Hydrogen Peroxide Emerges as a Regulator of Tone in Skeletal Muscle Arterioles During Juvenile Growth

Abstract

Objective

The endothelium-dependent dilation of skeletal muscle arterioles is mediated by factors that have not been identified in young rats, and partly mediated by an unidentified hyperpolarizing factor in maturing rats. This study was designed to determine if endogenous hydrogen peroxide (H₂O₂) contributes to this arteriolar dilation at either of these growth stages.

Methods

Gracilis muscle arterioles were isolated from rats at ages 24-26 days (“weanlings”) and 46-48 days (“juveniles”). We investigated the effects of catalase treatment on the endothelium-dependent dilation of these vessels to simvastatin and ACh. Catalase-sensitive 2′,7′-dichlorofluorescein (DCF) fluorescence also was measured as an index of H₂O₂ formation, and arteriolar dilation to exogenous H₂O₂ was pharmacologically probed in each age group.

Results

Responses to simvastatin and ACh were attenuated by catalase in juvenile, but not weanling, arterioles. Juvenile, but not weanling, arterioles also displayed catalase-sensitive DCF fluorescence that was increased by ACh. Exogenous H₂O₂ could induce dilation in juvenile, but not weanling, arterioles. In juvenile arterioles, this dilation was abolished by the K⁺ channel inhibitors TEA and glibenclamide, and attenuated by NOS inhibition or endothelial removal.

Conclusion

These findings suggest that endogenous H₂O₂ contributes to endothelium-dependent arteriolar dilation in juvenile rats but not in younger rats, and that H₂O₂ acts in juvenile rats by stimulating endothelial NO release and activating smooth muscle K⁺ channels.

Introduction

Although relatively high levels of hydrogen peroxide (H₂O₂) are sometimes responsible for the vascular dysfunction associated with certain disease states (2, 25), H₂O₂ at lower concentrations has been recognized as an important signaling molecule that can mediate normal vascular function (5, 19). For example, endothelium-derived H₂O₂ has been identified as one of the factors that mediate arteriolar dilation to some agonists in the cerebral (35), mesenteric (28) and coronary (27) vascular beds, and there is mounting evidence for a physiological role of arteriolar H₂O₂ in local coronary blood flow regulation (18, 29, 45). Our laboratory has recently reported that endogenous H₂O₂ can also contribute to the arteriolar dilation and hyperemia that accompanies skeletal muscle contraction (26)

Other studies from our laboratory have documented that postnatal growth of the microvasculature in skeletal muscle is accompanied by progressive changes in a number of factors that can influence arteriolar tone and blood flow (1, 23, 24, 32). For example, skeletal muscle arterioles from young rats exhibit endothelium-dependent dilation, but these responses are not mediated by either nitric oxide (NO) or cyclooxygenase metabolites, as they are in arterioles from mature rats (1). The specific endothelial factors that mediate this dilation in young arterioles remain obscure. Because H₂O₂ has been identified as an important endothelium-derived vasoactive factor in the cerebral microcirculation of newborn pigs (21), we undertook the current study to determine if H₂O₂ could also mediate endothelium-dependent dilation in skeletal muscle arterioles of young rats. A second aim of this study was to determine if there are any changes in the mechanism by which H₂O₂ can influence arteriolar tone during rapid juvenile growth.

Materials and Methods

Animals

All surgical and experimental procedures were approved by the West Virginia University Animal Care and Use Committee. Experiments were performed on gracilis muscle arterioles isolated from male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) at 3-4 weeks of age (“weanlings”) or 6-7 weeks of age (“juveniles”).

Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight), with supplemental anesthetic (10% of original dose) administered if needed. The right carotid artery was cannulated with polyethylene tubing (PE-10 for weanlings, PE-50 for juveniles) to measure mean arterial pressure before removal of the gracilis arteriole.

