrations produces relaxation followed by contraction, and that the relaxation is endothelium-dependent and is mediated by nitric oxide, an endothelium-derived relaxing factor.     

Angiotensin Converting Enzyme Inhibitors as Oxygen Free Radical Scavengers

The authors have compared the ability of two non-SH-containing angiotensin converting enzyme (ACE) inhibitors (enalaprilat and lisinopril) with an -SH containing ACE inhibitor (captopril) to scavenge the hydroxyl radical (OH). All three compounds were able to scavenge -OH radicals generated in free solution at approximately diffusion-controled rates (10¹⁰ M^-1s^-1) as established by the deoxyribose assay in the presence of EDTA. The compounds also inhibited deoxyribose degradation in reaction mixtures which did not contain EDTA but not so effectively. This later finding also suggests that they have some degree of metal-binding capability. Chemiluminescence assays of oxidation of hypoxanthine by xanthine oxidase in the presence of luminol, confirm that the three ACE inhibitors are oxygen free radical scavengers. Our results indicate that the presence of a sulphydryl group in the chemical structure of ACE inhibitors is not relevant for their oxygen free radical scavenging ability.     

Direct scavenging of free radicals by captopril, an angiotensin converting enzyme inhibitor

Captopril, an angiotensin converting enzyme (ACE) inhibitor, was hypothesized to be a potential scavenger of free radicals because of the presence of a thiol group. The scavenging action of captopril was examined against superoxide anion (O2-), hydroxyl radical (OH.), hypohalite radical (HOCL) either generated biochemically, or derived from activated polymorphonuclear leukocytes (PMN). Our results indicate that captopril is an extremely potent free radical scavenger, scavenging power being as effective as superoxide dismutase (SOD) against O2-, or dimethylthiourea against OH., but better than allopurinol against OCL. plus HOCL. Free radical scavenging action of captopril against PMN-derived free radical is equivalent to the combined effects of SOD, catalase and allopurinol.     

Augmentation of NO-mediated vasodilation in metabolic acidosis

Reduction of perivascular pH in acidemia produces hyporesponsiveness of vascular bed to vasoconstrictors. In the present study, we examined the effects of modest acidification on dilatory responses of isolated rat thoracic aorta. Acetylcholine produced endothelium-dependent relaxation in phenylephrine-precontracted aorta, which was markedly enhanced by acidification of Krebs-Henseleit solution from pH 7.4 to 7.0. A similar augmentation was observed in the relaxing responses to NO donors (SNP, SIN-1, SNAP), 8-Br-cGMP and NS-1619 (a putative K(Ca) channel opener and/or Ca channel inhibitor) in endothelium-denuded, phenylephrine-contracted aorta. However, papaverine-induced relaxation was not affected by the change in pH. At pH 7.4, the relaxing responses to acetylcholine and SNP were partially inhibited by charybdotoxin (K(Ca) channel inhibitor) but not glibenclamide (K(ATP) channel inhibitor), while at pH 7.0 the relaxation induced by either drug was not affected by K(+) channel inhibitors. Relaxation induced by 8-Br-cGMP or NS-1619 was not inhibited by charybdotoxin or glibenclamide. Acidification to pH 7.0 increased the cGMP production in response to acetylcholine in endothelium-intact aorta and to SNP in endothelium-denuded aorta. These results show that modest acidification augments NO-mediated relaxation in rat aorta, probably due to an enhancement of cGMP-dependent but K(+) channel-unrelated relaxation mechanisms.     

Inactivation of Rabbit Muscle Creatine Kinase by Hydrogen Peroxide

The effects of xanthine + xanthine oxidase-generated reactive oxygen species (ROS) on rabbit muscle creatine kinase (CK) were studied. Xanthine (0.1 mM) + xanthine oxidase (30 mU/ml) inhibited activity of rabbit muscle CK (1.2mU/ml). Catalase (100/ml), but not SOD (100 U/ml), deferoxamine (100μM) or mannitol (20 mM), protected CK from inactivation; suggesting that H₂O₂ was responsible for inactivation. These results were different from previously reported findings on bovine heart CK that superoxide radicals inactivate the enzyme. Thus, enzymes with homologous structures may have different reactivities to different ROS. H₂O₂-induced inactivation of rabbit muscle CK was accompanied by a decrease in its thiol group content, whereas no significant changes in the protein structure were detected by SDS-PAGE or carbonyl content. These results suggest that oxidation of -SH groups by H₂O₂ seems to be a major mechanism of activation of rabbit muscle CK by xanthine + xanthine oxidase. Such inactivation of CK by H₂O₂ may be important in ROS-induced pathology.     

