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Am J Physiol Heart Circ Physiol 288: H48-H54, 2005. First published September 2, 2004; doi:10.1152/ajpheart.00663.2004
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2004 CARDIOVASCULAR AND KIDNEY INVESTIGATORS MEETING

Role of hydrogen peroxide in ACh-induced dilation of human submucosal intestinal microvessels

Ossama A. Hatoum,1 David G. Binion,1 Hiroto Miura,1 Gordon Telford,2 Mary F. Otterson,2 and David D. Gutterman1

Cardiovascular Research Center, Digestive Disease Center, Departments of 1Medicine, 2Surgery, Froedtert Memorial Lutheran Hospital, Veterans Affairs Medical Center, Medical College of Wisconsin, Milwaukee, Wisconsin

Submitted 6 July 2004 ; accepted in final form 27 August 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several mediators of vasodilation, which include prostacyclin (PGI2), nitric oxide, and endothelium-derived hyperpolarizing factor (EDHF). We have recently defined the role of nitric oxide and PGI2 in the dilation of submucosal intestinal arterioles from patients with normal bowel function. However, significant endothelium-dependent dilator capacity to ACh remained after inhibiting both these mediators. The current study was designed to examine the potential role of EDHF in human intestinal submucosal arterioles. ACh elicited endothelium-dependent relaxation in the presence of inhibitors of nitric oxide synthase and cyclooxygenase (23 ± 10%, n = 6). This ACh-induced relaxation was inhibited and converted to constriction by catalase (–53 ± 10%, n = 6) or KCl (–30 ± 3%, n = 7), whereas 17-octadecynoic acid and 6-(2-propargylloxyphenyl) hexanoic acid, two inhibitors of cytochrome P450 monooxygenase, had no significant effect (3 ± 1% and 20 ± 8%, n = 5, respectively). Exogenous H2O2 elicited dose-dependent relaxation of intact microvessels (52 ± 10%, n = 7) but caused frank vasoconstriction in arterioles denuded of endothelium (–73 ± 8%, n = 7). ACh markedly increased the dichlorofluorescein fluorescence in intact arterioles in the presence of nitric oxide synthase and cyclooxygenase inhibitors compared with control and compared with catalase-treated microvessels (363.6 ± 49, 218.8 ± 10.6, 221.9 ± 27.9, respectively, P < 0.05 ANOVA, n = 5 arbitrary units). No changes in the dichlorofluorescein fluorescence were recorded in vessels treated with ACh alone. These results indicate that endothelial production of H2O2 occurs in response to ACh in human gut mucosal arterioles but that H2O2 is not an EDHF in this tissue. Rather, we speculate that it stimulates the release of a chemically distinct EDHF.

microcirculation; endothelium-derived hyperpolarizing factor; acetylcholine; cyclooxygenase; vasodilation

THE ENDOTHELIUM plays an important role in regulating the vascular tone by synthesizing and releasing several vasodilators, including prostacyclin (PGI2), nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF). We (15) have previously described an important role for NO and PGI2 in the vasodilation of human mucosal arterioles to ACh. However, after pharmacological blockade of both of these mediators, a significant dilation persisted. The purpose of the present study is to determine whether EDHF contributes to the regulation of intestinal perfusion and to examine the etiology of that dilator factor.

The nature of EDHF is disputed (3, 7), but it is widely accepted that EDHF plays an important role in modulating vasomotor tone, especially in the microcirculation (31, 42). Since the existence of EDHF (7) was first reported, several identities were proposed. In porcine and bovine coronary arteries, but not in other vessels (11, 48, 56), epoxyeicosatrienoic acid (EET), metabolites of cytochrome P-450 (CYP450) monooxygenase, were suggested to be EDHF (9, 17, 39). In fact, metabolites of CYP450 were among the most widely recognized compounds that act as EDHF. In rat hepatic arteries, K+ released from the endothelium was also suggested to be an EDHF (4), but a role for this mediator in other vascular territories was limited (19, 36). Gap junctions may also play a role in causing vasodilatation and hyperpolarization of the underlying vascular smooth muscle (5, 53). It was recently described in certain human vascular beds that EDHF may be H2O2 (22, 23, 27, 43) liberated from the endothelium in response to agonist activation or shear stress. H2O2 can hyperpolarize vascular smooth muscle and dilate arteries in several vascular beds (27, 49) in a catalase-sensitive fashion.