Preparation of Isolated Vessels

An arteriolar branch of the femoral artery supplying the gracilis muscle was removed, handling only the surrounding connective tissue to minimize vessel stretching or damage. The rat was sacrificed by intracardiac injection of sodium pentobarbital immediately after vessel removal. The vessel was placed in warmed physiological salt solution (PSS) equilibrated with 21% O₂, 5% CO₂, and 74% N₂, and having the following composition (mM): 119 NaCl, 4.7 KCl, 1.17 MgSO₄, 1.6 CaCl₂, 1.18 NaH₂PO₄, 24 NaHCO₃, 0.026 EDTA, and 5.5 glucose. After isolation, each vessel was prepared for in vitro video microscopy as previously described (12). Briefly, the vessel was mounted in a heated (37° C) chamber that allowed for its lumen and exterior surface to be perfused and superfused, respectively, with PSS from separate reservoirs. The vessel was cannulated at both ends with glass micropipettes (50- and 70-μm tip diameters for weanling and juvenile vessels, respectively) and secured to the inflow and outflow pipettes using 9-0 nylon suture. Any side branches were ligated with a single strand teased from 6-0 silk suture. The inflow pipette was connected to a reservoir perfusion system for control of intralumenal pressure and flow. The vessel was then extended to its in situ length and equilibrated at 80% of the animal's mean arterial pressure to approximate its normal in vivo perfusion pressure (10).

Vessel diameter was measured using an onscreen video micrometer. Any vessel that did not demonstrate endothelial viability, as judged by at least a 10% dilation in response to 10⁻⁷ M acetylcholine (ACh, Sigma Chemical, St. Louis, MO), was not used in the study. Using this criterion, approximately 1 in 10 vessels from each age group were discarded. In such cases, we then isolated and studied the anatomically similar arteriole from the animal's contralateral gracilis muscle, so that we were able to obtain a viable arteriole from each animal. Diameter measurements were made under static, zero-flow conditions after a 30-minute equilibration period with continuous perfusion. Resting vascular tone was calculated as (ΔD/D_max) × 100, where ΔD is the diameter increase from rest in response to Ca²⁺-free PSS (30-40 minute equilibration with Ca²⁺-free bath solution and no luminal flow), and D_max is the maximum diameter measured under these conditions.

Agonists

ACh, at bath concentrations of 10⁻⁵ or 10⁻⁷ M, was used to assess arteriolar capacity for Ca²⁺-dependent endothelial NO formation (42). Simvastatin (Merck Research Laboratories, Rathway, NJ) was used to assess arteriolar capacity for Ca²⁺-independent endothelial NO formation (32). Simvastatin stimulates endothelial NO release via activation of the phosphatidylinositol 3-kinase (PI₃K)/Akt pathway, which in turn leads to direct activation of eNOS through phosphorylation of its key serine residues (20). Simvastatin was activated by alkaline lysis (5.25 ml of 0.1 N NaOH per 140 mg, dissolved in 3.5 ml of ETOH) at 50°C for 2 h. The resulting solution was then diluted to a volume of 35 ml with PBS, and neutralized to pH 7.4 with HCl. One-ml aliquots of this solution were then serially diluted with phosphate-buffered saline (PBS), producing a final bath concentration of 10⁻⁵ or 10⁻⁷ M. The two concentrations of each agonist were applied in random order, separated by a washout period to allow the vessel to regain resting tone.

For exogenous H₂O₂ application, a 3% aqueous H₂O₂ solution was diluted with PSS to concentrations of 1×10⁻⁶, 5×10⁻⁶, 1×10⁻⁵, 5×10⁻⁵, and 1×10⁻⁴ M, and these increasing concentrations were sequentially added to the bath. After each application, the H₂O₂ was washed out of the chamber with PSS, and the vessel was allowed to return to its control diameter before the next H₂O₂ application.

Assessment of Arteriolar H₂O₂ Production

2′,7′ dichlorodihydrofluorescein diacetate (DCFH-DA) was used to detect intracellular H₂O₂ production (29, 33) in arterioles at rest or during exposure to 10⁻⁵ M ACh, either with or without simultaneous exposure to catalase (Sigma, from bovine liver, 80 U/ml bath concentration). To ensure maximal scavenging of H₂O₂, this concentration of catalase was higher than the concentration we have previously found to scavenge all H₂O₂ formed during stimulation of arteriolar superoxide (O₂ ⁻) generation in vivo (22).

