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Am J Physiol Renal Physiol 294: F719-F728, 2008. First published February 6, 2008; doi:10.1152/ajprenal.00506.2007
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Renal Hemodynamics: Biomolecular Control Mechanisms and Integration of Vascular and Tubular Function

Reactive oxygen species participate in acute renal vasoconstrictor responses induced by ETA and ETB receptors

Armin Just,1,2 Christina L. Whitten,1 and William J. Arendshorst1,2,3

1Department of Cell and Molecular Physiology, 2Carolina Cardiovascular Biology Center, and 3UNC Kidney Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina

Submitted 27 October 2007 ; accepted in final form 4 February 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reactive oxygen species (ROS) play important roles in renal vasoconstrictor responses to acute and chronic stimulation by angiotensin II and norepinephrine, as well as in long-term effects of endothelin-1 (ET-1). Little is known about participation of ROS in acute vasoconstriction produced by ET-1. We tested the influence of NAD(P)H oxidase inhibition by apocynin [4 mg·kg–1·min–1, infused into the renal artery (ira)] on ETA and ETB receptor signaling in the renal microcirculation. Both receptors were stimulated by ET-1, ETA receptors by ET-1 during ETB antagonist BQ-788, and ETB by ETB agonist sarafotoxin 6C. ET-1 (1.5 pmol injected ira) reduced renal blood flow (RBF) 17 ± 4%. Apocynin raised baseline RBF (+10 ± 1%, P < 0.001) and attenuated the ET-1 response to 10 ± 2%, i.e., 35 ± 9% inhibition (P < 0.05). Apocynin reduced ETA-induced vasoconstriction by 42 ± 12% (P < 0.05) and that of ETB stimulation by 50 ± 8% (P < 0.001). During nitric oxide (NO) synthase inhibition (N{omega}-nitro-L-arginine methyl ester), apocynin blunted ETA-mediated vasoconstriction by 60 ± 8% (P < 0.01), whereas its effect on the ETB response (by 87 ± 8%, P < 0.001) was even larger without than with NO present (P < 0.05). The cell-permeable superoxide dismutase mimetic tempol (5 mg·kg–1·min–1 ira), which reduces O2– and may elevate H2O2, attenuated ET-1 responses similar to apocynin (by 38 ± 6%, P < 0.01). We conclude that ROS, O2– rather than H2O2, contribute substantially to acute renal vasoconstriction elicited by both ETA and ETB receptors and to basal renal vasomotor tone in vivo. This physiological constrictor action of ROS does not depend on scavenging of NO. In contrast, scavenging of O2– by NO seems to be more important during ETB stimulation.

renal hemodynamics; vascular smooth muscle; renal vascular resistance; afferent arteriole; oxidative stress; redox signaling; nitric oxide; nitric oxide synthase; superoxide dismutase; endothelin

REACTIVE OXYGEN SPECIES (ROS) of vascular origin contribute importantly to peripheral vascular resistance and arterial pressure under pathophysiological conditions such as hypertension (34), ischemia (20), diabetes mellitus, and obesity (21). Popular models of established hypertension include those induced chronically (e.g., 1–6 wk) by mineralocorticoid (2, 3, 30, 36, 37), ANG II (33), ET-1 (53), or a high-sodium diet (12, 57). Published in vitro studies of oxidative stress on vascular cells and vessels are also characterized by relatively long-term treatment (e.g., 1–24 h) (10, 11, 33, 36, 37, 39, 53, 59).

Recent findings indicate that ROS participate in physiological signaling of rapid vascular responses occurring within sec to min. In particular, superoxide (O2–) mediates a significant portion of the acute vasoconstriction elicited by ANG II and NE in the kidney of normotensive as well as hypertensive animals (9, 29, 29, 40, 43, 50, 50, 54). Based on these findings, we queried whether ROS are more universally associated with vasoconstriction triggered by stimulation of other G protein-coupled receptors, in particular those activated by endothelin-1 (ET-1). This vasoconstrictor peptide is reported to stimulate ROS production in nonrenal vascular smooth muscle cells (VSMC) (33, 62, 63), isolated arteries (30, 36, 39, 53), and veins (37, 59), as well as endothelial (10, 11) and sympathetic ganglionic cells (8). Chronic administration of ET-1 to conscious rats enhances oxidative stress (53). Mineralocorticoid-induced hypertension is associated with elevated vascular levels of ET-1 and O2– (3, 30, 36, 37). Similarly, hypertension and ROS production depend on ET-1 in ANG II-induced hypertension (32, 33). In addition, ETB receptor-deficient rats consuming a high-sodium diet have hypertension with oxidative stress, both of which are attenuated by ETA receptor antagonism (12, 57).

Little is known about the role of ROS in the acute vasoconstrictor response to ET-1 in any vascular bed in a healthy animal. A recent study from our laboratory tested the role of O2– in initial changes in cytosolic calcium concentration ([Ca2+]i) stimulated by ET-1 in renal afferent arterioles isolated from normotensive rats (15). The results showed that ET-1 immediately stimulates O2– production and [Ca2+]I, both of which are markedly attenuated by the NAD(P)H oxidase inhibitors apocynin and DPI and by O2– degradation by the SOD mimetic tempol.