双环醇减轻超氧阴离子诱导的大鼠胸主动脉舒张功能损伤

目的：探讨双环醇（bicyclol）对超氧阴离子（O2）诱导的血管舒张功能损伤的影响。方法：采用离体器官灌流技术,观察bicyclol对离体大鼠胸主动脉环张力的影响。采用焦酚（O2的供体）建立O2损伤模型,观察bicyclol预孵育对氧化应激损伤后血管内皮依赖性舒张功能的改善作用。结果：bicyclol（10-8~10-5mol/L）对由苯肾上腺素预收缩的内皮完整主动脉环产生舒张作用,该作用可被NO合酶抑制剂L-NAME和环氧化酶抑制剂吲哚美辛阻断。500μmol/L焦酚可引起乙酰胆碱诱导的主动脉环内皮依赖性舒张反应减弱,bicyclol（10-5mol/L）预孵育45 min可减轻焦酚的损伤作用。对于吲哚美辛处理的主动脉环,bicyclol（10-5mol/L）可抑制焦酚所致的血管舒张反应降低,但这一效应未见于L-NAME处理的主动脉环。结论：bicyclol具有内皮依赖性舒血管作用,并能对抗O2引起的血管舒张功能损伤,该作用通过NO途径介导。     

Whey peptide Isoleucine-Tryptophan inhibits expression and activity of matrix metalloproteinase-2 in rat aorta

Aortic stiffness is an independent risk factor for development of cardiovascular diseases. Activation of renin-angiotensin-aldosterone system (RAAS) including angiotensin converting enzyme (ACE) activity leads to overproduction of angiotensin II (ANGII) from its precursor angiotensin I (ANGI). ANGII leads to overexpression and activation of matrix metalloproteinase-2 (MMP2), which is critically associated with pathophysiology of aortic stiffness. We previously reported that the whey peptide Isoleucine-Tryptophan (IW) acts as a potent ACE inhibitor. Herein, we critically elucidate the mechanism of action by which IW causes inhibition of expression and activity of MMP2 in aortic tissue. Effects of IW on expression and activity of MMP2 were assessed on endothelial and smooth muscle cells (ECs and SMCs) in vitro and ex vivo (isolated rat aorta). As controls we used the pharmaceutical ACE inhibitor – captopril and the ANGII type 1 receptor blocker – losartan. In vitro, both ANGII and ANGI stimulation significantly (P < 0.01) increased expression of MMP2 assessed with western blot. Similarly, to captopril IW significantly (P < 0.05) inhibited ANGI, but not ANGII mediated increase in expression of MMP2, while losartan also blocked effects of ANGII. Signaling pathways regulating MMP2 expression in ECs and SMCs were similarly inhibited after treatment with IW or captopril. In ECs IW significantly (P < 0.05) inhibited JNK pathway, whereas in SMCs JAK2/STAT3 pathway, assessed with western blot. In vitro findings were fully consistent with results in isolated rat aorta ex vivo. Moreover, IW not only inhibited the MMP2 expression, but also its activation assessed with gelatin zymography. Our findings demonstrate that IW effectively inhibits expression and activation of MMP2 in rat aorta by decreasing local conversion of ANGI to ANGII. Thus, similar to pharmaceutical ACE inhibitor captopril the dipeptide IW may effectively inhibit ACE activity and prevent the age and hypertension associated rise of aortic stiffness.     

