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Am J Physiol Heart Circ Physiol 293: H2085-H2092, 2007. First published July 20, 2007; doi:10.1152/ajpheart.00968.2006
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Cardiovascular-Renal Mechanisms in Health and Disease

Hydrogen peroxide mediates a transient vasorelaxation with tempol during oxidative stress

Yifan Chen, Adam Pearlman, Zaiming Luo, and Christopher S. Wilcox

Cardiovascular Kidney Hypertension Institute and Division of Nephrology and Hypertension, Georgetown University, Washington, District of Columbia

Submitted 6 September 2006 ; accepted in final form 17 July 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tempol catalyzes the formation of H2O2 from superoxide and relaxes blood vessels. We tested the hypothesis that the generation of H2O2 by tempol in vascular smooth muscle cells during oxidative stress contributes to the vasorelaxation. Tempol and nitroblue tetrazolium (NBT) both metabolize superoxide in vascular smooth muscle cells, but only tempol generates H2O2. Rat pressurized mesenteric arteries were exposed for 20 min to the thromboxane-prostanoid receptor agonist, U-46619, or norepinephrine. During U-46619, tempol caused a transient dilation (22 ± 2%), whereas NBT was ineffective (2 ± 1%), and neither dilated vessels constricted with norepinephrine, which does not cause vascular oxidative stress. Neither endothelium removal nor blockade of K+ channels with 40 mM KCl affected the tempol-induced dilation, but catalase blunted the tempol dilation by 53 ± 7%. Tempol, but not NBT, increased H2O2 in rat mesenteric vessels detected with dichlorofluorescein. To test physiological relevance in vivo, topical application of tempol caused a transient dilation (184 ± 20%) of mouse cremaster arterioles exposed to angiotensin II for 30 min, which was not seen with NBT (9 ± 4%). The vasodilation to tempol was reduced by 68 ± 6% by catalase. We conclude that the transient relaxation of blood vessels by tempol after prolonged exposure to U-46619 or angiotensin II is mediated in part via production of H2O2 and is largely independent of the endothelium and potassium channels.

superoxide dismutase; hypertension; angiotensin II; thromboxane

OXIDATIVE STRESS ACCOMPANIES all of the primary cardiovascular risk factors, which include hypertension, diabetes, hypercholesterolemia, smoking, and age (4). Moreover, the systemic administration of a membrane-permeable form of superoxide dismutase (SOD), or of nitroxide SOD mimetics, such as tempol, reduces blood pressure (BP) and peripheral vascular resistance and corrects parameters of oxidative stress in many animal models of hypertension (15), including spontaneously hypertensive rats (SHR) (30) and rats (42) or mice (20) infused with angiotensin II (ANG II). Tempol is effective in reversing hypertension and inducing vasodilation, whether given acutely by intravenous infusion (30) or over 2 wk (42). A recent study shows that tempol directly dilates mesenteric arteries in situ from deoxycorticosterone acetate-salt hypertensive rats (43). However, the role of H2O2 in the vasodilation by SOD or SOD mimetics is not established.1

Several factors complicate the interpretation of the antihypertensive or vasodilator actions of tempol. SOD or tempol not only reduce superoxide (O2bullet–), but also may increase H2O2 in blood vessels or the kidney medulla (7). H2O2 may produce vasodilation (11), vasoconstriction (12, 32), or a biphasic effect (8, 11), depending on the vascular bed and experimental conditions. Prolonged infusion of H2O2 into the renal medulla of the rat increases BP (24). Therefore, it is hard to predict the functional role, if any, of H2O2 formed in blood vessels after tempol administration.

The primary objective of this study is to develop a model of vascular oxidative stress to examine the role of H2O2 in the vasorelaxation response to tempol. We contrasted the effects of tempol and SOD with that of nitroblue tetrazolium (NBT), which we confirm in studies of vascular smooth muscle cells (VSMCs) metabolizes O2bullet– without the generation of H2O2 (26). We assessed the generation of H2O2 in rat isolated, perfused mesenteric resistance vessels using dichlorodihydrofluorescein (DCF) fluorescence and assessed the functional role of H2O2 from the effects of metabolism of H2O2 with polyethylene glycol (PEG)-catalase on the vasorelaxation response to tempol. Since oxidative stress is induced in vascular tissues after activation of thromboxane-prostanoid (TP) receptors with U-46619 (28, 36) or type 1 (AT1) receptors with ANG II (14, 40), but not after equally vasoconstrictive or hypertensive concentrations of norepinephrine (NE) (22, 40, 41), we contrasted the vasorelaxant effects of tempol and the role of H2O2 in vessels preconstricted with U-46619 or ANG II with those preconstricted with NE. Finally, we tested the physiological relevance of the role of H2O2 in vasorelaxation by tempol in vivo using mouse cremasteric resistance arterioles studied directly by intravital microscopy during contractions induced by ANG II.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Measurements of Chemiluminescence and Fluorescence of VSMCs

Preglomerular VSMCs (PGVSMCs, passages 5–15) were prepared from 13- to 15-wk-old SHR (1), seeded into 96-well plates (~5 x 105 cells/well), incubated in DMEM/F-12 medium (GIBCO) containing 10% of fetal bovine serum (ATCC), penicillin (100 U/ml), streptomycin (100 µg/ml), and glutamine (200 µg/ml), and maintained with 5% CO2 and 95% air at 37°C and 98% humidity to 80% confluence. Twenty-four hours before experiments, the medium was replaced with a serum-free supplement.