Based on the prominent role for EETs as EDHF in several animal models and the importance of H2O2 as an EDHF in human arterioles, including human mesenteric vessels (22, 23), we chose to examine the mechanism of the NO synthase (NOS)- and cyclooxygenase (COX)-independent dilation of human mucosal arterioles, focusing on the role of EET and H2O2 as potential EDHFs. It is important to note that ACh has a dual effect in many vascular beds, producing both an endothelium-dependent dilation as well as a direct smooth muscle constriction (26). Both effects were considered in these experiments.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The Medical College of Wisconsin's Institutional Review Board approved all protocols. Human submucosal arterioles were dissected from full-thickness intestinal specimens obtained from patients undergoing bowel operations. Tissues were not used from patients who had coronary artery disease, hypertension, hypercholesterolemia, and/or used tobacco. Demographic data and diagnoses were obtained from hospital records and recorded at the time of surgery. After resection, tissue samples were preserved as reported previously (15).

Video microscopy. Video microscopy was performed as previously reported (15, 29, 30, 46). Briefly, isolated microvessels (Table 1) were carefully dissected from the submucosal surface of the bowel tissue and transferred to a 20-ml organ chamber containing Krebs solution of the following composition (in mmol/l): 118 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 20 NaHCO3, 0.026 Na2EDTA, and 11 glucose; pH 7.4. Each end of the arteriole was secured to a separate glass micropipette (25–50 µm internal diameter) filled with Krebs buffer (20) and transferred to the stage of an inverted microscope (CK2, Olympus) coupled to a CCD camera (WV-BL200, Panasonic) and video micrometer (VIA-100K, Boeckeler Instruments). Internal vascular diameters were measured throughout the experiment with a manually adjusted video microscope as described previously (28). Micropipettes were connected to a hydrostatic reservoir at a pressure of 60 mmHg. The chamber solution was continuously recirculated at 30 ml/min, aerated with 20% O2-5% CO2-75% N2, and maintained at 37°C by an external heat changer. All pharmacological agents were added to the external bathing solution.


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  		Table 1. Demographics

 
Experimental protocols. After a 60-min stabilization period, vessels were constricted to 30–50% of maximal diameter with administration of endothelin-1 (ET-1, 10–10 to 5 x 10–10 M). Vascular responses to cumulative logarithmic increases in the concentration of ACh (10–9 to 10–4 M) or to H2O2 (10–7 to 10–3 M) in the external bathing media were examined. At the end of each experiment, maximal vessel diameter was determined by adding papaverine (10–4 M). After the cumulative dose was recorded, the chamber was washed with 300 ml of fresh buffer over 20–30 min. Inhibitors or vehicles were added to the circulating bath for an additional 30- to 60-min equilibration period. Vessels were again constricted to 30–50%, and a second dose-response relationship was generated. All chemicals were obtained from Sigma Chemical (St. Louis, MO).

Endothelial denudation. In some cases, experiments were performed following endothelial denudation. Endothelium was mechanically denuded as previously described (29). Approximately 2 ml of warmed air were passed through the vessel over a 2-min period. This was immediately followed by warmed physiological saline solution to fill the vessel and remove all air bubbles. The vessel was then pressurized and stabilized for 30 min before proceeding further. This results in functional denudation of isolated arterioles without damage to the vascular smooth muscle (26). Studies in denuded vessels were run in parallel with intact vessels from the same patient for comparison. Dilation to papaverine (10–4 M) was used to confirm integrity of the underlying vascular smooth muscle (25).

Fluorescence detection of H2O2. H2O2 generation was assessed using the membrane-permeate diacetate form of 2'7'-dichlorofluorescein (DCFH-DA) as described before (15). Briefly, in the cell, esterase cleaves the acetate groups on DCFH-DA, thus trapping the reduced probe (DCFH) intracellularly. H2O2 and peroxides oxidize DCFH yielding the fluorescent product dichlorofluorescein (DCF). DCFH-DA (5 µM) was added to the vessel and incubated at 37°C for 10 min. After being washed with fresh physiological saline solution, fluorescence was detected using confocal microscopy (excitation 490 nm, emission 526 nm). An untreated arteriole was used as a control to adjust for laser settings for all other vessels studied in parallel. Relative average fluorescence intensity was normalized for surface area and compared with control and experimental microvessels. The fluorescence intensity-to-vascular area ratio of the central portion of the vessel was normalized to that obtained from the control vessel using the public domain National Institutes of Health Image program (developed at the National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). The ratio of the fluorescence intensity between experimental vessel and control vessel was compared. Artifactual autofluorescent regions were manually eliminated from analysis.

