Journal of the American Society of Nephrology
2007 JASN IMPACT FACTOR 7.111 HOME   AUTHOR INFO   EDITORIAL BOARD   SUBSCRIBE   FEEDBACK   ALERTS   HELP 
    advanced
CURRENT ISSUE ARCHIVES JASN Express ONLINE SUBMISSION


Published ahead of print on November 15, 2006
J Am Soc Nephrol 17: 3347-3354, 2006
© 2006 American Society of Nephrology
doi: 10.1681/ASN.2006030212
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
ASN.2006030212v1
17/12/3347    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, D.
Right arrow Articles by Wilcox, C. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, D.
Right arrow Articles by Wilcox, C. S.

Hemodynamics and Vascular Regulation

beta1 Receptors Protect the Renal Afferent Arteriole of Angiotensin-Infused Rabbits from Norepinephrine-Induced Oxidative Stress

Dan Wang*, Pedro Jose{dagger} and Christopher S. Wilcox*

* Division of Nephrology and Hypertension and the Cardiovascular-Kidney Institute; and {dagger} Department of Pediatrics, Georgetown University, Washington, DC

Address correspondence to: Dr. Christopher S. Wilcox, Division of Nephrology and Hypertension, Georgetown University Medical Center, 3800 Reservoir Road, NW, PHC Suite F6003, Washington, DC 20007-2197. Phone: 202-444-9183; Fax: 202-444-7893; E-mail: wilcoxch{at}georgetown.edu

Received for publication March 8, 2006. Accepted for publication October 1, 2006.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Renal afferent arterioles (Aff) from angiotensin II (AngII)-infused rabbits have enhanced contractions to AngII that are normalized by tempol (superoxide dismutase mimetic), whereas contractions to norepinephrine (NE) are normal and unaffected by tempol. Tested was the hypothesis that beta-receptor stimulation with NE prevents enhanced reactivity and superoxide generation. Preconstricted Aff from AngII- or vehicle-infused rabbits were perfused at physiologic pressure. Aff from vehicle-infused rabbits had strong, endothelium-independent relaxations to dobutamine (beta1-receptor agonist; 78 ± 6%; P < 0.0001; mean ± SD) but only weak relaxations to salbutamol (beta2-receptor agonist; 13 ± 3%; P < 0.05) or BRL-37,344 (beta3-receptor agonist; 14 ± 3%; P < 0.05). Contractions to NE were similar in Aff from vehicle- and AngII-infused rabbits (–36 ± 5 versus –34 ± 3%; NS) and were unaffected by tempol (–32 ± 4%; NS). In contrast, phenylephrine contractions ({alpha}1 agonist) were enhanced in Aff from AngII-infused rabbits (–59 ± 6 versus –46 ± 4%; P < 0.05) and normalized by tempol. NE contractions in Aff from AngII-infused rabbits (–34 ± 4%) were enhanced (P < 0.01) by propranolol (nonselective beta antagonist; –53 ± 6%), CGP-20,712A (selective beta1-receptor antagonist; –61 ± 9%), or Rp-cAMP (competitive inhibitor of cAMP; –56 ± 4%); were normalized by tempol; but were unaffected by ICI-118,551 (selective beta2-receptor antagonist) or SR-59,230A (selective beta3-receptor antagonist). Superoxide generation in Aff from AngII-infused rabbits that were assessed from ethidium:dihydroethidium was enhanced by addition of CGP-20,712A to NE but was normalized by tempol. Aff have robust {alpha}1-receptor contraction and beta1-receptor dilation. NE elicits beta1 signaling via cAMP that moderates oxidative stress and contractions in Aff from AngII-infused rabbits.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Oxidative stress suggests an increased production or a decreased metabolism of reactive oxygen species (ROS). Superoxide (O2·–) mediates enhanced contractility in many models of hypertension (1,2). O2·– is metabolized by superoxide dismutase (SOD) to H2O2. Harrison and colleagues (1,3) showed that, whereas prolonged infusion of angiotensin II (AngII) generates O2·– in blood vessels, infusions of norepinephrine (NE) fail to induce vascular oxidative stress. Moreover, administration of permanent SOD reverses hypertension in the AngII-induced but not the NE-induced rat model (3). They concluded that AngII selectively induces vascular oxidative stress, which contributes to hypertension.

Relatively low rates of infusion of AngII into mice (4), rats (1,5,6), rabbits (7), or humans (8) increase BP gradually ("slow pressor response"). This is considered a model of human hypertension because the plasma levels of AngII increase only within a physiologic range of 50 to 100% (9). The hypertension entails a renal mechanism because it depends on salt intake (10) and an enhanced vascular reactivity of the renal afferent arteriole (Aff) to AngII (11). Unlike AngII, infusion of NE leads to tachyphylaxis and a decreased BP response (8). The AngII slow pressor response has been related to ROS because it is prevented by co-infusion of the permeant SOD mimetic nitroxide tempol (4,6).