ANG II (10–6 M), U-46619 (10–5 M), tempol (10–4 M), SOD (100 U/ml), and/or NBT (10–4 M) were applied 2 h before experiments, and catalase (3,000 U/ml) 4 h before experiments. The treated cells were washed twice with pH 7.4 salt solution containing 130 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 35 mM phosphoric acid, and 20 mM HEPES (3).

Lucigenin-enhanced (final concentration 5 µM) chemiluminescence (CL) was used to determine O2bullet– generation (40), while DCF (5 µg/ml) fluorescence was used to determine H2O2. These probes were added to the samples 20–30 min before experiments. The viability of the cells was >90%, as determined by Trypan blue exclusion. The generation of O2bullet– was initiated by adding NADPH (10–4 M). The CL or fluorescence signals were measured in a Multilabel Counter 1420 (PerkinElmer, Wellesley, MA) integrated each 30 s for 8 min.

Animals

Male adult Sprague-Dawley rats (body weight: 310–360 g) and mice (background: C57B6, body weight: 22–28 g) were maintained on standard diet and water ad libitum. All of the procedures were approved by the Georgetown University Animal Care and Use Committee.

Isolated Mesenteric Artery Preparation

Rats were anesthetized with inactin (50 mg/kg weight). The mesentery was removed through a longitudinal abdominal incision and placed immediately in ice-cold physiological salt solution (PSS). A second-order segment (internal diameter: 297–471 µm) was dissected free of fat and connective tissue and transferred to a pressure myograph bath with a video measurement system (Danish Myo Technology A/S, Danmark). A 3- to 5-mm segment was cannulated at both ends and mounted in the direction of flow. The vessel was superfused at 37°C, pH 7.4, with PSS containing the following in mM: 128 NaCl, 4.7 KCl, 2.0 CaCl2·2H2O, 1.2 MgSO4·7H2O, 17.8 NaHCO3, 0.02 EDTA, 1.2 NaH2PO4·H2O, 5.0 glucose, and 2.0 pyruvate, and gassed with 95% O2 and 5% CO2. The vessels were pressurized gradually to ~60 mmHg and tested for functional viability with 10–4 M acetylcholine and 10–5 M phenylephrine.

In Vivo Cremaster Arteriole

Mice were anesthetized with 1.5–2% isoflurane in air. The trachea was cannulated, and a catheter was placed into an external jugular vein for infusion of 1% albumin in 0.9% NaCl at ~300 µl/h to maintain hydration. The cremaster muscle was exteriorized for intravital microscopy. It was superfused at 34–35°C, pH 7.4, with PSS gassed with 10% O2 and 5% CO2. Body temperature was maintained at ~37°C by conductive and radiant heating. Third-order arterioles (maximal diameter: 35–41 µm) (6) were selected, which showed >70% vasodilation with 10–4 M acetylcholine and >70% constriction with 10–5 M phenylephrine. The preparation was equilibrated for 45–60 min to gain spontaneous tone (judged as <60% of maximum diameter).

The ex vivo mesenteric arteries and in vivo cremaster arterioles were transilluminated and examined by a microscope adjoined to a video camera, a video micrometer, and a video monitor detecting <2 µm in a ~400-µm mesenteric artery and <0.5 µm in a ~20-µm cremaster arteriole. The interior wall diameter was measured in real time from the video micrometer analog outputs digitized by personal computers programmed using LabView 7.1 (National Instruments, Austin, TX).

Measurement of H2O2 of Rat Mesenteric Arteries

Dynamic changes in H2O2 in mesenteric vessels were monitored with DCF (5 µg/ml) fluorescence (excitation: 485 nm; emission: 535 nm) (16, 29, 37, 38). Second-order segments of rat mesenteric arteries were dissected, cleaned, placed in ice cold, gassed PSS, transferred to a cuvette (Fisher Scientific), and incubated for 5 min in 1-ml PSS and DCF in a custom-built, light-tight box. The samples were excited with a Lambard LS/10-2 system (Sutter Instrument, Novato, CA), and the signals were recorded at 1 Hz with a photon-counting detector (Electron Tubes, Rockaway, NJ).

Steady-state levels of H2O2 of dissected mesenteric vessels were monitored with a horseradish peroxide-linked Amplex Red assay (Molecular Probes, Carlsbad, CA) (10). Vessels were protected from light and incubated at 37°C for 60 min on a microplate containing PBS with Amplex Red. H2O2 was determined spectrophotometrically (absorbance at 570 nM) using a microplate reader (Bio-Rad Laboratories, Hercules, CA) and corrected for total protein content.

Experimental Series

Series one: To contrast the effects of tempol, SOD, and NBT on O2bullet– and H2O2 generated by rat isolated VSMCs during stimulation with ANG II or U-46619. Experiments were conducted in this simplified system to contrast the effects of tempol and PEG-SOD with that of NBT on O2bullet– and H2O2 in ANG II-stimulated cells. First, the effects of tempol, NBT, and SOD in reducing NADPH-induced O2bullet– production in ANG II-stimulated rat PGVSMCs were compared using lucigenin CL. Lucigenin is sensitive to O2bullet– but not H2O2 (2). The PEG-SOD-inhibitable lucigenin CL was used to quantitate O2bullet–. Second, the effects of these drugs on H2O2 production were compared using catalase-inhibitable DCF fluorescence (37). The effects of NE (10–5 M), which does not cause vascular oxidative stress (22), on lucigenin CL and DCF fluorescence were also tested for further validation of the measurements.

Series two: To assess the generation of H2O2 by rat mesenteric arteries in response to tempol in vitro. The rat arterial segments from a single mesenteric bed were divided into four groups, which were incubated with a vehicle, NE (10–5 M), U-46619 (10–5 M), or U-46619 plus catalase (200 U/ml) for 15 min. Subsequently, tempol (10–3 M) or NBT (10–3 M) was applied. DCF fluorescence signals at baseline and during the tempol application were recorded.

