Hydrogen peroxide acts as an EDHF in the piglet pial vasculature in response to bradykinin

¹ Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157; and ² Institute of Human Physiology and Clinical Experimental Research, Semmelweis University, H1082 Budapest, Hungary

	ABSTRACT

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We investigated the mechanism of EDHF-mediated dilation to bradykinin (BK) in piglet pial arteries. Topically applied BK (3 µmol/l) induced vasodilation (62 ± 12%) after the administration of N-nitro-L-arginine methyl ester (L-NAME) and indomethacin, which was inhibited by endothelial impairment or by the BK₂ receptor antagonist HOE-140 (0.3 µmol/l). Western blotting showed the presence of BK₂ receptors in brain cortex and pial vascular tissue samples. The cytochrome P-450 antagonist miconazole (20 µmol/l) and the lipoxygenase inhibitors baicalein (10 µmol/l) and cinnamyl-3,4-dyhydroxy--cyanocinnamate (1 µmol/l) failed to reduce the BK-induced dilation. However, the H₂O₂ scavenger catalase (400 U/ml) abolished the response (from 54 ± 11 to 0 ± 2 µm; P < 0.01). The ATP-dependent K⁺ (K_ATP) channel inhibitor glibenclamide (10 µmol/l) had a similar effect as well (from 54 ± 11 to 16 ± 5 µm; P < 0.05). Coapplication of the Ca²⁺-dependent K⁺ channel inhibitors charybdotoxin (0.1 µmol/l) and apamin (0.5 µmol/l) failed to reduce the response. We conclude that H₂O₂ mediates the non-nitric oxide-, non-prostanoid-dependent vasorelaxation to BK in the piglet pial vasculature. The response is mediated via BK₂ receptors and the opening of K_ATP channels.

endothelium-derived hyperpolarizing factor; cerebral circulation; closed cranial window

	INTRODUCTION

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THERE IS CONSIDERABLE EVIDENCE showing that bradykinin (BK) is a mediator of pathophysiological reactions such as inflammation and edema formation (22, 46). All parts of the kininogen system have been described in the cerebral circulation (54), and kinins have been extensively studied for implications in the transmission of nociceptive information (34) and the control of blood pressure (9, 35). In the central nervous system, BK is found to be the main mediator of edema formation (46, 47, 55), and it is also a potent dilator of arteries (36, 41, 54, 58). However, the mechanisms that contribute to BK-induced cerebral vasodilation are not well understood (54).

The actions of BK are mediated via specific G protein-coupled receptors of which two different types have been identified so far. The BK₁ receptor is located mainly on neurons (44) and can also be found on the surface of adventitial fibroblasts of cerebral arteries (51). BK₂ receptors are represented in all layers of the cerebral vessel wall: their presence has been documented in cerebral endothelial as well as smooth muscle and adventitial cells (16, 44, 51, 53). In a variety of preparations, the vasodilator effect of BK is abolished by removal of the endothelium and is mediated by the BK₂ receptor subtype (54).

Vasorelaxation to BK is mediated in part by nitric oxide (NO) formation (6, 21, 27, 30, 41) and dilator prostanoids (8, 28, 41). Furthermore, BK is a widely used agent for evoking the release of the hypothetical endothelium-derived hyperpolarizing factor (EDHF) in different regions (12, 14, 24). Hyperpolarization induced by opening of the Ca²⁺-dependent K⁺ (K_Ca) channels is characteristic of EDHF (7, 12, 50, 56); however, EDHF-like actions can also be mediated via ATP-dependent K⁺ (K_ATP) channels (see Refs. 13, 40, 56). Arachidonic acid metabolites through the cytochrome P-450 (19) or the lipoxygenase pathways acting via K_Ca channels are widely accepted candidates for EDHF (40, 56). However, recent studies indicated that endothelial-derived H₂O₂ can also dilate arteries in an EDHF-like fashion.

The involvement of EDHF in agonist-induced cerebral vasodilation is not well understood. In isolated rat middle cerebral arteries, intraluminal application of uridine-5'-triphosphate induces vasodilation, which is mediated in part by EDHF (37, 62). In the same artery, BK is a potent vasodilator and its effect is still pronounced even after the blockade of the cyclooxygenase (COX) and guanylyl cyclase enzymes (Z. Benyó, Z. Lacza, and M. Wahl, unpublished observations). However, evaluation of the contribution of EDHF to BK-induced cerebral vasodilation has not yet been undertaken.

