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1Department of Internal Medicine, Cardiovascular Research Center, and Veterans Affairs Medical Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226; and 2Department of Cardiovascular Medicine, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan
Submitted 20 May 2003 ; accepted in final form 22 July 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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) by XA/XO is spontaneously converted to H2O2, which dilates HCA through vascular smooth muscle hyperpolarization. is also converted to H2O2 likely by superoxide dismustase within vascular cells and dilates HCA through a different pathway involving the activation of guanylate cyclase. These findings suggest that exogenously and endogenously produced H2O2 may elicit vasodilation by different mechanisms.
coronary microcirculation
) (17). Myocardial ischemia is a frequent pathological condition that stimulates production of ROS in both the vasculature and myocardium, contributing to myocardial cell injury (26). High levels of ROS have been shown to impair endothelial function both in vivo and in vitro (8, 11, 15). In contrast, ROS have also been reported to participate in endothelium-dependent vasodilation. Matoba et al. (24, 25) demonstrated that a major dilator factor released from the endothelium in mesenteric arteries from endothelial nitric oxide (NO) synthase (eNOS) knockout (KO) mice and humans is H2O2. Coronary arterial microvessels from the human heart also generate H2O2 from endothelial cells as a mechanism of dilation (30). Thus, in addition to inhibiting endothelial-generated NO, some ROS may actively participate in endothelium-dependent vasodilator mechanisms.
The mechanism of H2O2-mediated dilation varies. Some studies show that H2O2 hyperpolarizes vascular smooth muscle (VSM) membranes and therefore acts as an endothelium-derived hyperpolarizing factor (EDHF) (25, 30). Other studies, however, demonstrate that H2O2-induced vasodilation is mediated through the release of NO from the endothelium (12, 47) or is partially mediated by cGMP (9). Whether other ROS such as and the hydroxyl radical (OH–) directly affect vascular tone is not known, although OH– has been implicated in the mechanism of dilation to acetylcholine in the rabbit aorta (39). Such information is important to better understand the regulation of vascular tone in pathological conditions such as coronary disease or its risk factors where an increase in ROS is observed.
In this study, we tested the direct effect of ROS generated by exogenous application of xanthine (XA) and xanthine oxidase (XO) on vascular tone in human coronary arterioles (HCA). is spontaneously and enzymatically converted to H2O2. We hypothesized that exogenously generated and H2O2 contribute to vasodilation of HCA.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Experimental protocols. After 30 min of stabilization, basal tone was measured and endothelin-1 (ET-1, 10–10 to 10–9 M) was added if needed to increase resting tone to 30–50% of the estimated resting diameter at 60 mmHg. Inhibitors were added to the chamber 30 min before constriction with ET-1. None of the applied inhibitors altered vascular diameter. Furthermore, the dose of ET-1 required for subsequent constriction was similar among protocols, suggesting that there were no persistent vasomotor effects of the antagonists used. Diameter changes to XA (10–4 M) and XO (10 mU/ml) were examined. In some experiments, the endothelium was removed by injecting 2 ml of air through the lumen of the cannulated vessel as described previously (28). Denudation was confirmed by observation of preserved dilation to 10–4 M papaverine and elimination of the dilation to 10–4 M ADP (28). In a separate study, we tested the effects of superoxide dismutase (SOD, 150 U/ml), catalase (CAT, 500 U/ml), diethyldithiocarbamate trihydrate [DDC, inhibitor of both extracellular (EC) and Cu/Zn-SOD (37), 10–2 M], 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, a specific guanylate cyclase inhibitor, 5 x 10–5 M), and deferoxamine (DFX, a scavenger of OH–, 10–3 M). To determine whether membrane hyperpolarization contributes to the vasomotor effects of XA/XO, KCl (40 mM) was used to nonspecifically block K+ channels in HCA. To determine the contribution of H2O2 generated intracellularly, we used polyethylene glycol-catalase (PEG-CAT). In this case, dissected vessels were incubated in medium solution [Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum, 100 U/ml penicillin G, and 100 µg/ml streptomycin], with and without PEG-CAT (5,000 U/ml) at 37°C for 24 h, and then were used for pharmacological study. At the end of each experiment, papaverine (10–4 M) was administered to record the maximum (passive) diameter.