Materials. ET-1 (Peninsula Laboratories, San Carlos, CA) was prepared in saline with 1% bovine serum albumin to achieve the designated 30–50% vasoconstriction. All reagents were obtained from Sigma. Indomethacin was dissolved in saline with Na2CO3. 17-Octadecynoic acid (17-ODYA) and 6-(2-propargylloxyphenyl) hexanoic acid (PPOH) were dissolved in ethanol. Other agents were prepared in distilled water. Final molar concentrations of agents in the organ chambers are reported. The addition of pharmacological agents produced <1% change in the volume of the circulating bath. None of the pharmacological antagonists produced significant changes in baseline microvessel diameter.

Statistical analysis. Percent dilation was calculated as the percent change from the constricted diameter to the maximal passive diameter (maximal diameter in the experiment at 60 mmHg of luminal pressure) and was generally the diameter following papaverine. Percent constriction was determined by calculating the percent reduction from maximal diameter after the application of ET-1. Comparisons of percent vasodilation under different treatments were performed using a two-factor repeated-measures ANOVA utilizing SAS procedure mixed modules with autoregressive covariance assumptions (SAS software was used with the "proc mixed" procedure to run all analyses). Both computations were followed by a Bonferroni correction when significant differences were noted. To compare the sensitivities of the agents used, negative logarithm of the molar concentration of vasodilator that produced 50% of the maximal dilation to the agonist (ED50 values) were calculated. Maximal percent dilations and ED50 values were compared by Student's paired t-test. Statistical significance was defined as P < 0.05. All data are described as means ± SE; n indicates the number of microvessels and the number of patients.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Microvessel isolation. Submucosal microvessels from 35 patients were used. The average resting diameter was 155 ± 10 µm at 60-mmHg pressure. Patient demographics are summarized in Table 1.

Endothelium-dependent relaxation in normal intestine. We previously reported that the mechanism of dilation to ACh in the normal bowel microvasculature involved NO and COX pathways (15, 16). However, use of the NOS inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME) and the COX inhibitor indomethacin (Indo) did not fully eliminate the dilation to ACh, suggesting another mechanism was also operative [maximal dilation (MD): 23 ± 10%; n = 6]. The doses of each antagonist used were similar to those effectively used by others to inhibit the respective enzyme systems (6, 15, 51, 52). This dilation was abolished and replaced with frank vasoconstriction following endothelial denudation (15). To examine the mechanism of this remaining dilation, extracellular KCl (40 mM) was added to L-NAME and Indo. Figure 1 shows inhibition of the remaining dilation with a resultant vasoconstriction similar to that seen after denudation (MD: 82 ± 2%; –30 ± 3%; n = 7; in control and KCl, respectively; P < 0.05 ANOVA, n = 6). This finding implicates the involvement of a third mediator, possibly EDHF, as responsible for a substantial portion of the vasorelaxation in human gut submucosal arterioles.



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  		Fig. 1. Endothelium-dependent relaxation in normal intestine. Responses to ACh in human submucosal intestinal microvessels from control and after incubation with N{omega}-nitro-L-arginine methyl ester (L-NAME), indomethacin (Indo), and KCl using in vitro videomicroscopy are shown. Microvessels from control tissues dilate in response to increasing doses of ACh [maximal dilation (MD): 82 ± 2%; n = 7], whereas microvessels after incubation with L-NAME and Indo showed attenuation in their response to ACh (MD: 23 ± 10%; n = 7). Microvessels with L-NAME, Indo, and KCl (n = 7) showed frank vasoconstriction to ACh (MD: –30 ± 3%; n = 7; *P < 0.05 vs. control). The y-axis indicates the percent change from preconstricted diameter. Values are presented as means ± SE.