The steady-state generation of H2O2 was determined in a parallel series with the Amplex Red assay. The arterial segments were incubated with the following: 1) tempol (10–4 M) plus NE (10–5 M); or 2) tempol plus U-46619 (10–5 M); and 3) NBT (10–4 M) plus U-46619 at 37°C for 60 min. A submaximal concentration of tempol and NBT was used to minimize the spectral effects of their colors on the spectrophotometric signals.

Series three: To determine the role of H2O2 in the vasodilation to tempol in rat-perfused mesenteric arteries studied ex vivo during U-46619-induced oxidative stress. The vessels were stimulated by bath addition of U-46619 (10–5 M) for 20 min. U-46619 was selected rather than ANG II, since it causes a more robust and sustained constriction of mesenteric resistance vessels of ~48%, compared with ANG II (up to 10–5 M) responses of <12%. Vascular responses were contrasted following bath addition of tempol (10–3 M) or NBT (10–3 M). The response to tempol was repeated after bath addition of catalase (200 U/ml) for 15 min or after preconstruction with NE (10–5 M) in place of U-46619 for 20 min.

Series four: To test the role of the endothelium in the vasodilation to tempol and H2O2 in rat-perfused mesenteric arteries studied ex vivo during U-46619-induced oxidative stress. Two experiments were conducted on different sets of vessels. First, the responses to bath addition of tempol (1, 10, 20, 50 mM) were contrasted in endothelium-intact or -denuded vessels exposed to U-46619 (10–5 M) for 20 min. For endothelium removal, air bubbles were perfused slowly through the vessel lumen. Successful endothelium removal was confirmed by complete lack of vasodilation to acetylcholine (10–4 M). Second, the vessels were preconstricted with 10–6 M NE for 20 min. The responses to H2O2 (0.1, 1, 5, 10, 50 µM) were contrasted in endothelium-intact or -denuded vessels. H2O2 and tempol were applied in random order with washouts and time for equilibration between doses.

Series five: To test the role of potassium channels in the vasodilation to tempol in rat perfused mesenteric arteries studied ex vivo during U-46619-induced oxidative stress. The responses to tempol (10–2 M) were contrasted in endothelium-intact or -denuded vessels exposed to U-46619 (10–5 M) for 20 min, with or without bath addition of KCl (40 mM) during the last 5 min.

Series six: To determine the role of H2O2 in vasodilation with tempol in vivo in mouse cremaster arterioles during ANG II-induced oxidative stress. ANG II was selected since it caused a reproducible and sustained contraction of these vessels. The first study evaluated the response to a topical application (25 µl) of tempol (10–3 M) via a pipette to the target vessels superfused with vehicle or catalase (200 U/ml) at 30 min after 30 min of continuous superfusion with ANG II (5 x 10–8 M). The second study evaluated the response to a topical application (25 µl) of NBT (10–3 M) under similar conditions. The third study evaluated the response to a topical application (25 µl) of tempol (10–3 M) at 1 min during continuous superfusion with ANG II or at 30 min during continuous superfusion with NE (10–5 M).

Chemicals. U-46619 was purchased from Cayman Chemical (Ann Arbor, MI), ANG II from Bachem Bioscience (King of Prussia, PA), and DCF from Molecular Probes. All other drugs were purchased from Sigma-Aldrich (St. Louis, MO). All of the chemicals were dissolved in PSS.

Data processing and statistical analysis. Data are reported as means ± SD. Statistical analyses were performed using one-way or two-way analysis of variance with Dunnett's multiple-comparison or Bonferroni post hoc test. The results were considered significant at P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The absolute values for the baseline diameter of the rat mesenteric and mouse cremaster vessels were 400 ± 8 µm (n = 17) and 21 ± 2 µm (n = 11), respectively. There were no differences in the baseline diameter among the study series of either vessel.

Series One

Incubation of rat cultured PGVSMCs with U-46619 increased NADPH-stimulated O2bullet– production, as indexed by lucigenin CL, by 228 ± 11% (P < 0.01). Similar results were obtained with ANG II. However, no significant changes in CL were detected with NE. During ANG II stimulation, CL was reduced below control values by coincubation with tempol, NBT, or SOD (Fig. 1A). The reduction in CL by tempol was unaffected by catalase. Incubation of PGVSMCs with ANG II also increased H2O2, as indexed by DCF fluorescence. Coincubation with tempol or PEG-SOD further increased H2O2 (Fig. 1B). In contrast, NBT did not change H2O2 signals. Catalase reduced the DCF signal during tempol to the level of ANG II alone, indicating that tempol had increased DCF-sensitive H2O2 generation, although it is unclear why catalase did not reduce the fluorescence signal to the basal level. No significant changes in DCF fluorescence were found with NE. Thus tempol and NBT reduce O2bullet– in VSMCs similar to SOD, whereas tempol, but not NBT, increase H2O2.


Figure 1
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  		Fig. 1. A: relative signal intensity from lucigenin-enhanced chemiluminescence (CL) of rat pregomerular vascular smooth muscle cells (PGVSMCs) exposed to vehicle or ANG II in coincubation with 10–4 M tempol, 10–4 M nitroblue tetrazolium (NBT), or 100 U/ml PEG-SOD for 2 h (n = 3 per group). B: relative signal intensity from dichlorodihydrofluorescein (DCF)-sensitive fluorescence of PGVSMCs. Relative signal intensity values are normalized by values obtained in PGVSMCs not exposed to ANG II (=100, vehicle). NE, norepinephrine; PEG, polyethylene glycol. Significant difference compared with vehicle: *P < 0.05; compared with ANG II alone: {dagger}P < 0.05; and compared with tempol: {ddagger}P < 0.05.