Both ETA and ETB receptors mediate ET-1-induced vasoconstriction in the renal microcirculation (13–15, 17, 25, 27, 45). The extent to which these receptors acutely generate O2– in the renal resistance arterioles in vivo is not known. Earlier studies of isolated rat afferent arterioles suggest that O2– formation may be more strongly involved in ETA than ETB receptor activation (15). Similar results have been reported for other vascular beds and preparations. ETA receptor antagonism attenuates or abolishes ET-1-induced production of O2– by isolated carotid arteries (36) and vena cava (37, 59) and by cultured arterial VSMC (33, 59, 62, 63). Similarly, arterial pressure and oxidative stress during mineralocorticoid hypertension are susceptible to ETA receptor inhibition (3, 30). On the other hand, ETB receptors appear to be capable of inducing formation of ROS in isolated vessels and vascular cells. Examples include isolated arteries and veins (39, 59) and endothelial cells (10, 11).

The present study investigated whether ROS, in particular O2–, contribute to acute vasoconstriction induced by ET-1 in the kidney of anesthetized euvolemic rats and possible mediation by ETA and/or ETB receptors. We also assessed whether the renal vascular effects of O2– depend primarily on scavenging of ambient nitric oxide (NO) or occur in the absence of NO. To localize actions to the renal microcirculation, ET-1 and pharmacological agents were administered directly into the renal artery.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
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Experiments conducted on 30 male Sprague-Dawley rats (6–9 wk of age, 170–340 g body wt) from our local breeding colony were approved by the Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill. The study was performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the guidelines of the Animal Welfare Act. The animals were fed a standard lab chow with free access to tap water and kept on a 12:12-h light-dark cycle. The surgical preparation and general methods were similar to those described previously (28, 29).

Surgical Preparation

After induction of anesthesia by pentobarbital sodium (50–60 mg/kg body wt ip, Nembutal, Abbott, Chicago, IL), a rat was placed on a temperature-controlled table kept at 37°C. The depth of anesthesia was monitored by the response to ear or toe pinching. The left femoral artery was catheterized for measurement of mean arterial pressure (MAP), and two femoral venous catheters were used for infusion of volume replacement and injection of pentobarbital sodium. The trachea was cannulated to facilitate respiration. Via a midline abdominal incision, the aorta and left renal artery were exposed. A catheter inserted into the left common iliac artery and advanced until its tip faced the origin of the left renal artery was used for infusion of solutions into the renal artery. An ultrasound transit-time flowprobe (1RB, Transonic, Ithaca, NY) was placed around the left renal artery and filled with ultrasonic coupling gel (Surgilube, Fugera, Melville, NY). Urine was drained from the bladder by gravity via a 23-gauge needle. Isoncotic bovine serum albumin (4.75 g/dl) was infused initially at 50 µl/min to replace surgical losses (1.25 ml/100 g body wt), followed by a maintenance rate of 10 µl/min. The renal artery catheter was perfused with isotonic saline at 5 µl/min. Additional doses of pentobarbital sodium were given intravenously as required. All syringes and catheters used for solutions injected/infused into the renal artery (ira) were pretreated with albumin solution (0.5 g/dl) to reduce surface adhesion. At least 60 min were allowed after surgery before an experiment was started.

Measurements

Femoral arterial pressure (AP) was measured via a pressure transducer (Statham P23 DB). Renal blood flow (RBF) was measured by a flowmeter (T 420, Transonic, low-pass filter, 40 Hz). Zero offset was determined at the end of an experiment after cardiac arrest. AP and RBF were recorded on a computer (Pentium IV+DataTranslation A/D converter+Labtech Notebook-Pro 12.1) at 100 Hz; stored at 1 Hz as consecutive mean values over 1-s periods. AP was also stored at 100 Hz for determination of heart rate (HR).

Protocols

The RBF response to a bolus injection of ET-1 (10 µl x 0.075, 0.15, or 0.3 µM = 0.75, 1.5, or 3 pmol = 1.9, 3.7, or 7.5 ng), sarafotoxin 6C (S6C, 10 µl x 1.5 or 0.6 µM = 1.5 or 6 pmol = 3.8 or 15 ng), or ANG II (10 µl x 0.38 µM = 3.8 pmol = 4 ng) ira was measured during control and experimental conditions. Agonist doses were chosen to induce a reduction of RBF by 20–40%. Two minutes before each bolus injection, the renal arterial infusion rate was increased from 5 to 140 µl/min. A 10-µl bolus of ET-1, S6C, or ANG II was then injected into the infusion line through a microinjector valve (Valco Instruments, Houston, TX) and a new recording was started. The initial 25 s served as baseline values of MAP and RBF. Three hundred seconds after the injection of ET-1 or S6C and 150 s after injection of ANG II, the infusion rate was returned to 5 µl/min and the recording continued for another 4 min. At least 30 min were allowed for recovery after injection of ET-1, 15 min after S6C, and 5 min after ANG II.

Apocynin (acetovanillone; 4'-hydroxy-3'-methoxy-acetophenone) was used to inhibit NAD(P)H oxidase activity and 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (tempol) as a cell-permeable SOD mimetic to convert O2– to H2O2. Apocynin is known to block NAD(P)H oxidase subunit assembly and thus activity (56). Apocynin markedly reduces O2– production in the media of the aortic wall (6, 48), with complete inhibition noted in cultured aortic VSMC or cultured gluteal arterial VSMC stimulated by ANG II (60, 69). In our hands, apocynin blocks >90% of O2– produced (tempo-9AC fluorescence) in isolated afferent arterioles in response to stimulation by ANG II and ET-1 (15, 16). Tempol reduces intracellular O2– concentration > 90% in the same preparation.