To determine whether the endothelium contributed to the ROS-mediated responses, we tested the effect of XA/XO application before and after mechanical denudation of the endothelium. The technique used by us previously (28) involves injection of 1 ml of air into the perfusing catheter followed by a gentle wash using physiological saline. This procedure effectively disrupts the endothelium without altering dilation to papaverine or nitroprusside (28).
Measurement of H2O2 and 
. The amount of generated by the XA/XO reaction was measured using a spectrophotometer (DU 640; Beckman, Fullerton, CA) with or without SOD or CAT. Ferricytochrome c (5 x 10–5 M) reduction based on the molar extinction coefficient (
= 21,000 mol · l–1 · cm–1) was used to calculate generated by XA/XO (43). Absorbance at 550 nm before (10–4 M XA alone) and after application of XO (every minute for 30 min following application and every 5 min between 30 and 60 min after application) was measured by a spectrophotometer (Beckman DU 640) at room temperature.
The concentration of H2O2 generated by the reaction of XA and XO was determined by a modified version of the method of Staniek and Nohl (41). Aliquots (500 µl) of the solution, in which XA and XO reacts with and without SOD or CAT, were added to 500 µl 5 mM 3,3',5,5'-tetramethylbenzidine · 2HCl (TMB) containing 20 U/ml horseradish peroxidase. The absorption was read at 450 nm by a spectrophotometer, and the concentration of H2O2 was calculated from a standard curve using authentic H2O2. These measurements were done at 37°C. All solutions were made using HEPES buffer, and pH was adjusted to 7.4. HEPES was used in place of bicarbonate because the latter requires bubbling with CO2 to stabilize pH. In a separate study, we measured the concentration of H2O2 in the bathing solution simultaneously with the vasodilation study.
Detection of H2O2 in HCA. Arterioles dissected from atrial samples were incubated in HEPES (pH = 7.4) at room temperature for 60 min. HEPES was used because these experiments were performed in chambers where gas solutions could not be bubbled for pH buffering. In our laboratory, HEPES provides a stable pH at 7.4 under these conditions. Vessels were then placed on a glass slide equipped with a Secure-Seal spacer (Molecular Probes) filled with HEPES containing 10–4 M XA plus 5 µM 2',7'-dichlorodihydrofluoresein diacetate (DCFH). Exactly 15 min after the vessel was exposed to DCFH, laser confocal microscopy (Yokogawa Spinning Disk Confocal System; an excitation wavelength of 488 nm and emission wavelength of 522 nm at room temperature) was used to visualize ROS fluorescence. Fluorescence images of the endothelium and smooth muscle were separately obtained before and 5, 10, 15, 25, and 30 min after the application of XO (10 mU/ml) by using a Hamilton glass syringe and needle. Images were analyzed on a computer with the software program MetaMorph (Universal Imaging). The inhibitory effect of CAT (500 U/ml) or DDC (10–2 M) on XA/XO-induced increases in fluorescence was also determined.
Chemicals. ET-1 was obtained from Peninsula Laboratories. DCFH was purchased from Molecular Probes. All other chemicals were purchased from Sigma. ET-1 was prepared in saline with 1% bovine serum albumin. TMB was dissolved in 10% cold (4°C) acetic acid. DCFH and ODQ were dissolved in DMSO. Other agents were prepared in distilled water. All concentrations represent final steady-state values.
Statistical analysis. Percent dilation was calculated as the percent change from the constricted diameter to the maximal diameter (maximal diameter in the experiment at 60 mmHg luminal pressure), which was generally the diameter after papaverine (10–4 mol/l). Percent constriction was determined by calculating the percent reduction in maximal diameter after application of ET-1 or during myogenic tone if between 30 and 50%. Statistical comparisons of maximal percent vasodilation under different treatments were performed by Student's t-test. To compare time-dependent response relationships between treatment groups, a two-way ANOVA supported by a Bonferroni post hoc test was used.