Non-fluorescent DCFH-DA freely enters the cell, where it is de-acetated to DCFH and then oxidized by H₂O₂ to form fluorescent 2′,7′ dichlorofluorescein (DCF) (37). Under some conditions, DCFH can also be oxidized in vivo by cellular peroxidases, peroxynitrite, or even hypochlorous acid (33, 37). Therefore, we considered only the portion of DCF fluorescence that could be reduced by catalase as an indicator of H₂O₂ activity.

After the arteriole was isolated, cannulated and allowed to develop spontaneous tone at 80% of mean arterial pressure, 5×10⁻⁵ M DCFH-DA was added to the vessel bath under one of the conditions described above. After 20 minutes of exposure to DCFH-DA, the arteriole was quickly removed from the glass micropipettes and transferred to the stage of a fluorescence microscope coupled to a high-sensitivity black and white CCD video camera (Dage-MTI, Michigan City, IN), and then epi-illuminated for 1 second using a mercury lamp and the appropriate excitation and emission filters for detection of DCF fluorescence (460-490 nm excitation, 515-550 nm barrier). In all cases, an instantaneous image of the vessel was obtained immediately upon opening of the illumination diaphragm, with identical illumination settings and optical parameters used for each vessel. Metamorph software (v. 6.01, Universal Imaging, Downingtown PA) was used to acquire, digitize and store images for subsequent analysis (46). Average fluorescence intensity was determined for the entire vessel wall (V) as well as for the background (B) of each vessel. Fluorescence intensity, based on the standard 0-255 gray scale (17), was quantified using the following equation:

$$\%I_{\mathit{\text{MAX}}}=\stackrel{V-B}{}/\underset{255-B}{}$$

where %I_MAX is percent of maximum fluorescence intensity, V is the intensity of the vessel wall, and B is the background.

Scavengers and Inhibitors

To assess the role of reactive oxygen species in arteriolar dilation, vessels were pretreated with catalase (see above) or the membrane-permeable superoxide dismutase (SOD) mimetic 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (tempol) (39) (Sigma, 10⁻⁴ M), alone or in combination. These agents were added to the bath for 30 minutes before vessels were challenged with agonists. As a control for active catalase, arteriolar responses to ACh and H₂O₂ were also assessed after treatment with denatured catalase. For denaturization, catalase was boiled for 10 minutes. To determine the contribution of endothelial NO production to vessel responses, the NOS inhibitor N^ω-nitro-L-arginine methyl ester (L-NAME) was added to the bath at a concentration of 10⁻⁴ M for 20 minutes before agonist challenge (13).

Since many endothelium-derived relaxing factors reduce arteriolar tone at least in part through activation of smooth muscle K⁺ channels (6, 41, 43), the contribution of these channels to arteriolar dilation was assessed by using tetraethylammonium (TEA, Sigma) and the antidiabetic sulphonylurea, glibenclamide (Sigma), in both intact and endothelium-denuded vessels (see below). We used a 10⁻³ M bath concentration of TEA to selectively block Ca²⁺-activated K⁺ (K_Ca) channels (31), and 10⁻⁶ M glibenclamide to selectively block K_ATP channels (36).

Endothelial Denudation

To determine the role of the endothelium in mediating arteriolar responses to exogenous H₂O₂, the endothelium was removed from some vessels by mechanical abrasion (40). The pipette tip at each end of the vessel was gently advanced through the vessel lumen at least three times to ensure elimination of the endothelium. We have previously verified that this method successfully denudes the endothelium of gracilis muscle arterioles without affecting the underlying smooth muscle, as judged by the preservation of normal constrictor responses to phenylephrine and normal endothelium-independent dilator responses to sodium nitroprusside (1).

All data are presented as mean ± SE. For all analyses, a probability value of p < 0.05 was considered to be statistically significant. Dilation in response to Ca²⁺-free PSS is expressed as percent increase from control diameter. Differences between the means of individual experimental groups were determined by ANOVA/Newman-Keuls test, or by an unpaired Student's t-test when two independent means were compared.