 
Role of CYP450 metabolites. Previous studies indicate that CYP450 metabolites of arachidonic acid serve as EDHF in some vessels (9, 10, 17, 39). To examine the role of CYP450 metabolites, 17-ODYA and PPOH, both nonspecific CYP450 enzyme inhibitors (10), were used to treat microvessels (MD: 20 ± 8%; n = 5 and 3 ± 1%; n = 5; Fig. 2, A and B, respectively). These results demonstrate 17-ODYA had a minimal nonsignificant effect on the remaining dilation. PPOH had no affect on the relaxation, suggesting that CYP450 monooxygenase metabolites do not mediate the residual dilation to ACh in human submucosal gut arterioles.



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  		Fig. 2. Role of cytochrome P-450 metabolites. Responses to ACh in human submucosal intestinal microvessels from control (with L-NAME and Indo) and after incubation with 17-octadecynoic acid (17-ODYA) (A) and 6-(2-propargylloxyphenyl) hexanoic acid (PPOH) (B) are shown. Neither inhibitor had any effect on the dilation to ACh (MD: 82 ± 2%, 3 ± 1%, 20 ± 9%; n = 5, respectively). The y-axis indicates the percent change from preconstricted diameter. Values are presented as means ± SE.

 
Role of H2O2 in ACh-induced dilation. To examine the role of H2O2 in mediating relaxation of normal human intestinal submucosal arterioles, we first examined the inhibitory effect of catalase, which selectively cleaves H2O2 into oxygen and water (22, 23). Our data shows that catalase markedly inhibited the EDHF-mediated relaxation elicited by ACh in the presence of L-NAME and Indo, resulting in vasoconstriction similar to that seen after denudation (MD: 82 ± 2% in control to –53 ± 10% with catalase; P < 0.05 ANOVA, n = 6; Fig. 3). These data are consistent with H2O2 as an EDHF in human mucosal arterioles. However, if H2O2 is indeed an EDHF, then it should relax vascular mucosal segments directly (27).



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  		Fig. 3. Role of H2O2 in ACh-induced dilation. Effects of H2O2 inhibition on ACh-induced dilation are shown. Microvessels from control tissues dilate in response to increasing doses of ACh, whereas microvessels treated with L-NAME and Indo showed attenuation in their response to ACh (MD: 82 ± 2%; 23 ± 10%; n = 6). The addition of catalase (Cat) demonstrated frank vasoconstriction in response to ACh (MD: –53 ± 10% at 10–6; n = 6; *P < 0.05 vs. control), similar to that with KCl and after denudation. Values represent means ± SE.

 
We applied exogenous H2O2 dose dependently to isolated cannulated intestinal microvessels. In vessels with an intact endothelium, a significant dilation was observed (MD: 52 ± 10%; n = 7; Fig. 4A), but in vessels denuded of endothelium, a frank vasoconstriction was seen (MD: –73 ± 8%; n = 7; P < 0.05 ANOVA, Fig. 4B). These data suggest that H2O2 cannot be an EDHF but instead may act on the endothelium to release a vasodilator substance.



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  		Fig. 4. H2O2-induced response in human intact and denuded gut microvessels. Intact vessels and denuded vessels from control patients are shown. Top, concentration-dependent H2O2-induced vasodilation (MD: 52 ± 10%; n = 7). Bottom, concentration-dependent H2O2-induced vasoconstriction in normal denuded controls (MD: –73 ± 7%; n = 6; *P < 0.05 vs. control). Values represent means ± SE.

 
We next examined H2O2 generation in mucosal arterioles using DCF-DA fluorescence (41). In vessels with intact endothelium, basal production of H2O2 was minimal; however, exposure to ACh for 30 min elicited an increase in DCF fluorescence in arterioles treated with Indo and L-NAME. This increase in fluorescence was due to cellular production of H2O2 and not other reactive oxygen species because it has inhibited by catalase (Fig. 5). With the use of image analysis software to quantify the levels of H2O2, significant increases in DCF fluorescence (363.6 ± 49, 218.8 ± 10.6, 221.9 ± 27.9, n = 5, respectively, P < 0.05 ANOVA, Fig. 6A) were observed in arterioles incubated with ACh + Indo + L-NAME compared with either control vessels (L-NAME + Indo) or compared with vessels treated with ACh + L-NAME + Indo + catalase. Furthermore, exposure to ACh alone without L-NAME and Indo for 30 min elicited no change in the DCF fluorescence (Fig. 6B). Removal of the endothelium abolished the DCF signal in response to ACh (Fig. 7).