The aim of the present study was to characterize the vasodilation induced by topical application of BK in the porcine cerebral circulation in vivo. To exclude the participation of the NO and prostanoid pathways, all experiments were carried out in the presence of N-nitro-L-arginine methyl ester (L-NAME) and indomethacin. Using topical application of specific blocking agents, the involvement of K⁺ channels and putative EDHFs were studied in the BK-induced pial arterial dilation.

	MATERIALS AND METHODS

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Measurement of pial arterial diameter. All procedures were approved by the Animal Care and Use Committee of Wake Forest University. Newborn (1-7 days old) piglets of either sex that weighed 2.39 ± 0.06 kg were anesthetized with thiopental sodium (30 mg/kg ip), and anesthesia was maintained with -chloralose (75 mg/kg iv). The right femoral artery and vein were cannulated for blood pressure recordings, blood gas sampling, and drug administration. The piglets were intubated via a tracheotomy and artificially ventilated with room air. The respiratory parameters were set to maintain arterial pH, PCO₂, and PO₂ values in the physiological range (pH, 7.51 ± 0.01; PCO₂, 31 ± 1 mmHg; PO₂, 95 ± 2 mmHg). Body temperature was maintained at 37°C with a heating pad.

The head of the animal was fixed in a stereotaxic frame. The scalp was incised and the skin was removed along the connective tissue over the calvaria. A circular (19-mm diameter) craniotomy was made in the left parietal bone, and the dura was cut and reflected over the skull. A stainless steel cranial window with three needle ports was placed into the craniotomy, sealed with bone wax, and cemented with cyanoacrylate glue and dental acrylic.

The closed window was filled with artificial cerebrospinal fluid (aCSF) warmed to 37°C and equilibrated with 6% O₂-6.5% CO₂-87.5% N₂ to yield a pH of 7.33, a PCO₂ of 46 mmHg, and a PO₂ of 43 mmHg. The aCSF consisted of the following (in mmol/l): 132 NaCl, 2.9 KCl, 1.2 CaCl₂, 1.4 MgCl₂, 24.6 NaHCO₃, 6.7 urea, and 3.7 glucose. Diameters of pial arterioles were measured with a microscope (Wild M36) equipped with a digital videocamera and a video microscaler (model IV-550, For-A-Co). After a 45-min postsurgical equilibration period, a pial arterial segment with an initial diameter of ~100 µm was selected for the measurements. Only one segment was used from each animal. At the end of the experiments, the anesthetized animals were killed with an intravenous bolus of KCl.

After the determination of baseline diameter, a dose-response curve for topically applied bradykinin (0.03, 0.3, 3, and 30 µmol/l) was measured. BK was dissolved in aCSF and was given in 3-ml infusions under the window. Pial arterial diameter was monitored continuously and the peak dilation was used for further evaluation.

In another group of animals, 20 min after intravenous administration of 15 mg/kg L-NAME and 5 mg/kg indomethacin (a generous gift from Merck; Rahway, NJ), a dose-response curve for topically applied bradykinin (0.03, 0.3, 3, and 30 µmol/l) was measured. We have previously shown that these doses effectively block the respective enzymes in the experimental preparation (3, 4, 11). Repeated application of BK after the first dose-response curve had an inconsistently lower effect; therefore, BK was applied only for one response (3 µM) in each L-NAME plus indomethacin-treated animal.