Results

General characteristics of all rats from which vessels were removed for functional studies are reported in Table 1. Age, body weight, and mean arterial pressure were significantly greater in juvenile rats than in weanling rats. Table 1 also summarizes the general characteristics of all arterioles utilized for functional studies. Resting and passive diameters of arterioles from juvenile rats were significantly greater than those of arterioles from weanling rats, whereas there was no difference between groups in the level of spontaneous tone (resting vascular tone) that developed with vessel pressurization.

Table 1.

Animal Characteristics	Weanlings	Juveniles
N	42	39
Age (days)	25 ± 1	46 ± 1 *
Body Weight (g)	58 ± 1	170 ± 3 *
MAP (mmHg)	80 ± 2	96 ± 2 *
Vessel Characteristics	Weanlings	Juveniles
n	42	39
Resting Diameter (μm)	39 ± 1	56 ± 2 *
Passive Diameter (μm)	58 ± 2	83 ± 2 *
Resting Vascular Tone (%)	32 ± 2	32 ± 2

General characteristics of all rats and vessels used for functional studies. MAP = mean arterial pressure. Values are given as means ± SE. N = number of animals; n = number of vessels.

^*p<0.05 vs. weanling group.

There were no differences between age groups in overall arteriolar responsiveness to ACh or simvastatin (Figure 1). Treatment with catalase, tempol, or catalase + tempol had no effect on the resting diameter of either weanling or juvenile arterioles (Table 2). However, catalase dramatically reduced responses to ACh and simvastatin in juvenile arterioles, but not in weanling arterioles (Figure 2, left panels). Denatured catalase had no effect on responses to ACh in either group (Table 3), verifying that the effects of catalase on juvenile arterioles were specifically due to the enzymatic activity of the molecule. Tempol by itself was without effect on responses to ACh or simvastatin in either group. In the juvenile arterioles, treatment with catalase + tempol reduced ACh and simvastatin responses to levels that were not different from those with catalase treatment alone (top right panel). In weanling arterioles, catalase + tempol had no effect on responses to either agonist (bottom right panel).

Figure 1.

Responses of gracilis muscle arterioles from juvenile and weanling rats to acetylcholine and simvastatin. n = number of vessels. Values are given as means ± SE.

Table 2. Arteriolar Diameter (μm).

	Control	Catalase	Control	Tempol	Control	Catalase + Tempol
Weanlings	45 ± 4	43 ± 4	45 ± 4	43 ± 5	43 ± 3	43 ± 4
Juveniles	64 ± 4	62 ± 5	64 ± 4	58 ± 4	64 ± 4	60 ± 6

Summary of resting vessel diameters under control conditions and after treatment with 80 U/ml catalase and 10⁻⁴ M tempol, separately or in combination.Values are given as means ± SE.

Figure 2.

Effect of catalase (80 U/ml) and tempol (10⁻⁴ M), alone and in combination, on responses of juvenile and weanling arterioles to acetylcholine and simvastatin. n = number of vessels. Values are given as means ± SE. * p<0.05 vs. Control.

Table 3.

	Weanlings (n=3)		Juveniles (n=3)
	Control	Inactivated catalase	Control	Inactivated catalase
10−5M ACh	32 ± 4	41 ± 8	33 ± 3	40 ± 8
10−6 M H2O2	-22 ± 10	-22 ± 9	-13 ± 3	-12 ± 5
10−5 M H2O2	-22 ± 9	-27 ± 11	-1 ± 4	-2 ± 1
10−4 M H2O2	-7 ± 4	-3 ± 3	17 ± 4	18 ± 8

Magnitude of arteriolar diameter changes in response to ACh and H₂O₂ before and after treatment with denatured catalase (80 U/ml). Values are given as means (% maximum dilation) ± SE. n = number of vessels.

A total of 26 juvenile arterioles and 22 weanling arterioles were examined after DCFH-DA exposure for determination of relative H₂O₂ levels. Compared to their respective control levels, 10⁻⁵ M ACh significantly increased DCF fluorescence in juvenile arterioles (from 29 ± 4 to 44 ± 4% of maximum intensity), but not in weanling arterioles (Figure 3). Catalase reduced DCF fluorescence under control conditions as well as during ACh exposure in juvenile arterioles, but had no significant effect in weanling arterioles.