Low rates of infusion of the thromboxane-prostanoid receptor (TP-R) mimetic U-46,619 (12) or endothelin-1 (ET-1) (13) also elicit slow pressor responses that are accompanied by oxidative stress. Moreover isolated, pressurized Aff that are dissected from the kidneys of rabbits that are undergoing an AngII slow pressor response have an enhanced contractility not only to AngII but also to ET-1 and U-46,619 (11) that depend on O2·– because they are moderated or prevented by bath addition of the permanent SOD mimetic nitroxide tempol (11). In contrast, contractions to bath addition of NE or high [K+] are not enhanced in Aff from AngII-infused rabbits and are not affected by tempol. This suggests an alternative explanation for the previous studies: Prolonged AngII infusion generates vascular ROS that enhance the responses to AngII and also to a number of other G protein–coupled receptor agonists such as ET-1, U-46,619, and phenylephrine (PE). In contrast, activation of beta-adrenergic receptors by NE prevents the generation of ROS and consequently the enhanced contraction. This suggests that the unique response is to NE rather than to AngII. Therefore, these studies were undertaken to investigate the hypothesis that activation of a specific subtype of beta-adrenergic receptors by NE prevents ROS-induced enhanced contractility of Aff from AngII-infused rabbits.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal Protocols
The protocol was approved by the Georgetown University Animal Care and Use Committee. Male New Zealand white rabbits (1.8 to 2.1 kg) were maintained on tap water and standard diet (Na+ content 0.4 g/100g) and prepared as described previously (11). Under local anesthesia with EMLA cream (2.5% lidocaine and 2.5% prilocaine), sterile osmotic minipumps (Alzet, DURET Corp., Cupertino, CA) were implanted subcutaneously in rabbits (n = 6 per group) to infuse vehicle (0.154 M NaCl) or human AngII (Peninsula Laboratories, San Carlos, CA) at 200 ng/kg per min (AngII) for 12 to 14 d.

Isolation and Microperfusion of Aff
Rabbits were anesthetized with xylazine (10 mg/kg intramuscularly), ketamine (50 mg/kg intramuscularly), and pentobarbital sodium (10 mg/kg intravenously) followed by heparin (1000 USP intravenously) for anticoagulation. Microdissection and microperfusion at 60 mmHg of Aff were performed as described previously (14,15). Each experiment in each series used a separate arteriole.

Experimental Protocol
The first aim was to test the role of the beta-adrenoreceptor subtype and the endothelium in the relaxation of Aff from vehicle-infused rabbits (n = 6 per group). Aff were preconstricted by 60 to 70% from control luminal diameter with PE (10–6 M; {alpha}1-receptor agonist). Thereafter, dobutamine (10–7 M; beta1-receptor agonist) (16), salbutamol (10–6 M; beta2-receptor agonist) (17), or BRL-37,344 (BRL; 10–6 M; beta3-receptor agonist) (17) was added to the bath. These dosages were selected in pilot studies to show <90% of maximal responses. Vessels were observed for 3 to 5 min, which allowed for maximal responses. Parallel studies were undertaken in vessels (n = 6) that were denuded of endothelium by microperfusion with 0.1% goat anti-human von Willebrand factor antibody plus 2% guinea pig serum complement for 10 min (14,18). This entirely prevents relaxation responses to acetylcholine but leaves intact responses to sodium nitroprusside (14). A full dosage-response to the most effective agent, dobutamine (10–10 to 10–6 M) was performed with and without endothelium removal.

The second aim was to contrast the role of O2·– in contractile responses of Aff from AngII- versus vehicle-infused rabbits (n = 6 per group). Contractions to bath additions of NE (10–6 M, activating both {alpha}- and beta-adrenergic receptors) and PE (10–7 M, activating {alpha}1-adrenergic receptors) were related to a standard maximal contraction with NE (10–6 M) plus 40 mM KCl (NAK). These responses were maintained for 3 min. Thereafter, Aff were washed with buffer, recovered for 10 min, and incubated for 20 min with tempol (10–4 M) to assess the effects of metabolism of O2·– (19). Tempol at 10–4 M blocks fully the contractile responses of afferent arterioles to O2·– that are generated by a quinolone (19) but does not change basal tone or responses of vessels from normal rabbits that are infused with a vehicle to AngII, U-46,619, NE, or ET-1 (7).

The third aim was to contrast the effect of nonselective beta-adrenergic receptor blockade alone or with tempol (10–4 M) on contractions to NE or to high [K+] of Aff from AngII- or vehicle-infused rabbits (n = 6 per group). Aff were incubated for 20 min with a vehicle (MEM solution), propranolol (10–8 M; nonselective beta-adrenergic receptor antagonist) (20), tempol (10–4 M), or a combination of propranolol and tempol (order randomized). Luminal diameters were measured during basal conditions and after superfusion with NE (10–7 M) or KCl (40 mM).

The fourth aim was to test the role of cAMP in mediating the effects of beta-adrenergic receptor stimulation with NE in mitigating the enhanced contractions in Aff from AngII-infused rabbits (n = 6 per group). Contractions to NE (10–6 M) or KCl (40 mmol/L) were obtained before and after 20 min of incubation with vehicle, adenosine-3', 5'-cyclic monophosphorothioate Rp-isomer (Rp-cAMP, 5 x 10–6 M; cAMP antagonist) (21,22) or tempol (10–4 M) or a combination of Rp-cAMP and tempol (order randomized).

The fifth aim was to evaluate the beta-adrenergic receptor subtype that modulates NE contractions of Aff from AngII-infused rabbits (n = 6 per group). Aff were incubated for 30 min with CGP-20,712A (CGP; 10–8 M; selective beta1-adrenergic receptor agonist), ICI-118,551 (ICI; 10–8 M; selective beta2-adrenergic receptor agonist), or SR-59,230A (SR; 10–6 M, selective beta3-adrenergic receptor antagonist) (23) alone or in combination with tempol (orders randomized). Luminal diameters were measured during basal conditions and after super fusions with NE (10–7 M).