Doses for apocynin (4 mg·kg–1·min–1 ira) and tempol (5 mg·kg–1·min–1 ira) were chosen from our previous study showing dose-dependent inhibitory effects of both agents (29); employed doses inhibit ~50% of the acute renal vasoconstrictor responses to ANG II and NE. The dose of apocynin is ~50% of that used by Lopez et al. (40) in rats to raise baseline RBF, GFR, and sodium excretion rate and blunt ANG II-induced reductions in RBF and GFR. Based on the average baseline RBF (6 ml/min) and HCT (45%), in all experiments of the present study the dose of apocynin should lead to a plasma concentration in the renal artery of 1–2 mM. Previous studies found ROS or O2– production by isolated vessels, vessel segments, or smooth muscle cells to be reduced by apocynin at doses of 0.03–1 mM (15, 16, 36, 36, 38–40, 48). The tempol dose is 10 times greater than that reported (0.5 mg·kg–1·min–1 ira) to markedly reduce urinary excretion of 8-isoprostane in dogs (44) and 2 times greater than that infused into the renal artery of rats to increase baseline sodium excretion and blunt ANG II-induced reduction of GFR (40). The estimated plasma concentration of our dose was 2 mM. Studies in isolated vessels or cells reported doses of 0.1–1 mM to effectively reduce ROS (37, 40) or increase H2O2 levels (5).

Experimental Groups

Effect of apocynin on renal vasoconstrictor responses to ET-1 (n = 6 and n = 9). To test the effect of reducing the levels of both O2– and H2O2 on the reactivity to ET-1, RBF responses to ET-1 were tested in the absence and presence of the NAD(P)H oxidase inhibitor apocynin during control (vehicle, 8% ethanol ira), apocynin infusion (4 mg·kg–1·min–1 in 8% ethanol ira), and recovery (vehicle ira) periods. For the dose of the ET-1 bolus, 1.5 pmol/bolus was used in five rats and 3 pmol in two rats. In four rats, both doses were injected in two subsequent injections >30 min apart for each experimental period. No differences were observed between the effects of apocynin regardless of whether a single or both doses were used in an experiment, so the data were pooled. For comparison to previous results, ANG II (4 pmol) was injected in each of the experimental periods of four experiments.

Effect of apocynin on ETA receptor-mediated renal vasoconstriction (n = 7). Responses to ET-1 in the presence of the ETA receptor antagonist BQ-788 (7 nmol/min) were tested during control (BQ-788+8% ethanol ira), apocynin (BQ-788+apocynin in ethanol ira), and recovery (BQ-788+ethanol) periods. To achieve a 20–30% vasoconstriction to ET-1 during BQ-788, 0.75 pmol ET-1 was injected.

Effect of apocynin on ETB receptor-mediated renal vasoconstriction (n = 11). Responses to the ETB receptor agonist sarafotoxin 6C (S6C; 6 pmol) were tested during control (ethanol), apocynin (apocynin in ethanol), and recovery (ethanol) periods.

Effect of apocynin on ETA receptor-mediated renal vasoconstriction during NOS inhibition (n = 4). To assess whether the effect of NAD(P)H oxidase activity on ETA receptor-mediated vasoconstriction depends on the presence and activity of NO, the NO synthase (NOS) inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME) was injected (25 mg/kg iv); 15 min later, the reactivity to ET-1 (0.75 pmol) during BQ-788 was evaluated during control (BQ-788+ethanol), apocynin (BQ-788 +apocynin in ethanol), and recovery periods (BQ-788+ethanol).

Effect of apocynin on ETB receptor-mediated renal vasoconstriction in the absence of NO (n = 5). To assess whether the effect of NAD(P)H oxidase activity on ETB receptor-mediated vasoconstriction depends on NO, reactivity to S6C (1.5 pmol ira) was evaluated during control (ethanol), apocynin (apocynin in ethanol), and recovery periods (ethanol) 15 min after L-NAME (25 mg/kg iv).

Effect of tempol on responses to ET-1 (n = 5). To evaluate the involvement of ROS by scavenging of O2–, renal vascular reactivity was determined in the absence and presence of the SOD mimetic tempol. Such treatment is expected to reduce the level of O2– while raising that of H2O2. Reactivity to ET-1 (3 pmol ira) was tested during control (saline ira), tempol (5 mg/kg/min in saline ira), and recovery (saline) periods.

Drugs and Chemicals

ET-1, S6C, and BQ-788 were obtained from American Peptide (Sunnyvale, CA). ANG II, apocynin, tempol, L-NAME, and albumin were from Sigma (St. Louis, MO). Pentobarbital was from Abbott (Chicago, IL) or from Sigma.