 
Series Two

Tempol caused an ~45-s increase in DCF fluorescence of rat mesenteric arteries exposed to U-46619 for 15 min (Fig. 2A), which was prevented by catalase (Fig. 2B). NBT did not generate DCF fluorescence, and tempol did not generate DCF fluorescence in vessels exposed for 15 min to NE.


Figure 2
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  		Fig. 2. DCF fluorescence of rat mesenteric arteries. A: representative signals during addition of 10–5 M U-46619 and 10–3 M tempol. B: changes ({Delta}) in DCF fluorescence in response to 1) tempol 15 min after U-46619 given alone or with 200 U/ml catalase or 15 min after NE; or 2) NBT 15 min after U-46619. Significant difference compared with the response to tempol 15 min after U-46619 given alone: {ddagger}{ddagger}P < 0.01, {ddagger}{ddagger}{ddagger}P < 0.005.

 
Steady-state levels of H2O2 generated over a 60-min incubation of rat mesenteric vessels with tempol were assessed by Amplex Red. Coincubation with tempol did not significantly affect H2O2 generated by U-46619-treated vessels (5.2 ± 1.6 µmol/mg protein vs. U-46619 alone, 4.5 ± 0.5 µmol/mg protein) and by NE-treated vessels (4.3 ± 1.9 µmol/mg protein vs. NE alone, 3.8 ± 0.8 µmol/mg protein). We conclude that tempol and NBT both reduce O2bullet– in U-46619-stimulated vessels, but that tempol increases H2O2 transiently, whereas NBT does not.

Series Three

Addition of U-46619 to the bath of a rat isolated, perfused mesenteric vessel caused a sustained reduction in the internal diameter (from 426 ± 9 to 224 ± 14 µm, n = 6, P < 0.001). Tempol caused an abrupt increase in the diameter to 270 ± 15 µm (n = 5, P < 0.001) that persisted for 2–3 min (Fig. 3A) and was reduced significantly by coincubation with catalase (243 ± 13 µm, n = 5). In contrast, NBT tended to reduce the diameter of U-46619-constricted vessels further [198 ± 18 µm, n = 4, not significant (NS)]. A 20-min exposure to NE reduced the diameter of mesenteric vessels even more dramatically (426 ± 19 to 100 ± 6 µm, n = 4, P < 0.001), but this was not changed by tempol (100 ± 6 µm). A brief (1–3 min) application of U-46619 reduced the diameter from 417 ± 11 to 190 ± 5 µm (n = 4, P < 0.001), but this was not changed by tempol (194 ± 6 µm, NS). The relative changes in diameter in response to tempol or NBT during these different pretreatments are shown in Fig. 3B. We conclude that tempol induces a robust, but transient, vasodilation of vessels incubated with U-46619 for 20 min. This vasodilator response to tempol is not dependent on vasoconstriction itself, since it is not seen in vessels with a similar degree of vasoconstriction induced by a brief exposure to these agonists or after 20-min exposure to NE. Acute vasodilation with tempol apparently depends largely on H2O2, since catalase blocks 50–80% of the vasodilator response and NBT, which also metabolizes O2bullet– but does not generate H2O2, is not effective.


Figure 3
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  		Fig. 3. A: representative tracing of diameter of rat isolated, pressurized mesenteric arteries in response to U-46619 and tempol. B: percent changes in diameter of rat isolated, pressurized mesenteric arteries in response to tempol or NBT. Vessels were preincubated with U-46619 or NE for 20 min. Significant difference compared with the response to tempol with 20-min U-46619 pretreatment: {ddagger}P < 0.05, {ddagger}{ddagger}P < 0.01, {ddagger}{ddagger}{ddagger}P < 0.005.

 
Series Four

Applications of tempol to the bath of rat-isolated mesenteric arteries after a 20-min exposure to U-46619 led to a dose-dependent relaxation. The diameter increased from initial value of 153 ± 18 µm to maximal value of 374 ± 35 µm (n = 5) with 50 mM tempol. This response to tempol was unaffected by endothelium removal (Fig. 4). Applications of H2O2 to these vessels (n = 5), preconstricted with NE for 20 min, led to a quantitatively similar, dose-dependent vasodilation. H2O2 did not cause a detectable vasodilation until its concentration reached 5 µM (Fig. 5A). Both the duration and magnitude of the vasodilation caused by H2O2 increased rapidly with increasing concentrations of H2O2. The vasodilation to bath addition of 50 µM H2O2 lasted as long as ~35 min. The response to H2O2 also was unaffected by endothelium removal (n = 5, Fig. 5B). Thus tempol and H2O2 both cause graded, endothelium-independent vasodilation of rat preconstricted mesenteric arteries.


Figure 4
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  		Fig. 4. Percent changes in diameter of rat isolated, pressurized mesenteric arteries preconstricted for 20 min with 10–5 M U-46619 in response to different concentrations of tempol (n = 5 per group).

 

Figure 5
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  		Fig. 5. A: representative tracing of diameter of rat isolated, pressurized mesenteric arteries preconstricted for 20 min with 10–6 M NE during bath addition of different concentrations of H2O2. B: percent changes in diameter of rat mesenteric arteries preconstricted for 20 min with 10–6 M NE in response to different concentrations of H2O2 (n = 5 per group).