The sixth aim was to test the specificity of beta1-adrenergic receptor blockade by CGP in Aff from vehicle-infused rabbits (n = 6 per group). Aff were incubated for 30 min with vehicle (MEM solution) or the beta1-adrenergic receptor agonist CGP (10–8 M). Thereafter, they were preconstricted with PE (10–6 M), after which dobutamine (10–7 M), salbutamol (10–6 M), or BRL (10–6 M) (17) was added to the bath.

The seventh aim was to test the hypothesis that beta1-adrenergic receptor activation during NE prevents an increase in O2·– generation in Aff. O2·– generation was assessed by fluorescence microscopy of perfused Aff (n = 6) with dihydroethidium (DHE). DHE is freely permeable to cells. It is oxidized by O2·– to the highly fluorescence compound ethidium, which is trapped intracellularly and intercalated into DNA (24). The conversion of DHE to ethidium was quantified by a dual-wavelength determination using an excitation wavelength of 380 nm and an emission wavelength of 460 nm for DHE and an excitation wavelength of 535 nm and an emission wavelength of 605 nm for ethidium (25). Single-agent signal capture was achieved by cycling at 3-s intervals between a 460- and 605-nm filter. Changes in O2·– were expressed as the ratio of ethidium:DHE fluorescence (26). The system used an Olympus IX70 fluorescence microscope equipped with dual photomultipliers (PMT, Photon Technology Int., Lawrenceville, NJ). Excitation was provided by a 75-W xenon arc lamp using a 380/460 nm wavelength combination isolated with a computer-controlled monochromator. Ethidium and DHE emit blue and red light, respectively, that was directed to a dual PMT assembly by a beam splitter that directed light to the two separate PMT using a 400-nm dichroic mirror and using barrier filters centered at 460 and 605 nm, respectively. The ratio of ethidium:DHE was monitored in real time and recorded by software (Felix32; Photon Technology Int.).

For testing of beta1-adrenergic receptor activation of ROS generation during NE action, Aff from AngII-infused rabbits were cannulated and load with HBSS that contained DHE (10–5 M) and glutamine (10 mM). After 30 min for equilibration, the ratio of ethidium:DHE was assessed for 10-min periods during vehicle and after 30 min of incubation with NE (10–7M), 30 min of incubation with NE+CGP (10–8 M, beta1-adrenergic receptor blocker), and 30 min of incubation with NE plus a combination of CGP and tempol (10–4 M). A separate set of Aff were incubated throughout with a vehicle as a time control.

Drugs and Solutions
All solutions were prepared fresh daily. Reagents were purchased from Sigma (St. Louis, MO). Rp-cAMP was obtained from Biolog-Life Science Institute (La Jolla, CA). Earle’s Deficient BME Solution was used for dissection. It contains 8.89 g/L NaCl, 26 mM NaHCO3, 2 mM l-glutamine, and 5% BSA (pH 7.40 to 7.45). It was filtered (0.8 µm) and prepared fresh daily. MEM solution that contained 26 mM NaHCO3 and 5% BSA was used for perfusion, and MEM that contained 26 mM NaHCO3 and 0.15% BSA was used for superfusion. HBSS was purchased from Invitrogen (Carlsbad, CA).

Statistical Analyses
Statistical tests of the percentage of vasoconstriction or vasodilation used 2 x 2 factorial repeated-measures ANOVA (Statistical Software v.5.0; University of Hamburg, Hamburg, Germany). When appropriate, post hoc comparisons between groups were made with t test. Statistical significance was defined as P < 0.05. Data are presented as mean ± SD.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The diameter of Aff from AngII- and vehicle-infused rabbits was similar in the basal state (17.3 ± 1.2 versus 16.9 ± 1.2 µm; NS) and during NAK (6.5 ± 0.5 versus 6.3 ± 0.6 µm; NS)

beta-Adrenoreceptors Subtype Mediating Relaxation of Aff
Dobutamine (10–7 M; a selective beta1-adrenergic receptor agonist) potently relaxed PE-constructed Aff (78 ± 6%; P < 0.001), whereas salbutamol (10–6 M; selective beta2-adrenergic receptor agonist) had a significantly (P < 0.001) weaker effect (13 ± 3%; P < 0.05) as did BRL (10–6 M; selective beta3-adrenergic receptor agonist, 14 ± 3; P < 0.05). None of these relaxations was affected by endothelium removal (Figure 1A). Dobutamine induced a dosage-dependent, endothelium-independent relaxation, with an IC50 of 4.99 x 10–7± 2.03 x 10–9 M (Figure 1B). We conclude that beta-receptor vasodilation in rabbit Aff is mediated predominantly by beta1-adrenergic receptors via an endothelium-independent mechanism.


Figure 1
View larger version (18K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 1. Mean ± SD values (n = 6 per group) for percentage changes in luminal diameter of afferent arterioles (Aff) that were preconstricted with phenylephrine (10–6 M) from vehicle-infused rabbits with endothelium intact or endothelium denuded. (A) Relaxations to dobutamine (10–8 M; beta1-adrenergic receptor agonist), salbutamol (10–6 M; beta2-adrenergic receptor agonist), and BRL-37,344 (BRL; 10–6 M; beta3-adrenergic receptor agonist) in vessels with endothelium intact (endo+; {square}) or removed (endo–; {blacksquare}). ***P < 0.001 versus dobutamine. (B) Dosage-responses to dobutamine in vessels with endothelium intact (endo+; {blacksquare}) or removed (endo–; {triangleup}).