In all other experimental groups, specific inhibitors were applied topically 10 min before and in coapplication with BK. The role of the bradykinin receptor subtype involved was assessed by application of the specific BK₂ receptor blocker D-arg⁰-Hyp³-Thi⁵-D-Tic⁷-Oic⁸-BK (HOE-140, 0.3 µmol/l). This dose was sufficient to completely suppress the vasodilation that was induced by 10 µmol/l BK in rat cerebral arteries (21). To assess the role of cytochrome P-450 metabolites, arteries were pretreated with miconazole (20 µmol/l). The dose of miconazole applied was two times higher than what was enough to completely suppress EDHF-like responses in rat and piglet pial arteries (32, 42). The involvement of lipoxygenase products was tested by the application of baicalein (10 µmol/l) or cinnamyl-3,4-dyhydroxy--cyanocinnamate (CDC, 1 µmol/l, BioMol; Plymouth Meeting, PA). The applied dose of baicalein was sufficient to block the vasodilation to arachidonic acid in pial arteries in vivo (17). The chemically different lipoxygenase inhibitor CDC was applied in a dose that completely blocked dilatory responses in rat mesenteric and porcine coronary arteries (39, 63). H₂O₂ accumulation was prevented by the application of catalase (400 U/ml), which was previously shown to inhibit the vascular effects of endogenous H₂O₂ but not hypotension in the piglet (33). K_Ca channels were blocked by coapplication of charybdotoxin (0.1 µmol/l) and apamin (0.5 µmol/l), which are doses sufficient to block EDHF (26). K_ATP channels were inhibited by glibenclamide (10 µmol/l). This dose of glibenclamide was able to block the vasodilation induced by activators of the K_ATP channel but not by PGE₂ in piglet pial arteries (5). Baicalein and miconazole were prepared as stock solutions in DMSO and further diluted with aCSF; final DMSO content was 0.1 and 0.2%, respectively. All other drugs were diluted with aCSF. After the determination of BK-induced dilation, animals received 20 µmol/l sodium nitroprusside (SNP) topically to test smooth muscle responsiveness. To determine whether endothelial impairment alters the BK response, phorbol-12,13-dibutyrate (PDB) was infused into the left common carotid artery. PDB was dissolved in DMSO and diluted to the final concentration of 10 µM at a volume of 5 ml. This procedure of PDB injection has been shown to produce transient endothelial dysfunction for at least 15 min, whereas dilator responses to non-endothelium-dependent agents are intact (1, 3, 10). We tested bradykinin reactivity of arterioles 8-10 min after PDB infusion. Only one BK response was performed in each animal. All drugs were obtained from Sigma-Aldrich (St. Louis, MO) unless stated otherwise.

Western blot analysis. Tissue samples were isolated from piglet brain cortex or pial vasculature and were frozen immediately at 60°C. Protein was extracted by the addition of boiling lysis buffer (which contained 1% of 1 mol/l Tris and 1% sodium dodecyl sulphate). The samples were sonicated, heated at 95°C for 5 min, and centrifuged for 20 min at 12,000 rpm and 4°C. The supernatant was used for immunoblotting. Protein concentration was measured by a Bio-Rad (New York, NY) assay kit.

An equal volume of sample buffer (120 mmol/l Tris, 20% glycerol, 0.02% bromophenol blue, 4% sodium dodecyl sulphate, and 200 mmol/l dithiothreitol at pH 6.8) was added to each sample. Equal amounts of protein (50 µg) were separated on a 4-20% gradient mini gel (Bio-Rad) and transferred to nitrocellulose membrane. After samples were blocked with 5% milk, primary antibody against the BK₂ receptor (BD Transduction Laboratory; San Diego, CA) was applied in a 1:1,000 dilution and horseradish peroxidase-conjugated secondary antibody was subsequently applied. Chemiluminescence was used to visualize the bands. Standard for the protein (rat pituitary lysate, Transduction Laboratory) and molecular weight markers (Bio-Rad) were included on each blot. The visualized blots were scanned and evaluated by measuring pixel numbers in the respective bands with a computer protocol (NIH Image software).

Statistical analysis. Data are presented as means ± SE. Statistical analysis was carried out by one-way ANOVA with subsequent Duncan's post hoc test for comparing control and treated values; power of the calculations was 0.9961. For data presented in Fig. 1, ANOVA for repeated measures was performed with subsequent Fisher's least-significant-difference test for post hoc comparisons. Number of animals in the text and figures is indicated as n. P < 0.05 was considered significant.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Dose-response curves of bradykinin (BK)-induced vasodilation in naïve animals (n = 7) and after the blockade of the nitric oxide synthase and cyclooxygenase enzymes (n = 10) by systemic application of 15 mg/kg N-nitro-L-arginine methyl ester (L-NAME) and 5 mg/kg indomethacin (Indo). Initial vessel diameter was identical in the two groups (naïve, 105 ± 3 µm; L-NAME + Indo, 107 ± 4 µm). Data are presented as means ± SE arterial diameter. ** P < 0.01 vs. control.

	RESULTS

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Topically applied BK induced dose-dependent vasodilation. The dilation to each dose was transient, reached its peak within 1 min, and returned to baseline within 3 min. Administration of aCSF alone did not change vascular diameter. Systemic application of L-NAME and indomethacin increased blood pressure (from 68 ± 2 to 93 ± 3 mmHg; P < 0.01) and significantly constricted pial arteries (from 107 ± 2 to 87 ± 3 µm; P < 0.01). The dilator response of BK was significantly shifted to lower diameter values due to the constricted initial diameter in the presence of L-NAME and indomethacin (Fig. 1).