Separate statistical models were constructed to compare dose-treatment responses between vessels to adjust for the presence of other significant covariate disease/variables, e.g., hypertension versus no hypertension controlling for diabetes, age, etc. These analyses used two-factor repeated-measures modeling with an autoregressive covariance structure after we compared the model's Akaike's information criterion with several covariate models. All data were described as means ± SE. All computations were done using the proc-MIXED procedure in SAS. Statistical significance was defined as a value of P < 0.05.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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Production of and H2O2 by XA/XO. We measured the production of by XA/XO using cytochrome c. Figure 1A demonstrates that treatment with XA/XO increases the concentration of . Peak concentrations (32.3 ± 1.0 µM/min, n = 4) were observed 8 min after application of XA/XO. Subsequently, the concentration steadily declined. This decline seems to be due to reoxidization of (ferro)cytochrome c by H2O2 (43), because when CAT was also present, the decline was not observed; instead a persistent elevation in concentration was seen (29.4 ± 0.9 µM/min, 10 min after application of XA/XO, n = 4). When SOD was coincubated with XA/XO, the production of was abolished.
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As shown in Fig. 1B, application of XA/XO also caused H2O2 formation. The concentration of H2O2 increased to 6.2 ± 1 x 10–5 M (n = 4) by 10 min after application and was sustained for more than 50 min [concentration 60 min after the application was 7.9 ± 0.5 x 10–5 M; P = not significant (NS) vs. 10 min]. Treatment with SOD did not alter the maximal concentration of H2O2 (Concentration 20 min after the application was 7.9 ± 0.6 x 10–5 M; n = 4) or the time course of dilation. In contrast, cotreatment with CAT abolished the generation of H2O2. These data support the observation that exogenously applied XA/XO generates , which is spontaneously converted to H2O2.
Vasodilation by ROS in HCA. XO alone without XA did not alter diameter of HCA constricted with ET-1 (data not shown); however, XA alone without XO induced a small constriction (5 ± 2%, n = 32, P < 0.05 vs. baseline diameter). This constriction was prevented in the presence of the XO inhibitor allopurinol (10–5 M), which likely blocked endogenous XO (data not shown). Figure 2 demonstrates the time course of dilation to exogenously applied XA/XO. Maximal dilation was observed between 5 and 10 min after application. Endothelial denudation did not affect the dilation (Fig. 1A, dilations were 100 ± 0.01 in control and 99 ± 1 in denuded vessels, n = 5, P = NS), indicating that dilation to XA/XO is endothelium independent. This dilation was markedly attenuated by CAT (500 U/ml). The dilation was biphasic as seen after treatment with CAT. CAT added to the bath prevented dilation to XO during the first 5 min after application of XO (Fig. 1B, dilation 5 min after application of XA/XO was 88 ± 5% in control and 0 ± 1% in vessels with CAT, n = 6, P < 0.01). However, in the presence of CAT, a latent and gradual dilation (after 5 min) was revealed (dilation 20 min after application was 99.7 ± 0.3% in control and 51.6 ± 9.5% in vessels with CAT, n = 6, P < 0.01). Thus two observed distinct dilations to XA/XO were a CAT-sensitive dilation that peaked within a minute and lasted at least 20 min and a delayed CAT-insensitive dilation that began 5 min after application of XA/XO and gradually increased for an additional 15 min.
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Treatment with SOD (150 U/ml) did not affect dilation to XA/XO (Fig. 1C, dilation 20 min after application was 99.7 ± 0.3% in control and 99.5 ± 0.3% in vessels treated with SOD, n = 5, P = NS). These data suggest that H2O2, but not 
contributes to the dilation.
We simultaneously measured XA/XO-induced dilation and the concentration of H2O2 in the bathing solution as shown in Fig. 3. In the presence of 10–4 M XA, XO dose dependently increased the concentration of H2O2 in the bathing solution [Fig. 3A, calculated concentrations of H2O2 were 0.5 ± 0.1 x 10–5, 1.3 ± 0.1 x 10–5, 3.3 ± 0.4 x 10–5, 7.7 ± 0.7 x 10–5, and 7.9 ± 0.8 x 10–5 M in solutions with increasing concentrations of XO (10–4, 3 x 10–4, 10–3, 3 x 10–3, and 10–2 U/ml, respectively)]. The associated dilation revealed an ED50 of 3.3 ± 0.1 (–log U/ml) and maximal dilation of 99 ± 0.3% (Fig. 3B). Thus exogenously generated ROS by XA/XO dilate HCA, and H2O2 contributes to this vasodilation. Figure 3C demonstrates that in contrast to treatment with XA alone, treatment with XA/XO shows greater DCFH fluorescence in both the endothelium and smooth muscle layers in HCA (confocal microscopy), also supporting the generation of H2O2.