 
Series Five

Exposure of rat isolated mesenteric arteries to U-46619 for 20 min reduced the diameter from 373 ± 34 to 159 ± 16 µm in the endothelium-intact vessels and from 361 ± 11 to 155 ± 16 µm in the endothelium-denuded vessels (compared with the intact vessels: P > 0.05, n = 5). Subsequent application of tempol increased the diameter similarly to 290 ± 35 and 247 ± 28 µm in endothelium-intact and -denuded vessels, respectively. Incubation of U-46619-constricted vessels with KCl reduced the diameter further to 93 ± 8 and 133 ± 20 µm in the endothelium-intact and -denuded vessels, respectively (n = 5). Application of tempol increased the diameter of endothelium-intact vessels to 145 ± 10 µm and of endothelium-denuded vessels to 219 ± 28 µm. The fractional increase in diameter by tempol was similar in the presence or absence of endothelium or KCl (Fig. 6). We conclude that the vasodilation to tempol is independent of the endothelium or of K+ channels.


Figure 6
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  		Fig. 6. Effects of endothelial removal or 40 mM KCl on 10–2 M tempol-induced dilation (percent increases in diameter) of rat isolated, pressurized mesenteric arteries preconstricted for 20 min with 10–5 M U-46619 (n = 5 per group).

 
Series Six

As shown in Fig. 7A, superfusion with ANG II caused a sustained reduction in the internal diameter of mouse cremaster vessels in vivo (22 ± 2 to 7 ± 1 µm, n = 8, P < 0.001), which was significantly relaxed by tempol (20 ± 2 µm, P < 0.001, vs. ANG II alone). This vasodilation to tempol was not affected by blocking nitric oxide synthase (NOS) with 10–4 M N{omega}-nitro-L-arginine methyl ester (data not shown). Although catalase did not change the basal diameter of the ANG II-preconstricted vessels (7 ± 1 µm vs. catalase, 7 ± 1 µm, n = 4, NS), it attenuated the vasodilatory effect of tempol (with tempol alone, 21 ± 1 µm vs. with tempol and catalase, 14 ± 1 µm, n = 5). In contrast, NBT did not relax these vessels (7 ± 1 to 7 ± 1 µm, n = 4, NS). A brief (1 min) superfusion with ANG II reduced the vessel diameter (23 ± 1 to 8 ± 1 µm, n = 4, P < 0.001). In contrast to the 30-min ANG II superfusion, tempol did not increase the vessel diameter significantly after 1 min of ANG II (8 ± 1 to 10 ± 1 µm, NS). A 20-min superfusion with NE caused a contraction (22 ± 1 to 11 ± 1 µm, n = 4, P < 0.001), but, unlike prolonged ANG II, the NE constriction was not significantly attenuated by tempol (13 ± 2 µm, NS). The relative changes in diameter in response to tempol or NBT during these different pretreatments are shown in Fig. 7B. We conclude that tempol causes a relaxation of microvessels in vivo after a 30-min, but not a brief, contraction to ANG II, but not NE. This effect of tempol is apparently independent of NOS, but requires generation of H2O2, since it is not mimicked by NBT and is prevented by catalase.


Figure 7
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  		Fig. 7. A: representative tracing of diameter of a mouse cremaster arteriole during superfusion with 5 x 10–8 M ANG II and a topical application of 10–3 M tempol. B: percent changes in diameter of mouse cremaster arterioles in response to a topical application of 10–3 M tempol or 10–3 M NBT comparing vessels superfused with ANG II for 30 or 1 min, or NE for 30 min. Significant difference compared with the response to tempol with 30-min ANG II pretreatment: {ddagger}P < 0.05, {ddagger}{ddagger}P < 0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Tempol vasodilates mesenteric vessels in situ from deoxycorticosterone acetate-salt hypertensive rats (43). We extend this finding to a model of vascular oxidative stress that results from exposure of blood vessels ex vivo or in vivo to U-46619 or ANG II for 20–30 min. The development of oxidative stress in isolated, perfused afferent arterioles of the rabbit (36, 40) or mesenteric vessels of the mouse (39) requires stimulation of TP or AT1 receptors for 10–20 min, whereas brief (1–2 min) stimulation causes vasoconstriction without O2bullet– generation. Blood vessels preconstricted over 20–30 min with U-46619 or ANG II are briefly relaxed by tempol. However, a similar degree of vasoconstriction induced by 1- to 3-min exposure to these agonists or by 20-min exposure to NE, which does not cause oxidative stress in conduit vessels (22) or afferent arterioles (41), is not reversed by tempol. This indicates that tempol causes vasodilation only in blood vessels with oxidative stress, consistent with its primary mechanism of action as a catalytic SOD mimetic (34, 35).

Evidence obtained in vessels in vivo and ex vivo indicates that tempol reduces vasoconstriction of vessels with oxidative stress by generating H2O2. First, tempol briefly increases DCF fluorescence of blood vessels. This indicates that it generates H2O2. Second, pretreatment of vessels with catalase prevents 50–80% of the vasodilation to tempol, which indicates that most, but not all, of the vasorelaxation may be due to H2O2. Third, NBT, given in a dose that produces a comparable reduction in O2bullet– in isolated VSMCs but without generating H2O2, fails to dilate these vessels. The time course of the generation of DCF fluorescence and of relaxation of rat isolated, U-46619-pretreated mesenteric vessels with tempol lasts only for 1–4 min. This suggests that H2O2 generation with tempol is a non-steady-state effect. This is consistent with the finding that tempol did not change vascular H2O2 detected by Amplex Red in vessels stimulated with U-46619 for 60 min. Moreover, a catalase-like effect of tempol may become apparent after a few minutes (21). Our finding that 2-h incubation of PGVSMCs with ANG II caused an increase in H2O2 detected by DCF fluorescence is consistent with a previous report (45). Interestingly, tempol potentiated, but NBT prevented, this ANG II-induced H2O2. Since tempol reduced only 20% of the ex vivo constriction by U-46619 or 50% of the in vivo constriction by ANG II, we conclude that the vasoconstriction effects of U-46619 or ANG II are due only in part to oxidative stress.