Figure 3.

Representative microscopic images showing DCF fluorescence in arterioles exposed to DCFH-DA. Upper row: juvenile vessels. Lower row: weanling vessels. Bottom panel: Effect of 10⁻⁵ M ACh, alone or in the presence of catalase, on mean DCF fluorescence intensity in weanling and juvenile arterioles. * p<0.05 vs. Control in same age group. † p<0.05 vs. ACh in same age group. ‡ p<0.05 vs. Control in juvenile arterioles.

Application of exogenous H₂O₂ at bath concentrations ranging from 1×10⁻⁶ M to 1×10⁻⁴ M elicited concentration-dependent responses from juvenile arterioles, with the lower concentrations causing constriction and the higher concentrations causing dilation (Figure 4). Weanling arterioles also constricted in response to H₂O₂ at the lower concentrations, and these constrictions were significantly greater than those of the juvenile arterioles. However, whereas juvenile arterioles clearly dilated in response to higher concentrations of H₂O₂, weanling vessels did not.

Figure 4.

Responses of juvenile and weanling arterioles to exogenous hydrogen peroxide (H₂O₂). n = number of vessels. Values are given as means ± SE. * p<0.05 vs. juvenile arterioles.

To gain further insight into the contribution of H₂O₂ to ACh- and simvastatin-induced dilation of juvenile arterioles, we explored the mechanism(s) by which exogenous H₂O₂ induces the dilation of these vessels. Treatment with L-NAME had no significant effect on the resting diameters of either juvenile arterioles (51 ± 4 μm before vs. 48 ± 6 μm after L-NAME) or weanling arterioles (42 ± 3 μm vs. 39 ± 3 μm). However, L-NAME significantly reduced the H₂O₂induced dilation of juvenile arterioles, with subsequent addition of catalase completely abolishing the L-NAME-insensitive component of this dilation (Figure 5, top panel). Weanling arterioles remained unresponsive to these higher concentrations of H₂O₂ in the presence of L-NAME or L-NAME + catalase (bottom panel).

Figure 5.

Responses of juvenile and weanling arterioles to exogenous hydrogen peroxide (H₂O₂), before and after inhibition of nitric oxide synthase with N^ω-nitro-L-arginine methyl ester (L-NAME), alone and in combination with catalase. n = number of vessels. Values are given as means ± SE. * p<0.05 vs. Control.

Treatment with TEA + glibenclamide significantly reduced the resting diameters of juvenile arterioles (51 ± 5 μm before vs. 36 ± 3 μm after treatment) and weanling arterioles (36 ± 3 μm vs. 30 ± 2 μm). When expressed as percent reduction from control diameter, the magnitude of this effect was similar in the two groups (25-29%). TEA + glibenclamide also converted the H₂O₂ -induced dilation of juvenile arterioles into constrictor responses (Figure 6, top panel), and unmasked a constrictor response of weanling arterioles to these higher concentrations of H₂O₂ (bottom panel).

Figure 6.

Responses of juvenile and weanling arterioles to exogenous hydrogen peroxide (H₂O₂) before and after inhibition of K_Ca channels with TEA (10⁻³ M) and K_ATP channels with glibenclamide (10⁻⁶ M). n = number of vessels. Values are given as means ± SE. * p<0.05 vs. Control. † indicates significant difference from zero (p<0.05).

Endothelial removal tended to reduce the resting diameters of juvenile arterioles (54 ± 2 μm before vs. 47 ± 4 μm after de-endothelialization) and weanling arterioles (36 ± 3 μm vs. 29 ± 2 μm), but these reductions did not reach statistical significance. However, removal of the endothelium significantly reduced the H₂O₂ -induced dilation of juvenile arterioles, with subsequent exposure to TEA + glibenclamide completely abolishing the remaining endothelium-independent component of these responses (Figure 7, top panel). Weanling arterioles remained unresponsive to higher concentrations of H₂O₂ after endothelial removal, and in contrast to our findings in intact weanling arterioles, TEA + glibenclamide did not unmask a significant constrictor response to higher concentrations of H₂O₂ in these vessels after endothelial removal (bottom panel).