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Production of H2O2 from exogenous by intrinsic SOD in HCA. As shown in Fig. 2B, although CAT significantly reduced dilation to ROS, a small and latent dilation was still observed. This latent dilation was not affected by treatment with 5,000 U/ml of CAT for 24 h (data not shown). To examine the mechanism of this CAT-resistant vasodilation, we extended the observation time following administration of XA/XO from 20 to 30 min in subsequent experiments. This latent vasodilation was not observed when vessels were treated with SOD and catalase (dilation at 15 min in control = 99 ± 1.0% vs. SOD + CAT = –2.9 ± 2.3%), indicating that generated exogenously is critically involved in this latent dilation. When the vessels were treated for 24 h with 5,000 U/ml PEG-CAT, a cell-permeable CAT, the latent dilation was reduced as seen in Fig. 4B, where the maximum dilation 30 min after application of XA/XO was 72 ± 3% in the control group (time control that was treated with DMEM alone for 24 h) and 33 ± 9% in PEG-CAT-treated vessels (P < 0.01, n = 6), supporting the involvement of intracellular H2O2. When DDC, an inhibitor of EC-SOD and Cu/Zn-SOD, was applied together with CAT, the latent dilation was abolished (Fig. 4C, maximum dilations 30 min after application of XA/XO were 59.6 ± 7.1% in control and 4.5 ± 2.7% in vessels with DDC. P < 0.01, n = 5). These data suggest that endogenous SOD contributes to the latent dilation possibly by converting extracellular superoxide to intracellular H2O2. The same dose of DDC did not alter dilation to papaverine, indicating that the effect of DDC is specific (data not shown). Collectively, these data suggest that H2O2 generated intracellularly by endogenous SOD as well as H2O2 generated extracellularly by spontaneous conversion from exogenous both contribute to dilation in HCA. This latent dilation was not endothelium dependent because denudation had no effect (Fig. 4A, dilations 30 min after application of XA/XO were 65 ± 4% in control and 61 ± 6% in denuded vessels, P = NS, n = 4).
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We extended this observation by examining whether H2O2 was produced (DCFH fluorescence) in VSM when CAT is used in the external solution. Figure 5 shows that the fluorescence signal increased in the VSM layer 5–10 min after application of XA/XO even with CAT present and then declined close to the basal levels. When the vessel was treated with DDC in addition to CAT, the DCFH signal was completely abolished. These data indicate that even with extracellular treatment with CAT, extracellular generation results in an increase in H2O2 in the vascular cell, eliciting dilation of HCA.
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Mechanisms of H2O2-induced dilation. Recent evidence suggests that H2O2 is a prime candidate for EDHF. Thus we tested whether KCl, which depolarizes VSM membranes, interferes with dilation to XA/XO. As shown in Fig. 6A, although KCl completely abolished dilation to XA/XO for 5 min after administration, a latent dilation was subsequently observed even in the presence of KCl (dilations 30 min after application of XA/XO were 100 ± 0% in control and 76 ± 7% in vessels with KCl; P < 0.05, n = 5). However, the latent CAT-resistant dilation was not affected by KCl as shown in Fig. 6B (dilations 30 min after application of XA/XO were 67 ± 1% in vessels with CAT alone and 68 ± 3% in vessels with CAT plus KCl; P = NS, n = 6). Conversely, ODQ, an inhibitor of guanylate cyclase, did not affect the early dilation to ROS without CAT (Fig. 6C) but did markedly attenuate the CAT-resistant dilation (Fig. 6D, dilations 30 min after application of XA/XO were 67 ± 1% in vessels with CAT alone and 16 ± 4% in vessels with CAT plus ODQ, P < 0.01, n = 5). Failure of ODQ to alter the dilation to H2O2 in the absence of CAT reflects the much greater magnitude of the early and sustained dilation that overwhelms the loss of the cGMP-sensitive latent dilation. Only when the early and potent dilation is inhibited by KCl is the latent ODQ-sensitive dilation evident. These data indicate that exogenously generated H2O2 dilates HCA through membrane hyperpolarization, whereas intracellularly generated H2O2 dilates HCA through a different pathway involving activation of guanylate cyclase.