Effects of catechins on vascular tone in rat thoracic aorta with endothelium

The effects of eight catechin derivatives on vascular tone in rat thoracic aorta were examined. Catechin derivatives (10 microM) potentiated the contractile response to phenylephrine in endothelium-intact arteries. The potentiations produced by EGCg and EGC were almost absent in endothelium-denuded arteries and abolished by N(G)-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthesis. The catechin derivatives also inhibited endothelium-dependent relaxation in response to acetylcholine. The order of catechin derivatives ranked in terms of both increasing vascular reactivity and impairing endothelium-dependent relaxation was similar; (-)-gallocatechin (GC) >or= (-)-epigallocatechin (EGC) >or= (-)-gallocatechin gallate (GCg) >or= (-)-epigallocatechin gallate (EGCg) >or= (-)-catechin (C) >or= (-)-epicatechin (EC) >or= (-)-catechin gallate (Cg) >or= (-)-epicatechin gallate (ECg). In addition, EGC inhibited the endothelium-independent relaxation evoked by both sodium nitroprusside and NOC-7, a spontanous NO releaser, but EGCg inhibited only that by NOC-7. These findings indicate that catechin derivatives produce a potentiation of the contractile response and an inhibition of the vasorelaxant response, probably through inactivation of endothelium-derived nitric oxide (NO), and that the hydroxyl on C-5 of the B ring together with the stereoscopic structure between the C-3 group and the B ring of flavanols was of importance in mediating the above effects and that the substitution of a gallate group of C-3 attenuated the effects, probably due to a decreased response to solube guanylate cyclase in vascular smooth muscle cells.     

Effects of LY83583, nordihydroguaiaretic acid, and quinacrine on cyclic GMP elevation and inhibition of tension by muscarinic agonists in rabbit aorta and left atrium

Elevation of cyclic GMP by muscarinic agonists has been suggested to be responsible for the negative inotropic effects of these agents in cardiac muscle, and for the endothelium-dependent relaxation caused by these agents in vascular smooth muscle. These relationships were studied by monitoring the effects of muscarinic agonists on tension and cyclic GMP levels in rabbit left atrial strips and aortic rings, in the presence and absence of the cyclic GMP lowering agent, LY83583. LY83583 completely blocked both the cyclic GMP increase and the relaxation caused by acetylcholine in rabbit aortic rings with intact endothelial cells. Acetylcholine-induced cyclic GMP elevation and relaxation in these preparations were also blocked by quinacrine and nordihydroguaiaretic acid (NDGA), but neither response was blocked by the 5-lipoxygenase inhibitor U-60257. In the experiments with rabbit left atrium, LY83583 blocked the acetylcholine-induced cyclic GMP elevation but did not block the negative inotropic effects of the drug. Quinacrine, NDGA, and a guanylate cyclase inhibitor, methylene blue, failed to block either the cyclic GMP increase or the decrease in contractile force caused by carbachol in atrial strips. These results support the suggestion that an increase in cyclic GMP may be responsible for the endothelium-dependent relaxation of rabbit aorta by muscarinic agonists, but not for the direct negative inotropic effects of these drugs in rabbit atrium. Muscarinic agents appear to increase cyclic GMP levels in rabbit atrium and aorta by different mechanisms. Although both are blocked by LY83583, they differ not only in their requirements for endothelial cells, but also in their susceptibility to other blocking agents.     

亚慢性亚毒性有机磷酯类杀虫剂抑制抗氧化酶而致兔血管内皮功能损伤

有机磷酸酯类(OPs)是全球最广泛使用的杀虫剂之一．其除了抑制胆碱酯酶(AChE)活性外, 也抑制对氧磷酶(PON1)的活性．其急性中毒主要与抑制AChE有关．最近,OPs 对PON1的影响已引起学术界广泛关注．因为PON1除了有水解OPs的功能外,也有抗低密度脂蛋白(LDL)氧化和降解LDL中的脂质过氧化物的作用．因为血管内皮功能损伤是动脉粥样硬化形成的起始步骤．将探讨OPs对血管内皮功能的影响作为研究目的．研究结果表明,连续每天给兔灌胃敌百虫(18 mg/kg)70天,可导致其离体血管内皮依赖性舒张(EDR)反应和eNOS活性显著性降低,血浆超氧化物岐化酶活性、一氧化氮水平、PON1 和AChE 活性降低,脂质过氧化代谢产物丙二醛水平增加．OPs的活性成分——对氧磷在体外与兔胸主动脉环直接孵育也能浓度和时间依赖性地显著抑制EDR．研究结果提示,亚慢性-亚毒性剂量的敌百虫灌胃或对氧磷与离体血管环直接孵育,均可导致兔血管内皮功能损伤,其机制可能与OPs抑制氧化酶和诱发氧化应激反应有关．     