Data Analyses

The maximum RBF decrease following each injection was determined off-line by custom-built software from the 1-Hz data after smoothing by a sliding average over five values. The change was expressed as the percentage of the baseline value. Baseline RBF and MAP were determined from the average of the first 25 s of each recording immediately before injection. To obtain mean time courses, the original 1-Hz recordings (without smoothing) were averaged for each experimental period of all animals in a group. HR was determined from the 100-Hz recording of AP off-line. Data are expressed as means ± SE. Statistical significance among groups was tested by ANOVA in conjunction with a Holm-Sidak or Tukey test for multiple comparisons (SigmaStat 3.00, SPSS, Chicago, IL). In case of nonnormal distribution, data were transformed by square root before analysis. A paired t-test was used to detect changes within a group. P < 0.05 was considered statistically significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Baseline hemodynamic data at the beginning of the experiment and urine excretion rates during the observation period are presented in Table 1. In addition, hemodynamics after NOS inhibition are given. Baseline values at the beginning of the experiment did not differ. Urine excretion rate (UV) tended to be nominally higher in both protocols with NOS inhibition vs. the ET-1+tempol and ETB+apocynin groups. Note, however, that UV was determined over the entire observation period, including the hypertensive period after administration of L-NAME. HR in the control period of all experiments averaged 311 ± 9 beats/min.


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  		Table 1. Baseline hemodynamic and excretory data

 
The effect of O2– on basal renal vascular tone was determined from the pooled responses of RBF to ira infusion of apocynin to inhibit NAD(P)H oxidase and compared with the infusion of vehicle (8% ethanol) in the same animal. As shown in Fig. 1, apocynin induced a rapid increase in RBF of 10% (P < 0.001 from both vehicle and from zero). More pronounced renal vasodilation was observed in the absence of NO as apocynin elevated RBF 17% (P < 0.01 vs. vehicle, P < 0.05 vs. time 0) with a similar time course (Fig. 1, bottom) but larger magnitude (P < 0.01 vs. apocynin during control). Scavenging of O2– by the SOD mimetic tempol did not affect baseline RBF (+0.5 ± 3%, P > 0.9).


Figure 1
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  		Fig. 1. Time course of the acute vasodilatory effect of apocynin on baseline renal blood flow (RBF) of anesthetized rats in the presence and absence of nitric oxide production. The NAD(P)H oxidase inhibitor apocynin (4 mg·kg–1·min–1, filled circles) or the vehicle for apocynin (8% ethanol, n = 16, open circles) was infused separately into the renal artery during control conditions, starting at t = 0 s. Top: time course during control conditions. Bottom: time course during inhibition of nitric oxide synthase by N{omega}-nitro-L-arginine methyl ester (L-NAME). Data during control are pooled from all apocynin experiments (n = 25); data during L-NAME are from the experiments testing sarafotoxin 6C during L-NAME (n = 5). Stippled lines indicate ± SE. *, ***: P < 0.05, P < 0.001 of the average over the last 15 vs. 0 s and vs. vehicle.

 
To study the involvement of O2– in the acute renal vasoconstrictor responses to ET receptor stimulation, various combinations of ETA- and ETB-receptor agonists and antagonists were given before and during intrarenal infusion of apocynin or tempol. Injection of ET-1 (3 pmol ira.) to activate both ETA and ETB receptors induced a maximum RBF reduction of 28 ± 4% (Fig. 2, open circles). During apocynin inhibition of NAD(P)H oxidase, the same ET-1 dose caused a vasoconstriction of 14 ± 1% (Fig. 2, filled circles, P < 0.01 vs. control). This corresponds to apocynin inhibition of 49 ± 4% (P < 0.001 vs. zero, see Fig. 6). Such inhibition was reversible, as a subsequent injection of ET-1 in the recovery period reduced RBF by 27 ± 3%. A smaller dose of ET-1 (1.5 pmol) reduced RBF by 17% during control, 10% during apocynin, and 17% in the recovery period (Fig. 3, left). This corresponds to a 35% inhibitory effect of apocynin (see Fig. 6). For comparison with previous results, responses to ANG II were also tested. ANG II (4 ng ira) reduced RBF by 32 ± 4% during control, 14 ± 2% during apocynin (P < 0.01 vs. vehicle), and 40 ± 9% during recovery, thus being attenuated 52 ± 7% by apocynin (P < 0.001 vs. zero).


Figure 2
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  		Fig. 2. Effect of apocynin on the acute vasoconstrictor response of RBF to bolus injection of ET-1 into the renal artery. Shown are time courses of the RBF response of endothelin-1 (ET-1, 3 pmol) injected into the renal artery (ira) during control conditions (Control) and during subsequent infusion of the NAD(P)H oxidase inhibitor apocynin (4 mg·kg–1·min–1 ira). ET-1 was injected at t = 0. The infusion of vehicle (8% ethanol) or apocynin was started at least 2 min before and continued until 5 min after the ET-1 injection. Stippled lines indicate ± SE.

 

Figure 6
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  		Fig. 6. Summary of the inhibitory effects of apocynin and tempol on acute renal vasoconstrictor responses to ETA and ETB receptor activation in the presence and absence of NO. Percent attenuation by apocynin of the renal vasoconstriction elicited by ET-1 (left filled bar, n = 9), ET-1 during ETB antagonist BQ-788 (ETA, hatched bar, n = 8), and the ETB agonist S6C (ETB, vertically striped bar, n = 11) compared with respective control response is shown. Cross-hatched and cross-striped bars show apocynin-induced attenuation of the responses to ET-1+BQ-788 (ETA NO, cross-hatched, n = 4) and S6C (ETB NO, cross-striped, n = 5) during NO synthase inhibition. The right solid bar gives the inhibitory effect of tempol on the response to ET-1 (n = 5). Values are means ± SE. *, **, ***: P < 0.05, P < 0.01, P < 0.001 vs. zero. &P < 0.05 vs. ETB. #P < 0.05 vs. ETA NO.