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Finally, we tested DFX to assess whether OH– contributes to the dilation of HCA. This inhibitor did not affect dilation to ROS without CAT (Fig. 6E) but did reduce the CAT-resistant dilation (Fig. 6F, dilations 30 min after application of XA/XO were 68 ± 2% in vessels with CAT alone and 23 ± 6% in vessels with CAT plus DFX, P < 0.01, n = 5), indicating that OH– likely derived from H2O2 contributes to this latent dilation.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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is responsible for a portion of this vasodilation, whereas intrinsically generated H2O2 by SOD in the vascular cells also contributes to this vasodilation. Finally, extracellular H2O2-induced dilation is mediated by VSM membrane hyperpolarization, whereas intracellularly generated H2O2 dilates HCA via a different pathway involving activation of guanylate cyclase. Taken together, these findings indicate that ROS evoke HCA vasodilation through multiple pathways, possibly contributing to the regulation of coronary blood flow under pathological states such as ischemia, where high amounts of ROS are generated.
ROS-mediated vasomotion. The combination of XA (10–4 M) and XO (10 mU/ml) that we used in our study produces (22), which spontaneously dismutates to H2O2 (16). We confirmed generation of both ROS species by using cytochrome c and TMB, respectively. Our experimental design generated 
30 µM/min and 80 µM H2O2 (measured after 20 min of incubation). This concentration is similar to that of another study in which ROS altered colonic electrolyte transport in rats (16).
Treatment with XA plus XO produced very rapid vasodilation (initiated within 1 min, peaking between 10 and 20 min, and sustained for more than 30 min). This time course of vasodilation to ROS corresponds to the formation of H2O2 in solution. CAT but not SOD reduced this dilation. Additionally, in the presence of XA, XO showed a similar dose dependency between vasodilation and concentration of H2O2. Finally, confocal microscopy revealed strong DCFH fluorescence in the endothelial and VSM cell layers, indicating the presence of H2O2. From these data, we conclude that exogenously generated by XA/XO spontaneously converted into H2O2, thereby dilating HCA.
Similar findings were previously reported by Pomposiello et al. (38) who showed that XA/XO dilates porcine coronary arteries; however, in contrast to our study, they observed no latent dilation in the presence of CAT and observed a much slower time to peak dilation in the absence of CAT. On the other hand, XA/XO evokes vasoconstriction in aortas from WKY and SHR rats (47). Thus the response to ROS seems to be species or vessel specific. Our preliminary data in microvessels from human subcutaneous and omental fat show that XA/XO also elicits vasodilation (n = 3, data not shown).
Role of H2O2 in the vasodilation to ROS. Although CAT abolished the early ROS-induced vasodilation, a small latent dilation was observed. This CAT-resistant dilation was abolished by cotreatment with DDC, an inhibitor of Cu/Zn-SOD and EC-SOD. Because the cell membrane is relatively impermeable to CAT (2), we speculate that is acting to stimulate production of H2O2 intracellularly. This could occur by either small amounts of penetrating the cell membrane through anion channels (4) and being converted to H2O2, or possibly H2O2 generated near the cell surface activates intracellular NADPH oxidase to generate superoxide within the cell, which is subsequently converted to H2O2 (20). This CAT-resistant vasodilation was significantly attenuated by PEG-CAT, a cell membrane-permeable CAT, supporting this idea. Although we cannot exclude the possibility that H2O2 diffused into the VSM to produce the latent dilation, this is not likely because measured H2O2 levels in the bathing solution in the presence of CAT were far lower than those necessary for vasodilation. Confocal microscopy showed DCFH fluorescence in VSM cell even in the presence of CAT. DDC abolished this fluorescence as well as CAT-resistant vasodilation, suggesting intracellular generation of H2O2 from . From these data, we conclude that H2O2 generated both extracellularly and intracellularly from exogenous XO/XA contributes to dilating HCA.