AT1 receptors are expressed on VSMCs (46) and the endothelium of mouse cremaster arterioles (31), although their endothelial expression is variously reported to be rich (9, 27) or undetectable (46) in other vessels. TP receptors are expressed on VSMCs (18) and endothelial cells (25). Prolonged infusion of a suppressor dose of ANG II for 12–14 days increases O2bullet– production in blood vessels (22, 40), whereas similar infusions of NE that also raise BP fail to induce vascular oxidative stress (22). Administration of SOD encapsulated in PEG-modified liposomes lowers the BP in ANG II-infused rats, but is ineffective in NE-infused rats (22). Stimulation of TP receptors on pig pulmonary arteries for 30 min increases O2bullet–. This increase is blocked by diphenyleneiodonium or apocynin, which inhibit NADPH oxidase (28). Thus stimulation of blood vessels with U-46619 or ANG II for 20 min or more consistently produces oxidative stress.

Liochev and Fridovich (23) propose that there is normally sufficient SOD to metabolize ~90% of O2bullet– under steady-state conditions. Further increases in SOD should have little or no effect on H2O2 formation. Our results are generally consistent with this proposal, since we detect no increase in the steady-state tissue levels of H2O2 after an incubation with tempol for 60 min, even during development of oxidative stress. However, we do detect a transient increase in H2O2 lasting for 1 min after application of tempol.

A limitation of this study is that different vasculatures were used to study O2bullet–, H2O2, and vasoreaction. Rat PGVSMCs provide an in vitro system in which we confirm that both tempol and NBT reduce O2bullet– similarly to PEG-SOD, but only tempol generates H2O2. The rat isolated, perfused mesenteric vessel preparation stimulated with U-46619 was selected, since it has a robust vasoconstriction that permits parallel study of H2O2 levels and relaxation responses to tempol. Cremaster vessels stimulated with ANG II were selected, since ANG II produced a sustained contraction, which permitted testing of whether the mechanisms of tempol-induced vasodilation seen in vitro were accompanied by similar physiological responses in an intact preparation.

H2O2 can variously produce vasodilation (16), vasoconstriction (19), or biphasic vasomotion (8, 12), depending on the vessel and the experimental conditions. This suggests the involvement of multiple mechanisms. Indeed, vascular dilation by H2O2 can be mediated by increasing K+ conductance (17), prostaglandins (17), nitric oxide (5, 8, 13), or cGMP (44) via both endothelium-dependent and -independent mechanisms (8, 13, 16). Since both H2O2 and tempol produced dose-dependent, endothelium-independent vasodilation of rat isolated mesenteric vessels, we conclude that these agents did not induce the release of an endothelium-derived relaxing factor in this preparation and that the H2O2 that mediates the vasorelaxant effect of tempol does not derive from the endothelium. Consistent with this was the lack of effect of high bath K+ concentration or of blockade of NOS on tempol-induced vasodilation in this preparation. However, the finding that catalase largely prevents the transient vasodilation by tempol indicates that tempol enhances the generation of H2O2 within VSMC and that H2O2, or a substance released from VSMCs by H2O2, mediates much of the vasodilation. This is in contrast to the findings of Rubanyi and Vanhoutte (33) in canine coronary artery rings, where SOD accelerates the transformation of O2bullet– into H2O2, which triggers the release of an endothelium-dependent relaxing factor.

Using this model of vascular oxidative stress with stimulation of TP or AT1 receptors, we demonstrate a new mechanism by which H2O2, generated by tempol, can produce vasorelaxation. The tempol-induced vasorelaxation and H2O2 formation are transient and might contribute to the transient reduction in BP seen in SHR given bolus intravenous injections of tempol (30).


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This work was supported by National Institutes of Health Grants DK-36079 and DK-49870, HL-68686-01, and Nephrology Fellowship Training Grant DK 59274, and by funds from the George E. Schreiner Chair of Nephrology.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Chen, Division of Nephrology & Hypertension, Georgetown Univ., 4000 Reservoir Road, NW, Bldg. D-399, Washington, DC 20057 (e-mail: yc53{at}georgetown.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.

1 This paper was presented at the 9th Cardiovascular-Kidney Interactions in Health and Disease Meeting at Amelia Island Plantation, Florida, on May 26–29, 2006. Back