There was no significant difference in baseline vascular diameter values among the experimental groups. None of the applied additional treatments (HOE-140, baicalein, CDC, catalase, miconazole) affected baseline arterial diameter or smooth muscle responsiveness to SNP (data not shown).

The remaining substantial dilation to BK (62 ± 12%) after L-NAME and indomethacin was completely inhibited by the application of the selective BK₂ receptor antagonist HOE-140 (54 ± 11 vs. 3 ± 2 µm increase in vessel diameter; P < 0.01; Fig. 2). Furthermore, endothelial impairment by intracarotid application of PDB markedly decreased the BK response (54 ± 11 vs. 16 ± 6 µm increase in vessel diameter; P < 0.05; Fig. 2).

View larger version (11K): [in this window] [in a new window]   Fig. 2.   BK response is dependent on the endothelium and is mediated via BK2 receptors. Vasodilation was induced by 3 µmol/l BK in the presence of L-NAME and Indo (control). Effect was completely blocked by the BK2 receptor antagonist D-arg0-Hyp3-Thi5-D-Tic7-Oic8 (HOE-140) in a dose of 0.3 µmol/l. Endothelial impairment with phorbol-12,13-dibutyrate (PDB) also markedly attenuated the response. Data are presented as means ± SE changes from baseline diameter. * P < 0.05, ** P < 0.01.

Western blotting experiments using a monoclonal antibody against the BK₂ receptor showed a double immunoreactive band at 42 kDa in both brain cortex and pial vascular tissue samples (Fig. 3). The bands were doublets in the case of the samples as well as the company-provided standards, probably representing two slightly different splice variants of the BK₂ receptor (48).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Localization of BK2 receptors in the piglet brain. Bars show the intensity of the 42-kDa immunoreactive band (respective sample band shown) in the different tissues (n = 8) compared to the standard rat pituitary lysate. No significant difference could be observed between the protein amounts of the BK2 receptor in blood vessel and neural tissue.

The cytochrome P-450 antagonist miconazole as well as the lipoxygenase inhibitors baicalein and CDC had no effect on BK-induced dilation. In contrast, the H₂O₂ scavenger catalase abolished the BK response (54 ± 11 vs. 0 ± 2 µm increase in vessel diameter; P < 0.01; Fig. 4).

View larger version (10K): [in this window] [in a new window]   Fig. 4.   Participation of putative endothelium-derived hyperpolarizing factors (EDHFs) in the BK-induced dilation. Vasodilation was induced by 3 µmol/l BK in the presence of L-NAME and Indo (control). Additional application of the cytochrome P-450 inhibitor miconazole (Mic, 20 µmol/l), the lipoxygenase inhibitors baicalein (10 µmol/l) or cinnamyl-3,4-dyhydroxy--cyanocinnamate (CDC, 1 mmol/l) failed to reduce the response. H2O2 scavenger catalase (400 U/ml) abolished the BK-induced vasodilation. Data are presented as means ± SE changes from baseline diameter. ** P < 0.01.

The K_ATP channel inhibitor glibenclamide significantly reduced the BK response (54 ± 11 vs. 16 ± 5 µm increase in vessel diameter; P < 0.05). Conversely, the K_Ca channel inhibitors charybdotoxin (large- and intermediate-conductance K_Ca channels) and apamin (small-conductance K_Ca channels) in coapplication failed to reduce the BK-induced vasodilation (Fig. 5).

View larger version (11K): [in this window] [in a new window]   Fig. 5.   Contribution of K+ channels to the BK-induced vasodilation in the presence of systemic nitric oxide synthase and cyclooxygenase blockade. Ca2+-dependent K+ channels were blocked by coapplication of charybdotoxin and apamin (Ctx + Apa, 0.1 µmol/l or 0.5 µmol/l, respectively), whereas ATP-dependent K+ channels were inhibited by glibenclamide (Glib, 10 µmol/l). Data are presented as means ± SE changes from baseline diameter. * P < 0.05.

	DISCUSSION

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In the present study, we have shown that BK is a remarkably potent vasodilator in the piglet pial vasculature even after the blockade of NO synthase (NOS) and COX. This dilation is dependent on endothelium and requires the action of BK₂ receptors. The effect is mediated via H₂O₂ formation and the opening of K_ATP channels. To the best of our knowledge, this is the first indication of the role of an EDHF in the newborn cerebral circulation.