Figure 7.

Effect of endothelial removal on responses of juvenile and weanling arterioles to exogenous hydrogen peroxide (H₂O₂) before and after combined treatment with TEA and glibenclamide. n = number of vessels. Values are given as means ± SE. * p<0.05 vs. Control.

Discussion

Growth-related changes in microvascular endothelial function have been reported by our laboratory and others (1, 24, 32, 44). In a recent study (1), we found that skeletal muscle arterioles from mature rats exhibit endothelium-dependent dilations to ACh and simvastatin that are partially due to NO release, with vasodilator prostanoids also contributing to these responses. However, in that study, we also found that the endothelium-dependent dilation of arterioles from younger (weanling) rats is not mediated by any of these factors. Since H₂O₂ has been found to mediate endothelium-dependent dilation of the cerebral vasculature in newborn pigs (21), we undertook the current study in part to determine if H₂O₂ could be playing an analogous role in the skeletal muscle circulation of weanling rats. However, we found that catalase had no effect on the responses of weanling arterioles to ACh or simvastatin (Figure 2, bottom panel), or on DCF fluorescence in these vessels either at rest or during ACh exposure (Figure 3, bottom panel). These findings suggest that H₂O₂ does not contribute to the endothelium-dependent dilation of skeletal muscle arterioles in young rats. In further support of this conclusion, exogenous H₂O₂, at bath concentrations of up to 100 μM_, did not elicit a dilation of weanling arterioles (Figure 4). Due to the activity of catalase, glutathione peroxidase and other H₂O₂ -scavenging molecules in both smooth muscle and endothelial cells (5, 8), the extent to which vascular wall H₂O₂ actually increased during these exogenous applications is unknown. However, the concentrations that were achieved are most likely above normal, steady-state in vivo levels, which have been estimated to range from 1-100 nM within cells (14), and have been measured extracellularly at 1-10 nM within the vascular wall (7). During severe inflammation, tissue H₂O₂ levels can increase to as high as 100 μM (15). In the presence of K⁺ channel inhibitors, weanling arterioles did respond (i.e., constricted) to higher concentrations of exogenous H₂O₂ (Figure 6, bottom panel). This suggests that the signaling pathway for constriction that is activated by H₂O₂ at lower concentrations is overridden at higher concentrations of H₂O₂ by a dilator mechanism involving vascular smooth muscle hyperpolarization.

The current findings, in conjunction with those of our earlier study (1), rule out the contributions of NO, cyclooxygenase products and H₂O₂ to the endothelium-dependent dilation of skeletal muscle arterioles in rats between 3 and 4 weeks of age. Further studies will be necessary to identify the factor(s) that do mediate such responses at this early stage of life. It should also be noted that we did not find a significant difference between age groups in overall arteriolar responsiveness to ACh (Figure 1), which contrasts with our earlier findings that weanling arterioles exhibited a slightly greater responsiveness to ACh (1). Because this was found to be a relatively modest difference in the previous study, we may simply have been unable to statistically resolve that difference in the current study due to the much smaller number of vessels studied here (“n” values of 11-12 in the current study vs. 37-52 in the earlier study).

Contribution of H₂O₂ to endothelium-dependent responses of juvenile arterioles

In contrast to our findings in weanling arterioles, exogenous catalase greatly reduced the dilation of juvenile arterioles to both ACh and simvastatin (Figure 2). Furthermore, we found a large, catalase-sensitive component of DCF fluorescence in these vessels at rest (approximately 50% of total fluorescence) that was further increased (to approximately 63% of total fluorescence) when the vessels were challenged with ACh (Figure 3, bottom panel). These findings indicate that endogenous H₂O₂ does contribute to the dilation of these vessels to ACh and simvastatin, and in light of our findings in weanling arterioles, suggest that this mechanism becomes operative sometime between 4 and 6 weeks of age.