 

Figure 3
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  		Fig. 3. Effect of apocynin on the acute renal vasoconstrictor response to stimulation of ETA and ETB receptors in combination and alone. Maximum reduction of RBF in response to simultaneous activation of both ETA and ETB receptors (left, ETA+ETB, n = 9) or to selective activation of either ETA (middle, n = 8) or ETB receptors (right, n = 11) is shown. ET-1 (0.75 pmol) injected into the renal artery (ira) was used to activate both ETA and ETB receptors. ETA receptors were activated by injection of ET-1 (0.75 pmol) during intrarenal infusion of the ETB receptor antagonist BQ-788 (7 nmol/min ira). ETB receptors were stimulated by injection of ETB agonist sarafotoxin 6C (S6C) into the renal artery (6 pmol ira). In each group RBF responses are presented during infusion of vehicle (8% ethanol, open bars), during NAD(P)H oxidase inhibition (apocynin, 4 mg·kg–1·min–1 ira, filled bars), and during a subsequent recovery period with vehicle (hatched bars). Values are means ± SE. *, **, ***: P < 0.05, P < 0.01, P < 0.001 vs. initial control.

 
To specifically stimulate ETA receptors, ET-1 was injected in the presence of the ETB antagonist BQ-788 (7 pmol/min). BQ-788 alone did not change basal RBF (–0.6 ± 0.8%, P > 0.4), but the combined infusion of BQ-788 and apocynin elevated RBF by 8 ± 2% (P < 0.001 vs. 0, P < 0.01 vs. BQ-788). When coadministered with BQ-788, ET-1 (0.75 pmol) reduced RBF by 19% (note that a smaller dose of ET-1 was used than in the absence of BQ-788 to achieve similar degrees of vasoconstriction in both conditions). During additional infusion of apocynin, the response to ET-1+BQ788 was attenuated to 10% constriction, which recovered to 25% (Fig. 3, middle). This corresponds to an apocynin-inhibitory effect of 42% (see Fig. 6).

For selective activation of ETB receptors, the ETB agonist S6C was injected ira. S6C (6 pmol) reduced RBF by 17% during control conditions, 9% during apocynin (P < 0.001 vs. control), and 18% during the recovery period (Fig. 3, right). This corresponds to an apocynin-inhibitory effect of 50 ± 8% (see Fig. 6).

To determine the dependence of apocynin's effects on the presence of NO, ETA and ETB receptors were stimulated during NOS inhibition. During L-NAME, stimulation of ETA receptors by ET-1 (0.75 pmol) in the presence of BQ-788 caused a vasoconstrictor response of 27%, which is slightly larger than the effect of ET-1+BQ788 without L-NAME (19%, see above, P > 0.1). The ETA response during L-NAME was attenuated from 27 to 11% by additional infusion of apocynin, and recovered to 27% afterward (Fig. 4, left). This indicates an apocynin-attenuating effect of 60% (see Fig. 6), which is not different from that in the presence of NO (P > 0.3, see Fig. 6).


Figure 4
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  		Fig. 4. Effect of apocynin on the acute renal vasoconstriction produced by stimulation of ETA and ETB receptors in the absence of nitric oxide (NO). Maximum reduction of RBF in response to activation of ETA receptors (left, n = 4) or ETB receptors (right, n = 5) in the presence of NOS inhibition by L-NAME (25 mg/kg iv) is shown. ETA receptors were activated by injection of ET-1 (0.75 pmol) during BQ-788 (7 nmol/min) infusion into the renal artery (ira). ETB receptors were stimulated by injection of S6C (1.5 pmol ira). RBF responses are given during infusion of vehicle (8% ethanol, open bars), during NAD(P)H oxidase inhibition (apocynin, 4 mg·kg–1·min–1 ira, solid bars), and during a subsequent vehicle recovery period. Values are means ± SE. **, ***: P < 0.01, P < 0.001 vs. initial control.

 
Stimulation of ETB receptors by S6C (1.5 pmol) during L-NAME reduced RBF by 25% alone. Note that the magnitude of constriction tended to be larger than the constriction induced by a fourfold smaller dose of S6C during control conditions (17%, see above, P > 0.1). The S6C response during L-NAME was attenuated to a vasoconstriction of 3% during apocynin, and restored to 34% in the recovery period, indicating a 87% inhibitory effect of apocynin (P < 0.001 vs. zero). This influence of apocynin was greater than its action on ETB receptor-mediated vasoconstriction in the presence of NO and exceeded that on ETA-induced constriction in the absence of NO (P < 0.05 by t-test in each case, see Fig. 6).

To decide whether the above observations during NAD(P)H oxidase inhibition are predominantly due to reduction of O2– or rather to depression of H2O2 abundance, the SOD mimetic tempol was tested in a separate group of animals. By driving metabolism of O2– to H2O2, tempol is expected to reduce O2– while raising or maintaining levels of H2O2. In this group, ET-1 (3 pmol) reduced RBF by 16% during the control, 10% during the tempol, and 23% in the recovery period (Fig. 5). This corresponds to a tempol-inhibitory effect of 38% (Fig. 6).