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  		Fig. 5. Reactive oxygen species (ROS) (H2O2) production in human intestinal microvessels from control in the presence of ACh, L-NAME + Indo, and control in the presence of ACh, L-NAME, Indo, and catalase were assessed using fluorochromes and confocal fluorescence microscopy as described in MATERIALS AND METHODS. Relative fluorescence intensity (green) corresponds with ROS (H2O2) formation in the vessels. Relative fluorescence is measured in reference to freshly isolated vessels from the same surgical specimen pretreated with L-NAME + Indo. We observed minimal production of H2O2 in control arterioles; however, exposure to ACh for 30 min elicited increase in dichlorofluorescein fluorescence in arterioles. This increase in fluorescence was blocked by the addition of catalase.

 


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  		Fig. 6. ACh-induced H2O2 production in the presence and absence of L-NAME + Indo. Using image analysis software to quantify the levels of H2O2 in Fig. 5, we found significant increases in the dichlorofluorescein fluorescence (363.6 ± 49, 218.8 ± 10.6, 221.9 ± 27.9, n = 5, respectively, *P < 0.05 vs. control ANOVA) were measured in arterioles incubated with ACh, Indo, and L-NAME compare with control or to those treated with catalase (A). Values represent means ± SE. Moreover, using the same image analysis software to quantify the levels of H2O2, we observed no changes in the production of H2O2 in control, ACh, or in ACh with catalase-treated arterioles. No significant changes increases in the dichlorofluorescein fluorescence were measured in arterioles incubated with ACh compared with control or with those treated with catalase (222.8 ± 18.4, 225.6 ± 16.2, 217.6 ± 18.4, n = 3, respectively) (B). Values represent means ± SE. Values are in arbitrary units.

 


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  		Fig. 7. H2O2 production in denuded vessels. Removal of the endothelium abolished the dichlorofluorescein signal, confirming that H2O2 production in response to ACh was dependent on an intact endothelium (218.8 ± 10.6, 66.7 ± 10.5, n = 5, respectively, *P < 0.05 vs. control ANOVA). Values are in arbitrary units.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The major findings of this study are threefold. First, a vasodilator mechanism independent of NO and PGI2 but inhibited by KCl, plays an important role in the vasomotor response of submucosal intestinal microvessels to ACh. Second, this mediator, putatively EDHF, is dependent on an intact endothelium and H2O2. Unlike human mesenteric microvessels, H2O2 is not an EDHF in submucosal arterioles, and it constricts denuded mucosal arterioles. Finally, H2O2-induced dilation depends on the inhibition of both NOS and COX. This may be a novel example of NO inhibition of an EDHF response.

The vasoactive role of H2O2 in endothelium-dependent vasorelaxation was recently recognized to involve a multiplicity of responses. H2O2 can act as a vasodilator and/or vasoconstrictor depending on the vascular bed, species, and experimental conditions. H2O2 causes vasorelaxation of the canine coronary artery (40), bovine pulmonary artery (50), rat aorta (24, 35, 47), rabbit aorta (55), and mesenteric arteries of mice and humans (22, 23). In contrast, H2O2 has been shown to produce constriction in isolated the rat aorta, porcine pulmonary artery, and rabbit aorta (38). In the rabbit aorta, H2O2 accelerates NO release (40) and activates soluble guanylate cyclase thereby causing smooth muscle relaxation (55). This pathway is completely endothelium dependent and NO mediated. In our study, H2O2 endothelium-dependent dilation was only observed in the presence of an inhibitor of endothelial NOS, indicating that NO was not the mediator of dilation. The vasoconstrictor mechanisms of H2O2 could involve activation of phospholipase A2 and phospholipase C (2, 54), COX (37, 38), tyrosine kinase (37), and thromboxane A2 synthase pathways (12). In our study, we intentionally used indomethacin and L-NAME together to prevent the effects of COX products and NO from influencing the response to ACh. However, it will be important in future studies to examine the effect of ACh dilation in the presence of either inhibitor alone and to determine the effect of these inhibitors on the vasomotor responses to H2O2.