Our recent finding that endothelial removal completely abolishes the ACh- and simvastatin-induced dilation of juvenile arterioles (1) verifies that this H₂O₂ is endothelial in origin. The O₂⁻ from which H₂O₂ is formed could be generated during mitochondrial electron transport, or from the intracellular activity of membrane-bound NADPH oxidase, xanthine oxidase, cyclooxygenase, cytochrome P450 monooxygenase or nitric oxide synthase (3). Because any agonist-induced increase in H₂O₂ production must first trigger increased O₂⁻ formation, we initially expected that in the presence of the SOD mimetic tempol, ACh or simvastatin would lead to greater H₂O₂ production and therefore a larger dilation. The fact that this did not occur (Figure 2, top panel) suggests that the intrinsic SOD capacity of these vessels may be high enough to dismutate all of the additional O₂⁻ that is formed in response to these agonists, such that treatment with tempol would have no additional effect.

The responses of juvenile arterioles to exogenously applied H₂O₂ were complex, with lower concentrations of H₂O₂ eliciting constriction and higher concentrations of H₂O₂ eliciting dilation (Figure 4). This is consistent with a report by Cseko et al (9), who found that constriction of rat gracilis muscle arterioles to lower concentrations of H₂O₂ is completely abolished by prostaglandin H₂ /thromboxane A₂ (PGH₂/TxA₂) receptor blockade, but only reduced (not abolished) by endothelial removal. These authors concluded that at low concentrations, H₂O₂ increases arteriolar tone via the formation of constrictor prostanoids in both endothelial and vascular smooth muscle cells.

The dilation of juvenile arterioles to higher concentrations of H₂O₂ was reduced following endothelial removal (Figure 7), implying an important role for endothelium-derived mediators in these responses as well. Endothelial removal has also been shown to reduce H₂O₂ -induced dilation in mouse coronary arterioles (38), but not in human coronary arterioles, as reported by Miura and colleagues (29). Although this may reflect species-specific differences, it could also reflect the fact that the arterioles studied by Miura et al. were obtained from patients with various diseases (e.g., coronary artery disease, diabetes mellitus, hypertension), and therefore could have displayed some degree of endothelial cell dysfunction (34).

As shown in the top panel of Figure 5, inhibition of NOS in intact juvenile arterioles reduced the magnitude of H₂O₂- induced dilation to a level similar to that achieved by de-endothelialization, which implicates endothelium-derived NO as a major contributor to this response. We also found that blockade of K_Ca and K_ATP channels completely abolished the residual endothelium-independent portion of this dilation (Figure 7, top panel), suggesting a direct relaxing effect of H₂O₂ on the vascular smooth muscle due to K⁺ channel activation. This is consistent with previous findings that H₂O₂ can induce smooth muscle hyperpolarization and vasodilation by opening K⁺ channels, although the main contributing channel type (i.e., K_Ca vs. K_ATP channels) can vary with the species or vascular bed studied (4, 11, 16, 28, 30).

In conclusion, our results suggest that sometime between 4 and 6 weeks of age, H₂O₂ assumes an important role in the endothelium-dependent control of arteriolar tone through both an increase in the capacity of these vessels to produce H₂O₂ (Figure 3) and the onset of a vascular smooth muscle dilator response to H₂O₂ (Figure 4). The mechanisms that underlie these complex changes are unknown and will require further study. Our current findings seem to indicate that most or all of the elements of the cell signaling pathways that mediate H₂O₂ –induced dilation at 6 weeks of age are at least capable of being activated at 4 weeks of age. For example, because H₂O₂ and simvastatin both activate eNOS through AKT-dependent phosphorylation of its serine residues (2, 20), our finding that weanling and juvenile arterioles are both responsive to simvastatin (Figure 1) suggests that the signaling pathway through which H₂O₂ can induce endothelium-dependent smooth muscle relaxation is present in the younger animals. Furthermore, H₂O₂ can also activate smooth muscle potassium channels in weanling arterioles (Figure 6). However, the lack of any net dilator response to H₂O₂ in these vessels suggests that other unknown factors are involved.