Figure 5
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  		Fig. 5. Effect of tempol on the renal vasoconstrictor response to ET-1. Maximum reduction of RBF produced by simultaneous activation of ETA and ETB receptors by injection of ET-1 (3 pmol ira, n = 5) during ira infusion of saline (open bar), SOD mimetic tempol (5 mg·kg–1·min–1, filled bar), and saline during a recovery period with saline (hatched bar) is shown. Values are means ± SE. **P < 0.01 vs. initial control.

 
Because apocynin raised the baseline RBF, we also assessed the changes in RBF in absolute terms (ml/min). As Table 2 shows, apocynin and tempol produced significant inhibition of ET-1 and selective ETA or ETB receptor stimulation. The one exception was the 0.25 ml/min or 40% attenuation by apocynin when ETA receptors were stimulated during NOS inhibition that did not achieve statistical significance.


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  		Table 2. Renal vasoconstrictor responses to ET receptor stimulation

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present results demonstrate that ROS contribute importantly to the acute vasoconstrictor effect of ET-1 in the renal microvasculature under basal physiological conditions. This is the case for both ETA and ETB receptor-mediated constriction with a similar degree of ROS involvement following activation of each receptor. The results indicate that ROS contribute ~50% to the acute renal vasoconstriction mediated by ET receptors, a magnitude similar to our previous findings of ROS participation in acute ANG II- and NE-induced renal vasoconstriction (29). As in ANG II and NE signaling, the contribution of ROS to ET-1-induced vasoconstriction appears to be due to O2– rather than to H2O2 or other metabolites of SOD, as similar results were obtained by inhibiting NAD(P)H oxidase activity with apocynin and by the cell-permeant SOD mimetic tempol. Another important finding is that the acute renal vasoconstrictor action of O2– does not require scavenging of NO. On the contrary, in the case of ETB receptor stimulation, NO seems to buffer the constrictor influence of O2–, as the effect of NAD(P)H oxidase inhibition is magnified during NOS inhibition. In addition, our apocynin results demonstrate that ROS are tonically active and contribute to basal vascular tone in the intact kidney of anesthetized rats.

Impact of NAD(P)H Oxidase Metabolites on Basal Vasomotor Tone in the Kidney

Acute inhibition of NAD(P)H oxidase by apocynin consistently elevates baseline RBF by ~10% under resting conditions. This agrees with our previous finding that ROS actively contribute to basal vascular tone in Sprague-Dawley rats consuming a standard diet (29) and that of Lopez (40). Although the basal production of ROS may have been affected by anesthesia or surgical preparation, similar observations are reported by other authors: Mice deficient in gp91phox (NOX-2) have a higher baseline RBF (22). An increase in basal renal medullary blood flow is observed during local application of tempol (71), and a reduction of total RBF or medullary perfusion is induced by elevation of O2– via inhibition of SOD (42, 71). The RBF kinetics we observed suggest completion of the vasodilator effect of apocynin within less than 2 min. Thus basal O2– seems to be produced at a rapid turnover rate under resting conditions in normotensive rats.

Involvement of ROS in Renal Vasoconstrictor Responses to ET-1

NAD(P)H oxidase inhibition by apocynin attenuates (on average by ~50%) the renal vasoconstrictor response to a bolus ET-1 injected into the renal artery, indicating that NAD(P)H oxidase metabolites are involved in the vasoconstrictor signaling pathways of ET-1 in the normal kidney. The employed dose of apocynin was the highest we managed to keep in solution while infused at 140 µl/min and for the sufficiently inert composition of the vehicle (8% ethanol). The involvement of ROS in the ET-1 response might therefore be larger than 50%. The degree of inhibition in the present study was similar to that for renal vasoconstriction produced by ANG II, as well as that for ANG II and NE in a previous study (29). The consistent inhibition due to apocynin and tempol on vasoconstrictor agents utilizing three distinct receptor classes suggests that the contribution of ROS to renal vasoconstriction is a uniform feature generalized to other G protein-coupled receptors. It is tempting to speculate similar involvement with vasopressin V1 or thromboxane TP receptors in the kidney and possibly other vascular beds.

ROS may exert a mediating effect secondary to ET-1 receptor-stimulated release of ROS. Alternatively, ambient ROS may provide a modulatory action on common signaling pathways of vasoconstrictor agents. In this regard, ET-1 is known to stimulate immediate (in seconds) production of O2– that impacts on Ca2+ mobilization mediated by ADP ribosyl cyclase and ryanodine receptors in isolated afferent arterioles (15) as does ANG II (16). Such findings support the notion of active participation as a mediator.

Although apocynin attenuated the magnitude of maximum vasoconstriction elicited by ET-1, the general time course of RBF during the first 5 min after the ET-1 bolus was unaffected. Accordingly, the contribution of ROS to the constrictor response of ET-1 does not seem to be confined to a certain time window. This could mean that it impacts on the ET-1 signaling cascade at an early component, upstream of subsequent events that participate in the slow time course of the vasoconstriction. Consistent with this notion is a stimulatory influence of ROS on the production of cADP ribose that thereby sensitizes ryanodine receptors, which then augments inositol trisphosphate-mediated Ca2+ release induced by stimulation of a G protein-coupled receptor such as ANG II or ET-1 (15, 16, 31, 70). Such sensitization would be expected to maintain the time-course of the underlying response.