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  1. Andresen BT, Jackson EK, Romero GG. Angiotensin II signaling to phospholipase D in renal microvascular smooth muscle cells in SHR. Hypertension 37: 635–639, 2001.[Abstract/Free Full Text]
  2. Archer SL, Nelson DP, Weir EK. Detection of activated O2 species in vitro and in rat lungs by chemiluminescence. J Appl Physiol 67: 1912–1921, 1989.[Abstract/Free Full Text]
  3. Bhunia AK, Han H, Snowden A, Chatterjee S. Redox-regulated signaling by lactosylceramide in the proliferation of human aortic smooth muscle cells. J Biol Chem 272: 15642–15649, 1997.[Abstract/Free Full Text]
  4. Cai H, Harrison DG. Endothelial dysfunction in cardiovascular diseases: the role of oxidant stress. Circ Res 87: 840–844, 2000.[Abstract/Free Full Text]
  5. Cai H, Li Z, Dikalov S, Holland SM, Hwang J, Jo H, Dudley SC Jr, Harrison DG. NAD(P)H oxidase-derived hydrogen peroxide mediates endothelial nitric oxide production in response to angiotensin II. J Biol Chem 277: 48311–48317, 2002.[Abstract/Free Full Text]
  6. Chen Y, Rivers RJ. Arteriolar occlusion causes independent cellular responses in endothelium and smooth muscle. Microcirculation 9: 353–362, 2002.[CrossRef][Web of Science][Medline]
  7. Chen YF, Cowley AW Jr, Zou AP. Increased H2O2 counteracts the vasodilator and natriuretic effects of superoxide dismutation by tempol in renal medulla. Am J Physiol Regul Integr Comp Physiol 285: R827–R833, 2003.[Abstract/Free Full Text]
  8. Cseko C, Bagi Z, Koller A. Biphasic effect of hydrogen peroxide on skeletal muscle arteriolar tone via activation of endothelial and smooth muscle signaling pathways. J Appl Physiol 97: 1130–1137, 2004.[Abstract/Free Full Text]
  9. De PP, Porcellini A, Gigante B, Giliberti R, Lombardi A, Savoia C, Rubattu S, Volpe M. Modulation of the AT2 subtype receptor gene activation and expression by the AT1 receptor in endothelial cells. J Hypertens 17: 1873–1877, 1999.[CrossRef][Web of Science][Medline]
  10. Dikalov S, Griendling KK, Harrison DG. Measurement of reactive oxygen species in cardiovascular studies. Hypertension 49: 717–727, 2007.[Free Full Text]
  11. Gao YJ, Hirota S, Zhang DW, Janssen LJ, Lee RM. Mechanisms of hydrogen-peroxide-induced biphasic response in rat mesenteric artery. Br J Pharmacol 138: 1085–1092, 2003.[CrossRef][Web of Science][Medline]
  12. Gao YJ, Lee RM. Hydrogen peroxide induces a greater contraction in mesenteric arteries of spontaneously hypertensive rats through thromboxane A2 production. Br J Pharmacol 134: 1639–1646, 2001.[CrossRef][Web of Science][Medline]
  13. Gil-Longo J, Gonzalez-Vazquez C. Characterization of four different effects elicited by H2O2 in rat aorta. Vascul Pharmacol 43: 128–138, 2005.[CrossRef][Web of Science][Medline]
  14. Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res 74: 1141–1148, 1994.[Abstract/Free Full Text]
  15. Hahn SM, Sullivan FJ, DeLuca AM, Bacher JD, Liebmann J, Krishna MC, Coffin D, Mitchell JB. Hemodynamic effect of the nitroxide superoxide dismutase mimics. Free Radic Biol Med 27: 529–535, 1999.[CrossRef][Web of Science][Medline]
  16. Hatoum OA, Binion DG, Miura H, Telford G, Otterson MF, Gutterman DD. Role of hydrogen peroxide in ACh-induced dilation of human submucosal intestinal microvessels. Am J Physiol Heart Circ Physiol 288: H48–H54, 2005.[Abstract/Free Full Text]
  17. Hattori T, Kajikuri J, Katsuya H, Itoh T. Effects of H2O2 on membrane potential of smooth muscle cells in rabbit mesenteric resistance artery. Eur J Pharmacol 464: 101–109, 2003.[CrossRef][Web of Science][Medline]
  18. Huang JS, Ramamurthy SK, Lin X, Le Breton GC. Cell signalling through thromboxane A2 receptors. Cell Signal 16: 521–533, 2004.[CrossRef][Web of Science][Medline]
  19. Jones RD, Morice AH. Hydrogen peroxide–an intracellular signal in the pulmonary circulation: involvement in hypoxic pulmonary vasoconstriction. Pharmacol Ther 88: 153–161, 2000.[CrossRef][Web of Science][Medline]
  20. Kawada N, Imai E, Karber A, Welch WJ, Wilcox CS. A mouse model of angiotensin II slow pressor response: role of oxidative stress. J Am Soc Nephrol 13: 2860–2868, 2002.[Abstract/Free Full Text]
  21. Krishna MC, Samuni A, Taira J, Goldstein S, Mitchell JB, Russo A. Stimulation by nitroxides of catalase-like activity of hemeproteins. Kinetics and mechanism. J Biol Chem 271: 26018–26025, 1996.[Abstract/Free Full Text]
  22. Laursen JB, Rajagopalan S, Galis Z, Tarpey M, Freeman BA, Harrison DG. Role of superoxide in angiotensin II-induced but not catecholamine-induced hypertension. Circulation 95: 588–593, 1997.[Abstract/Free Full Text]
  23. Liochev SI, Fridovich I. The effects of superoxide dismutase on H2O2 formation. Free Radic Biol Med 42: 1465–1469, 2007.[CrossRef][Web of Science][Medline]
  24. Makino A, Skelton MM, Zou AP, Cowley AW Jr. Increased renal medullary H2O2 leads to hypertension. Hypertension 42: 25–30, 2003.[Abstract/Free Full Text]