Hydroxyl radicals mediate injury to endothelium-dependent relaxation in diabetic rat

The purpose of this study was to determine the radical species which mediates the toxic effects of exogenous oxygenderived free radicals on endothelial function of chronic diabetic rat aorta. Endothelium-dependent relaxation to acetylcholine was impaired in diabetic vessels. Exposure to the exogenous free radical generating system of xanthine plus xanthine oxidase selectively impaired endothelium-dependent relaxation to acetylcholine in control and diabetic aorta with relaxations essentially abolished in diabetic aorta. The loss of relaxation to acetylcholine in diabetic aorta was prevented or attenuated by pretreatment with catalase, dimethylthiourea or desferrioxamine, but not by mannitol or superoxide dismutase. These results suggest that hydroxyl radicals play an important role in the endothelial injury produced by oxygen-derived free radicals in chronic diabetic rat aorta. Furthermore, the site of the injury is likely due to intracellular generation of hydroxyl radicals.     

Differential Effect of Amylin on Endothelial-Dependent Vasodilation in Mesenteric Arteries from Control and Insulin Resistant Rats

Insulin resistance (IR) is frequently associated with endothelial dysfunction and has been proposed to play a major role in cardiovascular disease (CVD). On the other hand, amylin has long been related to IR. However the role of amylin in the vascular dysfunction associated to IR is not well addressed. Therefore, the aim of the study was to assess the effect of acute treatment with amylin on endothelium-dependent vasodilation of isolated mesenteric arteries from control (CR) and insulin resistant (IRR) rats and to evaluate the possible mechanisms involved. Five week-old male Wistar rats received 20% D-fructose dissolved in drinking water for 8 weeks and were compared with age-matched CR. Plasmatic levels of glucose, insulin and amylin were measured. Mesenteric microvessels were dissected and mounted in wire myographs to evaluate endothelium-dependent vasodilation to acetylcholine. IRR displayed a significant increase in plasmatic levels of glucose, insulin and amylin and reduced endothelium-dependent relaxation when compared to CR. Acute treatment of mesenteric arteries with r-amylin (40 pM) deteriorated endothelium-dependent responses in CR. Amylin-induced reduction of endothelial responses was unaffected by the H₂O₂ scavenger, catalase, but was prevented by the extracellular superoxide scavenger, superoxide dismutase (SOD) or the NADPH oxidase inhibitor (VAS2870). By opposite, amylin failed to further inhibit the impaired relaxation in mesenteric arteries of IRR. SOD, or VAS2870, but not catalase, ameliorated the impairment of endothelium-dependent relaxation in IRR. At concentrations present in insulin resistance conditions, amylin impairs endothelium-dependent vasodilation in mircrovessels from rats with preserved vascular function and low levels of endogenous amylin. In IRR with established endothelial dysfunction and elevated levels of amylin, additional exposure to this peptide has no effect on endothelial vasodilation. Increased superoxide generation through NADPH oxidase activity may be a common link involved in the endothelial dysfunction associated to insulin resistance and to amylin exposure in CR.     

Mechanisms of aging-induced impairment of endothelium-dependent relaxation: role of tetrahydrobiopterin

Oxidative stress has been implicated as an important mechanism of vascular endothelial dysfunction induced by aging. Previous studies suggested that tetrahydrobiopterin (BH4), an essential cofactor of endothelial NO synthase, could be a molecular target for oxidation. We tested the hypothesis that oxidative stress, in particular oxidation of BH4, may contribute to attenuation of endothelium-dependent relaxation in aged mice. Vasomotor function of isolated carotid arteries was studied using a video dimension analyzer. Vascular levels of BH4 and its oxidation products were measured via HPLC. In aged mice (age, 95 +/- 2 wk), endothelium-dependent relaxation to ACh (10(-5) to 10(-9) M) as well as endothelium-independent relaxation to the NO donor diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate, 10(-5) to 10(-9) M) were significantly reduced compared with relaxation detected in young mice (age, 23 +/- 0.5 wk). Incubation of aged mouse carotid arteries with the cell-permeable SOD mimetic Mn(III)tetra(4-benzoic acid)porphyrin chloride normalized relaxation to ACh and DEA-NONOate. Furthermore, production of superoxide anion in aorta and serum levels of amyloid P component, which is the murine analog of C-reactive protein, was increased in old mice. In aorta, neither the concentration of BH4 nor the ratio of reduced BH4 to the oxidation products were different between young and aged mice. Our results demonstrate that in mice, aging impairs relaxation mediated by NO most likely by increased formation of superoxide anion. Oxidation of BH4 does not appear to be an important mechanism underlying vasomotor dysfunction in aged mouse arteries.     