 
Role of O2·– in Contractions of Aff to NE and PE
Compared with Aff from vehicle-infused rabbits, Aff from AngII-infused rabbits had similar contractions to NE (–36 ± 5 versus –34 ± 3%; NS) but enhanced contractions to PE (–59 ± 6 versus –46 ± 4%; P < 0.05; Figure 2). Bath addition of tempol did not modify the diameter in the basal state or during NAK. Bath addition of tempol to Aff from AngII-infused rabbits did not affect their response to NE but reduced their contractions to PE to the level of Aff from vehicle-infused rabbits (49 ± 5 versus 46 ± 4; NS). We conclude that PE generates O2·– in Aff from AngII-infused rabbits, which enhances their responsiveness, whereas NE does not.


Figure 2
View larger version (18K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 2. Mean ± SD values (n = 6 per group) for contractile responses of Aff to norepinephrine (NE; 10–7 M) or phenylephrine (PE; 10–7 M) during addition to the bath of vehicle or tempol (10–4 M) comparing results of Aff from rabbits that were infused with vehicle ( Figure 2) or angiotensin II (AngII; {blacksquare}). *P < 0.05 versus vehicle-infused rabbits; aP < 0.05 versus PE alone.

 
Effect of beta-Adrenergic Receptor Blockade on Contractions of Aff to NE
Aff from rabbits that were infused with vehicle or AngII had similar contractions to KCl and NE (Figure 3). Bath addition of tempol, propranolol, or tempol plus propranolol did not modify contractions of Aff to KCl in either group and did not modify contractions to NE in vehicle-infused rabbits. However, propranolol enhanced contractions to NE in Aff from AngII-infused rabbits (–34 ± 3 versus –53 ± 6%; P < 0.01; Figure 3B). This effect of propranolol was prevented by tempol (53 ± 6%; NS versus propranolol alone). We conclude that activation of beta-adrenergic receptors in Aff from AngII-infused rabbits prevents enhanced O2·–-dependent contractions to NE.


Figure 3
View larger version (31K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 3. Mean ± SD values (n = 6 per group) for contractile responses of Aff to KCl (40 mmol/L) or NE (10–7 M) during addition to the bath of vehicle ({square}), propranolol (10–8 M; Figure 3), tempol(10–4 M; {blacksquare}), or propranolol plus tempol (lbox) of Aff from vehicle-infused rabbits (A) or AngII-infused rabbits (B). **P < 0.01 versus vehicle; aP < 0.05 versus propranolol alone.

 
Role of cAMP in Moderating NE Contraction of Aff from AngII-Infused Rabbits
KCl contractions of Aff from AngII-infused rabbits were unaffected by tempol, Rp-cAMP, or tempol plus Rp-cAMP (Figure 4). Whereas tempol did not affect NE contractions of Aff from AngII-infused rabbits, these contractions were enhanced by Rp-cAMP (–38 ± 3 versus –56 ± 4%; P < 0.01). This effect of Rp-cAMP was blocked by bath addition of tempol (–37 ± 6%; NS versus tempol alone). We conclude that cAMP that is generated in Aff from AngII-infused rabbits mediates the effects of beta-adrenergic receptors to inhibit O2·– generation and enhance NE contractions.


Figure 4
View larger version (24K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 4. Mean ± SD values (n = 6 per group) for contractile responses of Aff from AngII-infused rabbits to KCl (40 mmol/L) or NE (10–7 M; {blacksquare}) during addition to the bath of vehicle ({square}), Rp-cAMP (5 x 10–6 M; cAMP antagonist; Figure 4), tempol (10–4 M; {blacksquare}), and Rp-cAMP plus tempol (Figure 4). **P < 0.01 versus vehicle; bP < 0.01 versus Rp-cAMP alone.

 
Subtype of beta-Adrenergic Receptor Preventing Enhanced Contractions to NE in Aff from AngII-Infused Rabbits
NE contractions of Aff from AngII-infused rabbits were unaffected by tempol alone, ICI (selective beta2-adrenergic receptor antagonist), SR (selective beta3-adrenergic receptor antagonist), or ICI or SR plus tempol (Figure 5). However, CGP (10–8 M; selective beta1-adrenergic receptor antagonist) enhanced NE contractions (–40 ± 3 versus –61 ± 9%; P < 0.001). This effect was prevented by tempol (–39 ± 5%; NS versus tempol alone). We conclude that the beta-adrenergic receptors that inhibit O2·– generation and enhance contractions to NE in Aff from AngII-infused rabbits are subtype I.


Figure 5
View larger version (31K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 5. Mean ± SD values (n = 6 per group) for contractile responses to NE (10–7 M) of Aff from AngII-infused rabbits during addition to the bath of vehicle (V), tempol (T; 10–4 M), CGP-20,721A (CGP; 10–8 M; beta1-adrenergic receptor blocker), tempol plus CGP, ICI-118,551 (ICI; 10–6 M; beta2-adrenergic receptor blocker), ICI plus tempol, SR-59,230A (SR; 10–6 M; beta3-adrenergic receptor blocker), or SR plus tempol. **P < 0.01 versus vehicle; aP < 0.05 versus CGP.

 
Specificity of CGP as a beta1-Adrenergic Receptor Antagonist
The addition of CGP (10–8 M) to the bath of Aff from vehicle-infused rabbits prevented the relaxation to dobutamine (10–7 M; selective beta1-adrenergic receptor agonist; 78 ± 5 versus 3 ± 5%; P < 0.001) but not to salbutamol (selective beta2-adrenergic receptor agonist) or BRL (selective beta3-receptor agonist) (Figure 6). We conclude that CGP is a selective beta1-adrenergic receptor antagonist in this preparation.