  25. Miggin SM, Kinsella BT. Expression and tissue distribution of the mRNAs encoding the human thromboxane A2 receptor (TP) alpha and beta isoforms. Biochim Biophys Acta 1425: 543–559, 1998.[Medline]
  26. Mohazzab H, Kaminski PM, Fayngersh RP, Wolin MS. Oxygen-elicited responses in calf coronary arteries: role of H2O2 production via NADH-derived superoxide. Am J Physiol Heart Circ Physiol 270: H1044–H1053, 1996.[Abstract/Free Full Text]
  27. Morrell NW, Grieshaber SS, Danilov SM, Majack RA, Stenmark KR. Developmental regulation of angiotensin converting enzyme and angiotensin type 1 receptor in the rat pulmonary circulation. Am J Respir Cell Mol Biol 14: 526–537, 1996.[Abstract]
  28. Muzaffar S, Shukla N, Lobo C, Angelini GD, Jeremy JY. Iloprost inhibits superoxide formation and gp91phox expression induced by the thromboxane A2 analog U46619, 8-isoprostane F2alpha, prostaglandin F2alpha, cytokines and endotoxin in the pig pulmonary artery. Br J Pharmacol 141: 488–496, 2004.[CrossRef][Web of Science][Medline]
  29. Nowicki PT, Flavahan S, Hassanain H, Mitra S, Holland S, Goldschmidt-Clermont PJ, Flavahan NA. Redox signaling of the arteriolar myogenic response. Circ Res 89: 114–116, 2001.[Abstract/Free Full Text]
  30. Patel k Chen Y, Dennehy K, Blau J, Connors S, Mendonca M, Tarpey M, Krishna M, Mitchell JB, Welch WJ, Wilcox CS. Acute antihypertensive action of nitroxides in the spontaneously hypertensive rat. Am J Physiol Regul Integr Comp Physiol 290: R37–R43, 2006.[Abstract/Free Full Text]
  31. Petnehazy T, Stokes KY, Russell JM, Granger DN. Angiotensin II type-1 receptor antagonism attenuates the inflammatory and thrombogenic responses to hypercholesterolemia in venules. Hypertension 45: 209–215, 2005.[Abstract/Free Full Text]
  32. Rodriguez-Martinez MA, Garcia-Cohen EC, Baena AB, Gonzalez R, Salaices M, Marin J. Contractile responses elicited by hydrogen peroxide in aorta from normotensive and hypertensive rats. Endothelial modulation and mechanism involved. Br J Pharmacol 125: 1329–1335, 1998.[CrossRef][Web of Science][Medline]
  33. Rubanyi GM, Vanhoutte PM. Oxygen-derived free radicals, endothelium, and responsiveness of vascular smooth muscle. Am J Physiol Heart Circ Physiol 250: H815–H821, 1986.[Abstract/Free Full Text]
  34. Samuni A, Krishna CM, Mitchell JB, Collins CR, Russo A. Superoxide reaction with nitroxides. Free Radic Res Commun 9: 241–249, 1990.[Web of Science][Medline]
  35. Samuni A, Krishna CM, Riesz P, Finkelstein E, Russo A. A novel metal-free low molecular weight superoxide dismutase mimic. J Biol Chem 263: 17921–17924, 1988.[Abstract/Free Full Text]
  36. Schnackenberg CG, Welch WJ, Wilcox CS. TP receptor-mediated vasoconstriction in microperfused afferent arterioles: roles of O2bullet– and NO. Am J Physiol Renal Physiol 279: F302–F308, 2000.[Abstract/Free Full Text]
  37. Su B, Mitra S, Gregg H, Flavahan S, Chotani MA, Clark KR, Goldschmidt-Clermont PJ, Flavahan NA. Redox regulation of vascular smooth muscle cell differentiation. Circ Res 89: 39–46, 2001.[Abstract/Free Full Text]
  38. Sundaresan M, Yu ZX, Ferrans VJ, Irani K, Finkel T. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science 270: 296–299, 1995.[Abstract/Free Full Text]
  39. Wang D, Chabrashvili T, Borrego L, Aslam S, Umans JG. Angiotensin II infusion alters vascular function in mouse resistance vessels: roles of O and endothelium. J Vasc Res 43: 109–119, 2006.[CrossRef][Web of Science][Medline]
  40. Wang D, Chen Y, Chabrashvili T, Aslam S, Borrego-Conde LJ, Umans JG, Wilcox CS. Role of oxidative stress in endothelial dysfunction and enhanced responses to angiotensin II of afferent arterioles from rabbits infused with angiotensin II. J Am Soc Nephrol 14: 2783–2789, 2003.[Abstract/Free Full Text]
  41. Wang D, Jose P, Wilcox CS. beta1 Receptors protect the renal afferent arterioles of angiotensin-infused rabbits from norepinephrine-induced oxidative stress. J Am Soc Nephrol 17: 3347–3354, 2006.[Abstract/Free Full Text]
  42. Welch WJ, Blau J, Xie H, Chabrashvili T, Wilcox CS. Angiotensin-induced defects in renal oxygenation: role of oxidative stress. Am J Physiol Heart Circ Physiol 288: H22–H28, 2005.[Abstract/Free Full Text]
  43. Xu H, Bian X, Watts SW, Hlavacova A. Activation of vascular BK channel by tempol in DOCA-salt hypertensive rats. Hypertension 46: 1154–1162, 2005.[Abstract/Free Full Text]
  44. Yang M, Yang Y, Zhang S, Kahn AM. Insulin-stimulated hydrogen peroxide increases guanylate cyclase activity in vascular smooth muscle. Hypertension 42: 569–573, 2003.[Abstract/Free Full Text]
  45. Zafari AM, Ushio-Fukai M, Akers M, Yin Q, Shah A, Harrison DG, Taylor WR, Griendling KK. Role of NADH/NADPH oxidase-derived H2O2 in angiotensin II-induced vascular hypertrophy. Hypertension 32: 488–495, 1998.[Abstract/Free Full Text]
  46. Zhuo J, Moeller I, Jenkins T, Chai SY, Allen AM, Ohishi M, Mendelsohn FA. Mapping tissue angiotensin-converting enzyme and angiotensin AT1, AT2 and AT4 receptors. J Hypertens 16: 2027–2037, 1998.[CrossRef][Web of Science][Medline]



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