H2O2 is a reasonable candidate for endogenous EDHF, because it is produced by endothelial cells and causes vascular smooth muscle relaxation through activation of Ca2+-dependent K+ (KCa) channels (12, 23). Recently, Matoba et al. (12, 13) showed that H2O2 is a primary EDHF in mesenteric arteries of mice and humans. Miura et al. (27) also demonstrated H2O2 to be an EDHF in human coronary arterioles. ACh-induced production of superoxide has been demonstrated in the aorta where ACh-generated reactive oxygen species may contribute to dilation via activation of KCa channels (8). Thus in multiple human vasculatures H2O2 functions as an EDHF.

The source of endothelial production of H2O2 is not known. It is likely that H2O2 is produced from the dismutation of superoxide anions. Superoxide may be derived from several sources in endothelial cells including NOS, COX, lipoxygenase, CYP450 enzymes, mitochondria (21), and NAD(P)H oxidase (12). Relatively specific inhibitors of these enzymatic pathways are available to decipher the source of reactive oxygen species generation in response to ACh. In preliminary studies, we (14) have recently identified xanthine oxidase as an important source of reactive oxygen species in gut mucosal vessels. This enzyme might also be involved in the mechanism of dilation to ACh.

In the present study, CYP450 metabolites of arachidonic acid did not appear to play a role in the mucosal dilation to ACh. These experiments were performed in the presence and of L-NAME + Indo, therefore optimizing the chance to observe an effect because NO can inhibit the CYP450 expoxygenase enzyme (1). Thus EETs are not likely an important physiological EDHF in human mucosa. However, it is known that EDHF-mediated responses can often be uncovered in the presence of disease states (30, 34). Therefore, it remains possible that EETs play a role in regulating vasomotor tone in disease states such as inflammatory bowel disease or chronic enteritis.

Catalase abrogated the ACh-induced, EDHF-mediated relaxation and caused vasoconstriction similar to that with KCl and denuded vessels. Furthermore, in a concentration-dependent manner, exogenous H2O2 elicited vasodilation in intact microvessels. H2O2 caused profound vasoconstriction in denuded arterioles likely through a direct effect on vascular smooth muscle cells. In addition, ACh markedly increased the catalase-sensitive DCF fluorescence in the presence of L-NAME and Indo compared with untreated controls and compared with catalase-treated vessels, as illustrated in Fig. 5. In contrast, ACh had no effect on DCF fluorescence in the absence of L-NAME and Indo (Fig. 6). These data emphasize a primary role of H2O2 in mediating ACh-induced dilation of submucosal gut arterioles. It indicates that under physiological conditions, NO reduces effective concentrations of H2O2 possibly by quenching superoxide or by inhibiting enzymatic formation of reactive oxygen species. When NO synthesis is inhibited, restoration of H2O2 production may help to preserve endothelial vasodilator function.

Study limitations. DCFH is sensitive to H2O2 but is not specific because other peroxide radicals may also be detected with this method (18, 32, 33, 44, 45). Our use of catalase, which is highly specific for H2O2, ensures specificity of the DCFH fluorescence. Patients enrolled in this study were not in a normal physiological state, underwent surgery for a variety of reasons, and may have been on different medications. It is impossible to control for these variables, but we did exclude subjects with risk factors known to influence vasomotor responses, including coronary artery disease, hypertension, hypercholesterolemia, and tobacco use.

ACh was the only pharmacological endothelium-dependent dilator used in this study. It will be important to determine whether other agonists also utilize endothelial production of H2O2 as a mechanism to release EDHF. Nevertheless ACh is an important vascular stimulus being released in response to parasympathetic nerve stimulation, which is vital in regulating gut function (13). Future investigations will evaluate the nature of the EDHF released in human mucosal arterioles in response to ACh.

In summary, the present study demonstrates that H2O2 plays a critical role in the EDHF-mediated dilation of submucosal arterioles in the human gut. However, the link between endothelial generation of H2O2 and smooth muscle vasodilation remains unclear and requires further investigation.


   ACKNOWLEDGMENTS
 
This study was supported by the Cardiovascular Center and Impulse Dynamics and the Digestive Disease Center, Medical College of Wisconsin.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. D. Gutterman, Cardiovascular Research Center, Medical College of Wisconsin, Milwaukee, WI 53226 (E-mail: dgutt{at}mcw.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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