Figure 6
View larger version (14K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 6. Mean ± SD values (n = 6 per group) for effect of CGP (10–8 M; {blacksquare}) compared with vehicle ({square}) on the relaxation responses of Aff from vehicle-infused rabbits that were preconstricted with 10–6 M PE to dobutamine (10–7 M; beta1-adrenergic receptor agonist), salbutamol (10–6 M; beta2-receptor agonist), or BRL (10–6 M; beta3-adrenergic receptor agonist). ***P < 0.001 versus vehicle.

 
Role of beta1 Adrenergic Receptors in Preventing O2·– Generation with NE in Aff from AngII-Infused Rabbits
NE at 10–7 M slightly but significantly increased the ethidium:DHE fluorescence ratio (before 0.01 ± 0.08 versus during NE 0.28 ± 0.10; P < 0.05). After Aff were incubated with CGP (10–8 M; beta1-adrenergic receptor blocker), NE increased the ethidium:DHE ratio significantly (1.33 ± 0.5 CGP+NE versus 0.28 ± 0.1 NE alone; P < 0.001). This was restored to the level of NE alone by addition of tempol (10–4 M) to vessels that were incubated with CGP and NE (0.23 ± 0.11; NS versus NE+vehicle; Figure 7). The ratio of ethidium:DHE did not change in Aff that were incubated with a vehicle (time control).


Figure 7
View larger version (15K):
[in this window]
[in a new window]
[as a PowerPoint slide]
  		Figure 7. Mean ± SD values (n = 6) for reactive oxygen species (ROS) generation from the ratio of fluorescence of ethidium:dihydroethidium (Eth:DHE) during incubation of Aff from AngII-infused rabbits with vehicle, 10–7 M NE, NE plus CGP (10–8 M; beta1-adrenergic receptor antagonist), or NE plus CGP plus 10–4 M tempol (TMP). *P < 0.05, ***P < 0.001 versus vehicle; bP < 0.01 versus NE alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We confirm that prolonged infusion of AngII does not modify NE contractions of Aff, in contrast to the enhanced responses reported with AngII, ET-1, and U-46,619 (7,11). The new findings are that the absence of an enhanced response to NE in these vessels cannot be ascribed to a protective role of {alpha}1-adrenergic receptor stimulation because the contractile response to phenylephrine is enhanced similarly to AngII, ET-1, and U-46,619. Blockade of beta-adrenergic receptors unmasks an enhanced NE response in Aff from AngII-infused rabbits, similar to PE. This implicates beta-adrenergic receptors in moderating the responsiveness. Because this effect is reversed by incubation with tempol, it can be ascribed to beta-adrenergic receptor-dependent inhibition of vascular O2·– generation. The beta-adrenergic receptor subtype that is responsible is identified as type I because the beta1-adrenergic receptor agonist dobutamine causes a much more powerful relaxation of preconstricted Aff than beta2- or beta3-adrenergic agonists and the beta1-adrenergic receptor antagonist CGP enhances NE contractions in Aff from AngII-infused rabbits, whereas beta2- or beta3-adrenergic receptor antagonists are ineffective. Fluorescence microscopy demonstrated that blockade of beta1-adrenergic receptors during stimulation with NE enhanced O2·– in the perfused Aff, which was restored by incubation with tempol. The effects of beta1-adrenergic receptors to moderate contractions are independent of the endothelium. Because this effect is blocked by Rp-cAMP, it seems to be mediated by generation of cAMP. Therefore, the absence of an enhanced response to NE in Aff from AngII-infused rabbits is related to beta1-adrenergic receptors that activate cAMP and counteract {alpha}1-adrenergic receptor stimulation of O2·–.

Tempol is freely membrane permeable and acts as a catalytic SOD mimetic (15). Tempol fully prevents the graded vasoconstriction of isolated, perfused Aff to the O2·–-generating quinolone paraquat (19). Studies with fluorescence indicators of O2·– have confirmed that tempol, in the dosage used in this study, fully prevents the generation of O2·– in isolated, perfused vasa recta that are stimulated with AngII (27). This was confirmed in this study, in which enhanced vascular O2·– generation by NE during beta1-adrenergic receptor blockade was normalized by bath addition of 10–4 M tempol.

Contractions to NE in Aff are mediated via {alpha}-adrenergic receptor–dependent transmembrane increases in intracellular [Ca2+] (28) and release of Ca2+ from intracellular stores (29). The finding that PE seemed to have enhanced ROS generation in Aff from AngII-infused rabbits is consistent with its effect to increase ROS generation in vitro in vascular smooth muscle cells (30) and in vivo in rat aorta, where the effect is accompanied by upregulation of p47phox expression and mediated via NADPH oxidase (31).