Mechanisms by which ROS dilates HCA. Recently, H2O2 has been reported to be an EDHF (24, 25, 30). Agonists (acetylcholine or bradykinin) and sheer stress have been shown to evoke both vasodilation and VSM cell membrane hyperpolarization through the formation of H2O2 in endothelium. In our study, the early XA/XO-induced vasodilation, which is mediated by exogenous H2O2, is inhibited by KCl, supporting an EDHF mechanism. However, the inhibitory effect of KCl was not effective on the later dilation. This KCl-resistant dilation resembled the CAT-resistant vasodilation (Fig. 6B). A possible explanation is that intracellularly generated H2O2, which is responsible for the later dilation, operates through a different mechanism than extracellular H2O2. Interestingly, ODQ, an inhibitor of guanylate cyclase, and deferoxamine (DFX), a scavenger of OH–, abolished this CAT-resistant vasodilation (Fig. 6, D and F) but did not affect the early dilation (Fig. 6, C and E). These data suggest that intracellular H2O2 may dilate HCA through conversion to OH– and activation of guanylate cyclase. This idea is in accord with a previous study that shows that OH– activates guanylate cyclase (29). Furthermore, OH– has been implicated in vasodilation in the rabbit aorta (3). We also speculate that the transient increase in DCFH fluorescence by XA/XO in the presence of CAT (Fig. 5) is due to intracellular conversion of H2O2 to OH– in vascular smooth muscle cells. The induction of cGMP-mediated signal transduction is much more prolonged than the half-life of the OH– radical.
Study limitations. We observed that in the presence of CAT and DDC, XA/XO administration did not induce DCFH oxidation and fluorescence. DDC inhibits both EC-SOD and Cu/Zn-SOD by chelating the copper ion; however, it can also quench free iron (18). Because ferrous ions increase the rate of DCFH oxidation by H2O2 (40, 42), it is plausible that this mechanism contributes to the inhibition of the DCF signal in the presence of DDC.
DCFH is sensitive to H2O2 but is not specific, because other peroxide radicals may also be detected with this method (42, 43). NO can oxidize DCFH, thereby directly producing fluorescence (14, 34, 35), although not all studies support this (1). We used CAT, which is highly specific for H2O2, to ensure specificity of the DCFH fluorescence. Neither the CAT-sensitive nor the CAT-insensitive dilations (Figs. 2A and 4A) were affected by endothelial denudation, suggesting that NO derived from eNOS does not contribute to the dilation. NO from other isoforms of NOS (inducible NOS or neuronal NOS) may have been generated, although we believe this is unlikely because inducible NOS produces large amounts of NO, which would be expected to be vasoactive. Nevertheless, we cannot rule out the possibility that ROS such as NO might contribute to DCFH fluorescence.
We show that PEG-CAT, a cell-permeable CAT, reduced the latent CAT-resistant vasodilation to XA/XO. Previous studies (5, 13) have demonstrated that although PEG-CAT is cell permeable, it needs more than 12 h to achieve adequate CAT activity in the cell culture. Thus we incubated HCA with PEG-CAT for 24 h. We expected that PEG-CAT would completely eliminate the latent dilation; however, this treatment was only partially effective. It is possible that the penetration rate of PEG-CAT into intact vascular cell membranes is less than that in cell culture monolayers where it is traditionally used. Because we did not measure CAT activity before and after treatment of PEG-CAT in HCA, we cannot rule out this possibility. Furthermore, we did not perform control experiments to determine whether this application of PEG-CAT exerted nonspecific effects on dilation, although dilation to maximal doses of papaverine at the end of the experiment were not different in vessels incubated with or without PEG-SOD.
EDHF appears to play the major role in endothelium-mediated dilation in human coronary arterioles (28, 31–33). In tissue from subjects without evident coronary disease, EDHF still seems to predominate, although a clear role for NO-mediated dilation has been observed in these subjects in response to flow-mediated dilation (33). We have also previously demonstrated that endothelium-dependent dilation to adrenomedullin in human coronary arterioles is mediated by NO (44). Thus under physiological and certain pathological circumstances, a role for NO-mediated dilation is evident and may be affected by the presence of superoxide. Because it only requires small doses of NO-releasing agents to inhibit cytochrome P-450, the characteristic of the dilator response may be significantly influenced by the loss of even small amounts of NO (36). More importantly, the human vessels we studied provide a relatively "clean" system in which to test the direct vasomotor properties of superoxide and hydrogen peroxide. With minimal influences of NO and prostaglandins in this system, it is easier to determine the direct effects of ROS on vasomotor tone.
ROS concentrations within cells are tightly regulated by short half-lives of many ROS and by the presence of numerous cellular antioxidant systems. Whether is dismutated to H2O2 to induce cell signaling or dilation or whether it reacts with NO to form peroxynitrite likely depends on the subcellular site of formation. The purpose of this study was to determine whether formed or its dismutase product have direct vascular effects in coronary arterioles from humans with CAD, where ambient levels of ROS are elevated. The identification of such an action indicates that formation of H2O2 is an important confounding factor when contemplating the effect of on vasomotor tone in any vessel system.