Observed ROS Effects are More Likely Mediated by O2– Than by H2O2

The ability of apocynin to reduce renal vasoconstriction clearly indicates a net constrictor action of ROS in the immediate response to ET-1. Considering apocynin's known primary action to inhibit NAD(P)H oxidase activity, intrarenal levels of O2– and H2O2 were likely reduced, not elevated. The ability of apocynin to attenuate constriction therefore implicates either O2–, H2O2, or other metabolites of NADPH oxidase as a mediator, provided they have vasoconstrictor influence. The preservation of the inhibitory influence of apocynin in the absence of NO excludes ONOO– as being critical in the acute response. There is general agreement that O2– acts as a vasoconstrictor by acting on VSMC, as well as quenching NO in the chronic setting. The vascular actions of H2O2 are less certain. In some vascular beds under certain conditions, H2O2 may act as a vasoconstrictor (5, 19, 59), vasodilator (4, 16, 51), or both (41, 58). With regard to the kidney, H2O2 appears to exert vasoconstrictor actions in the renal medulla (5) and main renal artery (19). On the other hand, H2O2 appears to be without effect or exert vasodilator-like effects at high concentrations in preglomerular resistance arterioles (16). The inhibitory effect of tempol in the present study is most readily explained by increased SOD activity that scavenges O2– and reduces its vasoconstrictor activity. Alternatively, or in addition, tempol may elevate H2O2 levels, with the observed inhibitory effect of tempol attributed to augmentation of a possible vasodilator effect of H2O2 in the renal cortical circulation. Many investigators have used the cell-permeable SOD mimetic tempol to chronically reduce ROS activity and attenuate peripheral and renal vasoconstriction (52). In addition to increasing SOD activity, tempol has been reported to have other vasodilator-like actions, including inhibiting sympathetic nerve activity (55, 67), activating large-conductance K+ channels (68), and inducing catalase-like activity of heme proteins (46). Our preparation utilizing short-term local application of agents into the renal circulation seems to exclude reduced sympathetic drive due to an action on the central nervous system. Increased K+ channel activity would tend to produce hyperpolarization and vasodilation that might exaggerate the blunted constriction during tempol due to reduced O2– and increased H2O2 levels. Increased catalase activity may reduce H2O2 levels or have no net effect; it could attenuate ET-1-induced constriction, if H2O2 were a renal vasoconstrictor. In either case, the tempol effect is most simply explained by reduced O2– responsible for weaker vasoconstriction. Overall, the bulk of evidence presented in the present study favors the interpretation that vasoactive ROS mediate ET-1-induced acute renal vasoconstriction. The ability of both apocynin and tempol effects to blunt vasoconstriction implicate a primary constrictor action of O2–. Whether H2O2 and downstream SOD metabolites also play a role supporting or opposing the action of O2– awaits further investigation. We conclude that the tendency of apocynin to reduce and SOD to increase H2O2 favors O2– over H2O2. Some uncertainty remains about the source of ROS responsible for our observations. The generally assumed action of apocynin is to inhibit NAD(P)H oxidase assembly and activity (56, 65, 66), and the compound has been shown to inhibit O2– production in several preparations (16, 36–40, 62). However, its action may involve inhibition of other ROS-producing enzymes (65) or a more direct influence on O2– levels through an antioxidant effect (23). In any event, the present data indicate that ROS, regardless of the source, exert a net constrictor influence in the renal circulation and contribute significantly to acute ET-1-induced renal vasoconstriction.

Contribution of ROS to Both ETA and ETB Receptor-Mediated Vasoconstriction

We determined whether the impact of O2– on ET-1-induced renal vasoconstriction is unique to ETA or ETB receptors, or both. ETA receptors were stimulated by ET-1 during ETB receptor inhibition with BQ-788, and ETB receptors were activated by the ETB agonist S6C. Doses were adjusted to achieve similar degrees of constriction before apocynin. Our results indicate that both ETA and ETB receptor-mediated responses are mediated by ROS and that the degree of contribution by NAD(P)H oxidase activity is similar (~50%). This is the case for ETA or ETB receptor stimulation separately as well as for combined stimulation of both receptors. This agrees with a previous report of equal inhibition of ET-1-induced ROS production by either ETB or ETA receptor antagonists in strips of vena cava (59). On the other hand, in aortic rings, selective stimulation of either ETA or ETB receptors had no effect on O2– production in aortic rings, although costimulation of both ET receptors had a positive effect (39).

Our RBF result varies quantitatively from a previous report on isolated afferent arterioles, in which both augmentation of [Ca2+]i transients and production of O2– induced by ET-1 were predominantly mediated through ETA (15). The reason for the difference in results of differing preparations is not clear. It is possible that ETB receptor function is preferentially disturbed by an in vitro preparation. In the in vivo setting, both receptors encounter ET agonists and antagonists, including the endothelial ETB receptor that has complex interactions with the smooth muscle ET receptors (7, 27). Another difference in preparations is that transient stimulation by endothelial-derived or blood-borne ET-1 in vivo initially acts from the vascular lumen, whereas the isolated arterioles encounter a large and continuous dose of ET-1 applied to the abluminal bath. Since ETB receptors are thought to contribute to clearance of ET-1 (18), and because ETA and ETB receptors show different receptor-internalization cycles (1), the characteristics of in vitro application of ET-1 might lead to a different activation pattern of the receptor subtypes than in vivo. In addition, the whole kidney in vivo response reflects the integrated vasoconstriction of both pre- and postglomerular arterioles.