In this study, the specific BK₂ receptor antagonist HOE-140 abolished the vasodilation induced by BK, which indicates the prominent role of this receptor subtype in the mediation of vasorelaxation to BK. A similar exclusive participation has already been described in other species including the rat (21) and cat (58). The BK₁ receptor appears to have no contribution to vasorelaxation, at least in the preparations studied so far (54, 58). BK₂ receptors were found in both neural and vascular tissue in the present study. Receptors at each location may have a role in the mediation of vasodilation; however, the BK-induced response is primarily endothelium dependent in various preparations (21, 43, 54). In the present study, endothelium impairment with PDB markedly reduced the BK response. This in vivo approach of endothelial impairment has been shown to reduce endothelium-mediated vasodilation and constriction, whereas the smooth muscle responsiveness remained intact. Accordingly, in the present study, the endothelium-independent vasodilation to SNP remained intact after PDB infusion. Furthermore, in a recent study by Willis and Leffler (59), endothelial impairment with the light dye technique also abolished the BK response, making it less likely that neural or direct smooth muscle action can account for the effect. Although endothelial denudation cannot be adequately performed in vivo, two independent approaches provided the same results. Therefore, this substantial, non-NO, non-prostanoid-mediated vasodilation appears to be the specific action of BK₂ receptors of the endothelial cell layer. Because BK induces EDHF release in different vascular beds, our next aim was to test which of the various EDHF candidates could be responsible for the observed vasodilation.

There is considerable evidence that cytochrome P-450 products dilate cerebral arteries and thus act like EDHF (2, 15, 20, 32) In the present study, blocking arachidonic acid metabolism at the lipoxygenase or the cytochrome P-450 pathways failed to reduce the BK response, making it unlikely that vasodilator arachidonic acid derivates contribute to the response.

Previous in vivo observations showed that BK relaxes the pial arteries via H₂O₂ formation in mice, rats, and cats (28, 29, 45, 60), as well as in porcine coronaries (43). Furthermore, H₂O₂ has recently been shown to fulfill the criteria to be an EDHF in mouse isolated vessels (38, 52). Because H₂O₂ is also a potent vasodilator in the piglet cerebral circulation (31), we hypothesized that it may contribute to the BK-induced vasodilation. Catalase, the endogenous H₂O₂ scavenger, specifically breaks down H₂O₂ to less vasoactive products. In the present study, the BK-induced vasodilation was abolished by catalase, which indicates the primary role of H₂O₂ in the mediation of this vasodilator response. Considering factors such as the dose of BK used and the initial baseline diameter, we estimate that H₂O₂-driven mechanisms provide at least one-half of the dilator response to BK.

The dilator action of H₂O₂ is reported to be mediated by hyperpolarization through K⁺ channels (23, 38, 49, 57, 61). The main contributing channel type is dependent on the species; for example, in the cat pial vasculature, K_ATP channels mediate the response, whereas in rats or dogs, K_Ca channels are responsible (25, 49, 57). Patch-clamp studies provided direct evidence that H₂O₂ induces hyperpolarization of the cell membrane, which was inhibited by the K_ATP channel blocker glibenclamide (18). In the rat cerebral circulation, the dilator effect of BK was inhibited to a similar extent by either catalase or the K_Ca channel blocker iberiotoxin (49). In this study, both catalase and glibenclamide markedly reduced the response to BK in the piglet pial vasculature, which indicates that the H₂O₂-induced vasodilation is mediated by K_ATP channel-derived hyperpolarization. Because glibenclamide did not totally abolish the BK-induced dilation, it seems likely that other mechanisms, including cyclic nucleotides, also contribute to the dilator response.

In conclusion, H₂O₂ has been found to be an EDHF-like mediator of the BK-induced vasodilation in the piglet cerebral circulation. Together with NOS and COX metabolites, EDHF appears to be an important mediator of vasodilation in the neonatal cerebral circulation.

	ACKNOWLEDGEMENTS

This study was supported by National Heart, Lung, and Blood Institute Grants HL-30260, HL-50557, HL-46558 (to D. W. Busija), and HL-66074 (to A. W. Miller), American Heart Association MidAtlantic Grant 99512724 (to D. W. Busija), Bugher Foundation Award 0270114N (to D. W. Busija), and EI Award 0140212N (to A. W. Miller). Z. Lacza was partially supported by a Hungarian National Eötvös Fellowship.

	FOOTNOTES

Address for reprint requests and other correspondence: Z. Lacza, Wake Forest Univ. School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157 (E-mail: zlacza{at}wfubmc.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.

First published March 21, 2002;10.1152/ajpheart.00007.2002

Received 8 January 2002; accepted in final form 8 March 2002.

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