Risk factors for CAD such as diabetes (6, 23) and other medical conditions may alter the activity or expression of endogenous antioxidants such as SOD, CAT, and glutathione peroxidase. Changes in endogenous antioxidant levels would be expected to modulate the vasomotor effects of applied ROS. Although the distribution of risk factors for CAD were similar between groups, we cannot exclude a potential influence from changes in endogenous antioxidant levels within the vessels studied.
Summary and physiological implications. Previous studies demonstrate that sheer stress and agonist (acetylcholine, bradykinin)-induced formation of H2O2 in endothelium is inhibited by CAT. Because these studies used cell-impermeable CAT, H2O2 presumably formed in the endothelial cell, traverses the intracellular space, and diffuses to the underlying VSM where it activates potassium channels to elicit hyperpolarization and dilation. We speculate that this H2O2-induced vasodilation is identical to the CAT-sensitive dilation we observed to XA/XO. Extracellular or H2O2 also stimulates production of H2O2 within the VSM layer. H2O2 formed this way either directly or through conversion to OH– mediates dilation through activation of guanylate cyclase.
ROS have previously been considered negative modulators of vasomotor function. Excess quenches NO thereby impairing vasodilation. Peroxynitrite inhibits key enzymes (prostacyclin synthase) (48) and proteins (Ca2+-activated K+ channels) (21) responsible for endothelium-dependent dilation. However, beneficial functions for ROS have also been described in the cardiovascular system. For example, redox-sensitive transcription factors are important in vascular cell function (27). Preconditioning is in part dependent on ROS (19). We describe a prominent vasodilator effect of H2O2 generated from exogenous in HCA. This dilation may be regulated differentially depending on whether the H2O2 is generated from within or outside the cell. Modulation of antioxidant enzymes [e.g., upregulation of SOD by shear stress (19) or increased expression of CAT by atorvastatin (46)] may be a novel way to regulate intrinsic vasodilator capacity.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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 Y. Liu, A. H. Bubolz, Y. Shi, P. J. Newman, D. K. Newman, and D. D. Gutterman Peroxynitrite reduces the endothelium-derived hyperpolarizing factor component of coronary flow-mediated dilation in PECAM-1-knockout mice Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2006; 290(1): R57 - R65. [Abstract] [Full Text] [PDF]
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M. Ohashi, F. Faraci, and D. Heistad Peroxynitrite hyperpolarizes smooth muscle and relaxes internal carotid artery in rabbit via ATP-sensitive K+ channels Am J Physiol Heart Circ Physiol, November 1, 2005; 289(5): H2244 - H2250. [Abstract] [Full Text] [PDF]
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C. S. Wilcox Oxidative stress and nitric oxide deficiency in the kidney: a critical link to hypertension? Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2005; 289(4): R913 - R935. [Abstract] [Full Text] [PDF]
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 T. Fujiki, H. Shimokawa, K. Morikawa, H. Kubota, M. Hatanaka, M.A. H. Talukder, T. Matoba, A. Takeshita, and K. Sunagawa Endothelium-Derived Hydrogen Peroxide Accounts for the Enhancing Effect of an Angiotensin-Converting Enzyme Inhibitor on Endothelium-Derived Hyperpolarizing Factor-Mediated Responses in Mice Arterioscler Thromb Vasc Biol, April 1, 2005; 25(4): 766 - 771. [Abstract] [Full Text] [PDF]
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D. D. Gutterman, H. Miura, and Y. Liu Redox Modulation of Vascular Tone: Focus of Potassium Channel Mechanisms of Dilation Arterioscler Thromb Vasc Biol, April 1, 2005; 25(4): 671 - 678. [Abstract] [Full Text] [PDF]
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C. S. Wilcox and D. Gutterman Focus on oxidative stress in the cardiovascular and renal systems Am J Physiol Heart Circ Physiol, January 1, 2005; 288(1): H3 - H6. [Full Text] [PDF]
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B. Kalyanaraman and D. D. Gutterman Prologue: Vascular effects of free radicals Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2253 - H2254. [Full Text] [PDF]
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