Coronary reperfusion in dogs inhibits endothelium-dependent relaxation: role of superoxide radicals

Previous studies indicate that release of superoxide radicals during coronary reperfusion following occlusion may relate to the loss of endothelium-dependent coronary arterial relaxation. We examined coronary arterial ring relaxation in dogs subjected to temporary circumflex (Cx) coronary artery occlusion and treated with saline or the superoxide radical scavenger superoxide dismutase (SOD). In dogs treated with saline, Cx coronary ring relaxation in response to leukotriene D4 (LTD4) and acetylcholine (ACh) was attenuated (p less than 0.01), but coronary relaxation in response to nitroglycerin was preserved, suggesting loss of endothelium-dependent relaxation following coronary reperfusion. In contrast, Cx coronary relaxation in response to LTD4 and ACh was preserved in the SOD-treated dogs (p less than 0.01 compared to saline-treated dogs). To further examine the role of superoxide radicals in the loss of endothelium-dependent relaxation, normal nonischemic canine coronary artery and rat aortic rings were exposed to a superoxide radical generating system of xanthine and xanthine oxidase in vitro. Xanthine plus xanthine oxidase treatment caused a significant (p less than 0.01) decrease in the relaxant effects of ACh. Pretreatment of rat aortic rings with SOD protected against the loss of ACh-induced relaxation. These observations suggest that release of superoxide radicals during reperfusion is the basis of loss of endothelium-dependent coronary arterial relaxation. Treatment with superoxide radical scavengers prior to coronary reperfusion protects against this loss.     

A relaxing factor released by phospholipase A2 in the absence of endothelium

Summary Phospholipase A₂ (PLA₂) produced slow dose dependent relaxation in intact and endothelium-deprived precontracted rabbit aortic strips. In endothelium-deprived preparations, relaxation induced by PLA₂ is inhibited by hemoglobin, methylene blue and parabromophenacylbromide (PBPB), and is potentiated by superoxide dismutase (SOD). Indomethacin has no effect. Relaxation is accompanied by a rise in c-GMP. Phospholipase C causes a significant increase in tension, while Phospholipase D has no effects. In intact aortic strips PLA₂ causes a biphasic response with no elevation in c-GMP. The results indicate several common features of the PLA₂ released factor with endothelium-derived relaxing factor (EDRF). However PLA₂ induced relaxation is not dependent on endothelial cells. Apparently in addition to nitric oxide which may be the endothelium-derived relaxing factor, a second smooth muscle relaxing factor exists which is initiated by PLA₂ and is independent of endothelium. The production of the PLA₂ produced relaxation is dependent on its specific hydrolytic activity. We call this relaxing factor the phospholipid-derived relaxing factor (PDRF).     

三羟异黄酮对离体家兔股动脉张力的影响及其机制

植物雌激素三羟异黄酮（genistein,GST)使离体的预先收缩的动脉舒张,其舒张的机制仍然不完全清楚。本研究旨在观察植物雌激素三羟异黄酮对离体家兔股动脉的作用及其机制,结果如下：（1）在苯肾上腺素（PE,1umol/L)引起血管收缩的基础上,GST（10－40umol/L)剂量依赖性地舒张离体家兔股动脉;（2）去除血管内皮显著地抑制GST引起的舒张;（3）在内皮完整情况下,预先应用NOS抑制剂L－NAME（100umol/L)也可显著地抑制GST引起的舒张,提示GST的舒血管作用是内皮依赖的,并与一氧化氮有关;（4）在内皮完整的扣去除内皮的股动脉环,预先应用L－型钙通道激动剂Bay M8644(0.5umol/L)也显著抑制由GST引起的血管舒张,以上结果表明,GST引起的兔股动脉的舒张是部分内皮依赖的,且与拮抗钙有关。     