ROS Modify ET-1 Responses Independently of NO

To determine the extent to which the acute ET-1 responses depend on scavenging of the vasodilator NO by O2–, we evaluated the ability of apocynin to inhibit ETA and ETB receptor-induced vasoconstriction in the presence and absence of NO. During NOS inhibition, apocynin attenuated the ETA response by 60%, an inhibitory effect that was clearly not smaller, but even slightly larger than before L-NAME (42%). Similarly, the inhibitory effect of apocynin on the ETB response was not diminished in the absence of NO but was markedly enhanced (87 vs. 50%). The elevated baseline vascular tone following L-NAME may have contributed to larger constrictor responses or might have sensitized the vasculature to the inhibitory influence of apocynin. However, changes in baseline tone alone cannot explain the more pronounced augmentation of vasoconstriction as well as apocynin-induced inhibition in the case of ETB vs. that of ETA receptor stimulation, as both receptor subtypes were assessed during the same dose of L-NAME and with a similar rise in baseline vascular tone. Note also that the same conclusion of enhanced inhibition by apocynin on ETB-induced vasoconstriction is obtained when constrictor responses are assessed in absolute terms without normalization to baseline RBF.

Collectively, our findings indicate that the contribution of ROS to the immediate renal vasoconstrictor response to either the ETA or ETB receptor does not depend on the presence of NO and thus does not require quenching of NO. O2– seems to be exerting a direct action on VSMC in the renal microcirculation of normal animals. This agrees with our previous observations of the NO-independent influence of O2– on the acute renal constrictor responses to ANG II and NE (29). Our findings also are congruent with the observations of other investigators that the attenuating effect of tempol on the acute renal vasoconstrictor response to ANG II is not impaired in the absence of NO in dogs (43) or rats (9). It is important to appreciate that the latter two studies as well as our work were conducted using intact kidneys of anesthetized animals and that acute vasoconstrictor responses were recorded over a short-term time range of a few seconds to 30 min. This contrasts to the long-term vasoconstrictor effects of ROS in chronic disease states such as ANG II-induced (35, 61), renovascular (26), mineralocorticoid-dependent (36), or genetic hypertension (47, 64), and diabetes mellitus (49), which are well established to be mediated by scavenging of NO by O2– and subsequent endothelial dysfunction accounting for exaggerated vasoconstriction.

In the case of ETB-induced vasoconstriction, we found that the absence of NO not only failed to disturb, but markedly enhanced, the inhibitory influence of apocynin. This suggests that under these conditions NO reduces the availability of O2– rather than vice versa. The chemical reaction thought to underlie the scavenging effect (O2–+NO ONOO–) (24) involves both substrates in stoichiometrically equal amounts. Therefore, scavenging of O2– by NO is inevitably linked to that of NO by O2–. The determination of whether and to what extent the quenching will have functional consequence for either reaction partner depends on the relative magnitude of ambient pools of O2– and NO compared with the amounts reacting. Under basal conditions, there appears to be a relative abundance of NO in the vasculature of normal healthy kidneys based on the observations that NOS inhibition produces an acute ~30% decrease in RBF compared with a 10% increase in RBF produced by NAD(P)H oxidase inhibition. The present results suggest that scavenging of O2– by NO occurs in vivo in the acute setting in normal animals, most clearly evident during ETB receptor stimulation. The balance between preferential scavenging of NO and O2– is likely to change with ambient levels of either of the two reaction partners under different conditions.

Summary and Perspectives

In summary, our study provides new evidence that ROS, probably O2–, contribute importantly to acute renal vasoconstriction produced by ET-1 as well as to basal renovascular tone under control conditions in a normal rat. Both ETA and ETB receptor signaling is mediated by ROS to similar degrees (at least 50%). The magnitude of ROS involvement in short-term renal vasomotor responses to ET-1 is similar to its role in acute responses to ANG II and NE, suggesting the participatory role of O2– is a general feature of G protein-coupled receptors in the renal microcirculation and may extend to other vascular beds. The effect of ROS on ET-1 responses is independent of NO, suggesting a direct constrictor effect of ROS on VSMC in renal resistance arterioles distinct from quenching of NO. In the case of ETB receptor stimulation, the influence of ROS is augmented in the absence of NO, pointing to the functional relevance of scavenging of O2– by NO in acute regulation of hemodynamics in the intact kidney. Although this direction of scavenging is opposite that in many chronic pathophysiological conditions such as hypertension, diabetes mellitus, or atherosclerosis, in which quenching of NO by O2– leads to endothelial dysfunction, there might be a continuum between preferential scavenging of one or the other depending on their relative abundance dictated by experimental and clinical conditions. The detailed underlying mechanisms of direct and indirect influences of O2– and other ROS, both acute and chronic, on VSMC of resistance arterioles require further investigation.


   GRANTS
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 ABSTRACT
 METHODS
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-02334 and a gift from the Thomas Maren Foundation.


   ACKNOWLEDGMENTS
 
We thank Andrea R. Richardson for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Just, Dept. of Cell and Molecular Physiology, 6341 Medical Biomolecular Research Bldg., CB#7545, School of Medicine, Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7545 (e-mail: just{at}med.unc.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.


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