Within the cardiovascular system, beta1-adrenergic receptors are expressed predominantly in cardiomyocytes, where they mediate chronotropic and inotropic effects (32), whereas beta2-adrenergic receptors are expressed predominantly in peripheral blood vessels, where they mediate vasorelaxation (32). beta3-Adrenergic receptors can cause endothelium-dependent relaxation of coronary microvessels but have a less defined role presently in the cardiovascular system (33). Therefore, the finding that vasorelaxation of Aff was much more prominent with a beta1- than with a beta2- or beta3-adrenergic receptor agonist would not be expected from Land’s hypothesis. This may relate to a selective action of beta1-adrenergic receptors on the terminal renal Aff, where specialized granular smooth muscle cells are stimulated to release renin predominantly by beta1-adrenergic receptors (34,35). It seems that a similar receptor activation also induces vasorelaxation of this blood vessel. Moreover, recent studies have shown predominant beta-adrenergic receptor regulation of vascular tone by the beta1 subclass in some small vessels, depending on species and site (36). Recent studies in the rat have concluded that beta1, not beta2-adrenergic receptors predominate on vascular smooth muscle cells of mesenteric resistance vessels (17) and renal microvessels and macula densa epithelium (37). Functionally, the beta1-adrenergic receptor is the predominant class mediating vasodilation in the small blood vessels of the kidney and mesentery of rats or mice (17,38). In an in vivo analysis in humans, Wellstein et al. (39) estimated that 77% of beta-adrenergic receptor–related hypotension is mediated via beta1-adrenergic receptors and 23% via beta2-adrenergic receptors. Studies in the rabbit have confirmed that beta1-adrenergic receptor blockade with atenolol inhibits the renin release in response to angiotensin receptor blockade, which is considered to be an effect that is mediated at the terminal Aff (40).

The effect of beta1-adrenergic receptors to inhibit ROS generation seems to depend on protein kinase A (PKA) because it is blocked by Rp-cAMP. Studies in the isolated mesenteric resistance vessel of the rat have shown that beta1-adrenogenic receptors induce vasorelaxation via separate PKA-dependent and -independent pathways (22).

Within the peripheral circulation, beta-adrenergic receptor stimulation can lead to endothelium-dependent or -independent vasorelaxation. Removal of the endothelium from the terminal renal Aff did not modify the vasorelaxation to beta1-, beta2-, or beta3-adrenergic receptor stimulation, indicating that relaxation at this site is endothelium independent. This conclusion held for the entire dosage-response range for the beta1-adrenergic receptor agonist dobutamine. This is consistent with recent conclusions from studies with fluorescence covalent ligands for beta1-adrenergic receptors that locate these receptors on vascular smooth muscle cells of rat mesenteric resistance vessels in the plasma membrane, Golgi, endoplasmic reticulum, and perinuclear spaces (17).

Whereas epinephrine is a potent agonist of beta1- and beta2-adrenergic receptors, NE activates predominantly beta1-adrenergic receptors. This is consistent with the finding that the beta1-adrenergic receptor antagonist CGP increased NE vasoconstriction markedly in Aff from AngII-infused rabbits, whereas a beta2- or beta3-adrenergic receptor antagonist was not effective. CGP was validated as a selective beta1-adrenergic receptor antagonist because it prevented the response to the beta1-adrenergic receptor agonist dobutamine without affecting the response to a beta2- or beta3-adrenergic receptor agonist.

Whereas there is much evidence of receptor-dependent stimulation of vascular ROS by agonists such as PE, AngII, ET-1, or U-46, 619, less is known concerning receptor-mediated antioxidant defense, for example by catecholamines. Activation of dopamine D1 and D5 receptors in rat renal vascular smooth muscle cells inhibits ROS generation (41). D5 receptor activation in human embryonic kidney HEK-293 cells inhibits ROS generation by activation of heme oxygenase-1 (42) and inhibition of the assembly and activity of NADPH oxidase (43). Because the D5 receptor–mediated inhibition of ROS generation is independent of cAMP in HEK-293 cells, whereas D1-like receptor–mediated inhibition of ROS in renal vascular smooth muscle cells is mediated in part by cAMP, the D1 dopamine receptor, like the beta1-adrenergic receptor, may exert their antioxidant effects via cAMP.

If our finding that beta1 receptors protect the renal Aff of AngII-infused rabbits from NE-induced oxidative stress and associated enhanced contractility is relevant to other microvessels, then this may help to explain why prolonged treatment of hypertension with a beta1-adrenergic receptor antagonist, atenolol, in contrast to the antioxidant calcium channel blocker, amlodipine (44), fails to restore endothelial function or reverse remodeling of subcutaneous microvessels (45). Moreover, atenolol fails to provide effective cardiovascular protection in trials of hypertensive patients (46). This has led some authorities to recommend that beta1-adrenergic receptor antagonists no longer be prescribed as primary therapy for hypertension (47). A potential explanation that requires study is that such therapy prevents the counterbalance of {alpha}-adrenergic receptor–mediated ROS generation during NE stimulation by activated beta1-adrenergic receptors.


   Acknowledgments
 
This work was support by grants from the National Institute of Diabetes and Digestive and Kidney Disease (DK-36079 and DK-49870) and the National Heart, Lung, and Blood Institute (HL-68686) and by funds from the George E. Schreiner Chair of Nephrology.

We are grateful to Margaret Brierton for preparing the manuscript.