Chronic ouabain treatment increases the contribution of nitric oxide to endothelium-dependent relaxation

The aim of this study was to analyze the contribution of nitric oxide, prostacyclin and endothelium-dependent hyperpolarizing factor to endothelium-dependent vasodilation induced by acetylcholine in rat aorta from control and ouabain-induced hypertensive rats. Preincubation with the nitric oxide synthase inhibitor N-omega-nitro-l-arginine methyl esther (L-NAME) inhibited the vasodilator response to acetylcholine in segments from both groups but to a greater extent in segments from ouabain-treated rats. Basal and acetylcholine-induced nitric oxide release were higher in segments from ouabain-treated rats. Preincubation with the prostacyclin synthesis inhibitor tranylcypromine or with the cyclooxygenase inhibitor indomethacin inhibited the vasodilator response to acetylcholine in aortic segments from both groups. The Ca²⁺-dependent potassium channel blocker charybdotoxin inhibited the vasodilator response to acetylcholine only in segments from control rats. These results indicate that hypertension induced by chronic ouabain treatment is accompanied by increased endothelial nitric oxide participation and impaired endothelium-dependent hyperpolarizing factor contribution in acetylcholine-induced relaxation. These effects might explain the lack of effect of ouabain treatment on acetylcholine responses in rat aorta.     

Gene transfer of superoxide dismutase isoforms reverses endothelial dysfunction in diabetic rabbit aorta

Increased production of oxygen free radicals is an important mechanism of endothelial dysfunction in diabetes mellitus. Our goal was to test whether adenovirus (Ad)-mediated gene transfer of copper/zinc (CuZn) or manganese superoxide dismutase (Mn SOD) improves relaxation of diabetic vessels. The aortas from 9 alloxan-induced diabetic mellitus (DM) and 16 control rabbits were used. Control and DM rings were transduced ex vivo with Ad vectors encoding Mn SOD (AdMn SOD), CuZn SOD (AdCuZn SOD), beta-galactosidase (Ad(beta)gal), or diluents. In the absence of gene transfer, SOD activity was significantly increased in DM aortas. Transgene expression in DM AdCuZn SOD and DM AdMn SOD-transduced vessels was confirmed by Western blot analysis and by increased SOD activity (DM AdCuZn SOD, 76.2 +/- 9.3; DM AdMn SOD, 65.2 +/- 4.8; P < 0.05 vs. DM Ad(beta)gal; 50.9 +/- 4.4 U/mg protein). Superoxide production was increased in DM Ad(beta)gal-transduced aorta and relaxations to acetylcholine were impaired in these vessels. Gene transfer of CuZn SOD and Mn SOD corrected both of these defects. Thus Ad-mediated gene transfer CuZn and Mn SOD to the diabetic aorta improves endothelium-dependent relaxation.     

Is hydrogen peroxide an EDHF in human radial arteries?

In human radial arteries, a nitric oxide/prostanoid-independent mechanism that has the pharmacological characteristics of an EDHF contributes to endothelium-dependent relaxation. H2O2 can act as an EDHF in some vascular beds. We examined the hypothesis that endogenously produced H2O2 mediated the nitric oxide/prostanoid-independent relaxation to carbachol in radial arteries obtained from patients undergoing coronary artery bypass surgery. Superoxide levels, measured by chemiluminescence, were similar in radial and internal mammary arteries, but immunohistochemical staining for Cu/Zn superoxide dismutase (SOD) was lower in endothelium from radial arteries. In organ chamber studies, neither addition of catalase nor addition of SOD to the bathing fluid modified nitric oxide/prostanoid-independent relaxations to carbachol in radial arteries. However, nitric oxide-dependent vasorelaxation was enhanced in the presence of SOD. Thus the nitric oxide/prostanoid-independent relaxation to carbachol is not due to H2O2 and, unlike nitric oxide-mediated vasorelaxation, is not attenuated by superoxide. Blood vessels showing EDHF-mediated relaxations resistant to oxidative stress may provide favorable outcomes in revascularization surgery.