   Footnotes
 
Published online ahead of print. Publication date available at www.jasn.org.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. 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]
  2. Wilcox CS: Oxidative stress and nitric oxide deficiency in the kidney: A critical link to hypertension? Am J Physiol Regul Integr Comp Physiol 289 : R913 –R935, 2005[Abstract/Free Full Text]
  3. Rajagopalan S, Kurz S, Munzel T, Tarpey M, Freeman BA, Griendling KK, Harrison DG: Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. Contribution to alterations of vasomotor tone. J Clin Invest 97 : 1916 –1923, 1996[Medline]
  4. 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]
  5. Ortiz MC, Sanabria E, Manriquez MC, Romero JC, Juncos LA: Role of endothelin and isoprostanes in slow pressor responses to angiotensin II. Hypertension 37 : 505 –510, 2001[Abstract/Free Full Text]
  6. 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]
  7. 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]
  8. Ames RP, Borkowski AJ, Sicinski AM, Laragh JH: Prolonged infusions of angiotensin II and norepinephrine and blood pressure, electrolyte balance, and aldosterone and cortisol secretion in normal man and in cirrhosis with ascites. J Clin Invest 44 : 1171 –1186, 1965[Medline]
  9. Atkinson AB, Brown JJ, Fraser R, Lever AF, Morton JJ, Riegger AJ, Robertson JI: Angiotensin II and renal hypertension in dog, rat and man: Effect of converting enzyme inhibition. Clin Exp Hypertens 2 : 499 –524, 1980[Medline]
  10. Csiky B, Simon G: Synergistic vascular effects of dietary sodium supplementation and angiotensin II administration. Am J Physiol 273 : H1275 –H1282, 1997[Medline]
  11. Wang D, Chabrashvili T, Wilcox CS: Enhanced contractility of renal afferent arterioles from angiotensin-infused rabbits: roles of oxidative stress, thromboxane prostanoid receptors, and endothelium. Circ Res 94 : 1436 –1442, 2004[Abstract/Free Full Text]
  12. Welch WJ, Ahlstrom NG, Wilcox CS: Mechanism of hypertension during prolonged infusion of thromboxane mimetic. Eur J Int Med 2 : 277 –280, 1992
  13. Sedeek MH, Llinas MT, Drummond H, Fortepiani L, Abram SR, Alexander BT, Reckelhoff JF, Granger JP: Role of reactive oxygen species in endothelin-induced hypertension. Hypertension 42 : 806 –810, 2003[Abstract/Free Full Text]
  14. Wang D, Borrego-Conde LJ, Falck JR, Sharma KK, Wilcox CS, Umans JG: Contributions of nitric oxide, EDHF, and EETs to endothelium-dependent relaxation in renal afferent arterioles. Kidney Int 63 : 2187 –2193, 2003[CrossRef][Medline]
  15. Schnackenberg CG, Wilcox CS: The SOD mimetic tempol restores vasodilation in afferent arterioles of experimental diabetes. Kidney Int 59 : 1859 –1864, 2001[CrossRef][Medline]
  16. Randall MD, McCulloch AI: The involvement of ATP-sensitive potassium channels in beta-adrenoceptor-mediated vasorelaxation in the rat isolated mesenteric arterial bed. Br J Pharmacol 115 : 607 –612, 1995[Medline]
  17. Briones AM, Daly CJ, Jimenez-Altayo F, Martinez-Revelles S, Gonzalez JM, McGrath JC, Vila E: Direct demonstration of beta1- and evidence against beta2- and beta3-adrenoceptors, in smooth muscle cells of rat small mesenteric arteries. Br J Pharmacol 146 : 679 –691, 2005[CrossRef][Medline]
  18. Juncos LA, Ito S, Carretero OA, Garvin JL: Removal of endothelium-dependent relaxation by antibody and complement in afferent arterioles. Hypertension 23 : I54 –I59, 1994[Medline]
  19. Schnackenberg CG, Welch WJ, Wilcox CS: TP receptor-mediated vasoconstriction in microperfused afferent arterioles: Roles of O(2)(-) and NO. Am J Physiol Renal Physiol 279 : F302 –F308, 2000[Abstract/Free Full Text]
  20. Takagawa Y, Berger ME, Tuck ML, Golub MS: Impaired endothelial alpha-2 adrenergic receptor-mediated vascular relaxation in the fructose-fed rat. Hypertens Res 25 : 197 –202, 2002[CrossRef][Medline]
  21. Zhang X, Hintze TH: cAMP signal transduction cascade, a novel pathway for the regulation of endothelial nitric oxide production in coronary blood vessels. Arterioscler Thromb Vasc Biol 21 : 797 –803, 2001[Abstract/Free Full Text]
  22. White R, Bottrill FE, Siau D, Hiley CR: Protein kinase A-dependent and -independent effects of isoproterenol in rat isolated mesenteric artery: Interactions with levcromakalim. J Pharmacol Exp Ther 298 : 917 –924, 2001[Abstract/Free Full Text]
  23. Pourageaud F, Leblais V, Bellance N, Marthan R, Muller B: Role of beta2-adrenoceptors (beta-AR), but not beta1-, beta3-AR and endothelial nitric oxide, in beta-AR-mediated relaxation of rat intrapulmonary artery. Naunyn Schmiedebergs Arch Pharmacol 372 : 14 –23, 2005[CrossRef][Medline]
  24. Fiordaliso F, Cuccovillo I, Bianchi R, Bai A, Doni M, Salio M, De Angelis N, Ghezzi P, Latini R, Masson S: Cardiovascular oxidative stress is reduced by an ACE inhibitor in a rat model of streptozotocin-induced diabetes. Life Sci 79 : 121 –129, 2006[CrossRef][Medline]
  25. Vanden Hoek TL, Li C, Shao Z, Schumacker PT, Becker LB: Significant levels of oxidants are generated by isolated cardiomyocytes during ischemia prior to reperfusion. J Mol Cell Cardiol 29 : 2571 –2583, 1997[CrossRef][Medline]
  26. Mori T, Cowley AW Jr: Angiotensin II-NAD(P)H oxidase-stimulated superoxide modifies tubulovascular nitric oxide cross-talk in renal outer medulla. Hypertension 42 : 588 –593, 2003[Abstract/Free Full Text]
  27. Zhang Z, Rhinehart K, Kwon W, Weinman E, Pallone TL: Ang II signaling in vasa recta pericytes by PKC and reactive oxygen species. Am J Physiol Heart Circ Physiol 287 : H773 –H781, 2004[Abstract/Free Full Text]
  28. Salomonsson M, Arendshorst WJ: Calcium recruitment in renal vasculature: NE effects on blood flow and cytosolic calcium concentration. Am J Physiol 276 : F700 –F710, 1999[Medline]
  29. Facemire CS, Arendshorst WJ: Calmodulin mediates norepinephrine-induced receptor-operated calcium entry in preglomerular resistance arteries. Am J Physiol Renal Physiol 289 : F127 –F136, 2005[Abstract/Free Full Text]
  30. Nishio E, Watanabe Y: The involvement of reactive oxygen species and arachidonic acid in alpha 1-adrenoceptor-induced smooth muscle cell proliferation and migration. Br J Pharmacol 121 : 665 –670, 1997[Medline]
  31. Bleeke T, Zhang H, Madamanchi N, Patterson C, Faber JE: Catecholamine-induced vascular wall growth is dependent on generation of reactive oxygen species. Circ Res 94 : 37 –45, 2004[Abstract/Free Full Text]
  32. Lands AM, Arnold A, McAuliff JP, Luduena FP, Brown TG Jr: Differentiation of receptor systems activated by sympathomimetic amines. Nature 214 : 597 –598, 1967[CrossRef][Medline]
  33. Skeberdis VA: Structure and function of beta3-adrenergic receptors. Medicina (Kaunas) 40 : 407 –413, 2004[Medline]
  34. Kopp UC, DiBona GF: Interaction between neural and nonneural mechanisms controlling renin secretion rate. Am J Physiol 246 : F620 –F626, 1984[Medline]
  35. Buhler FR, Burkart F, Lutold BE, Kung M, Marbet G, Pfisterer M: Antihypertensive beta blocking action as related to renin and age: A pharmacologic tool to identify pathogenetic mechanisms in essential hypertension. Am J Cardiol 36 : 653 –669, 1975[CrossRef][Medline]
  36. Chruscinski A, Brede ME, Meinel L, Lohse MJ, Kobilka BK, Hein L: Differential distribution of beta-adrenergic receptor subtypes in blood vessels of knockout mice lacking beta(1)- or beta(2)-adrenergic receptors. Mol Pharmacol 60 : 955 –962, 2001[Abstract/Free Full Text]
  37. Boivin V, Jahns R, Gambaryan S, Ness W, Boege F, Lohse MJ: Immunofluorescent imaging of beta1- and beta2-adrenergic receptors in rat kidney. Kidney Int 59 : 515 –531, 2001[CrossRef][Medline]
  38. Taira N, Yabuuchi Y, Yamashita S: Profile of beta-adrenoceptors in femoral, superior mesenteric and renal vascular beds of dogs. Br J Pharmacol 59 : 577 –583, 1977[Medline]
  39. Wellstein A, Belz GG, Palm D: Beta adrenoceptor subtype binding activity in plasma and beta blockade by propranolol and beta-1 selective bisoprolol in humans. Evaluation with Schild-plots. J Pharmacol Exp Ther 246 : 328 –337, 1988[Abstract/Free Full Text]
  40. Hajj-Ali AF, Wong PC: Beta adrenoceptor blockade in rabbit inhibits the renin-releasing effect of AT1 receptor antagonist losartan. J Pharmacol Exp Ther 267 : 1423 –1427, 1993[Abstract/Free Full Text]
  41. Yasunari K, Kohno M, Kano H, Minami M, Yoshikawa J: Dopamine as a novel antioxidative agent for rat vascular smooth muscle cells through dopamine D(1)-like receptors. Circulation 101 : 2302 –2308, 2000[Abstract/Free Full Text]
  42. Lu Q, Asico LD, Jones JE, Bai R, Li H, Maines MD, Jose PA: Impaired heme oxygenase activity, increased reactive oxygen species, and high blood pressure in D5 dopamine receptor deficient mice [Abstract]. J Am Soc Nephrol 16 : 163A –2308, 2005[CrossRef]
  43. Yang Z, Asico LD, Yu P, Wang Z, Jones JE, Escano CS, Wang X, Quinn MT, Sibley DR, Romero GG, Felder RA, Jose PA: D5 dopamine receptor regulation of reactive oxygen species production, NADPH oxidase and blood pressure. Am J Physiol Regul Integr Comp Physiol 290 : R96 –R104, 2006[Abstract/Free Full Text]
  44. Mason RP, Walter MF, Trumbore MW, Olmstead EG Jr, Mason PE: Membrane antioxidant effects of the charged dihydropyridine calcium antagonist amlodipine. J Mol Cell Cardiol 31 : 275 –281, 1999[CrossRef][Medline]
  45. Schiffrin EL, Pu Q, Park JB: Effect of amlodipine compared to atenolol on small arteries of previously untreated essential hypertensive patients. Am J Hypertens 15 : 105 –110, 2002[CrossRef][Medline]
  46. Lindholm LH, Carlberg B, Samuelsson O: Should beta blockers remain first choice in the treatment of primary hypertension? A meta-analysis. Lancet 366 : 1545 –1553, 2005[CrossRef][Medline]
  47. Carlberg B, Samuelsson O, Lindholm LH: Atenolol in hypertension: Is it a wise choice? Lancet 364 : 1684 –1689, 2004[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
Y. Chen, A. Pearlman, Z. Luo, and C. S. Wilcox
Hydrogen peroxide mediates a transient vasorelaxation with tempol during oxidative stress
Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2085 - H2092.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
M. Hultstrom, L. Jansson, B. Bodin, and O. Kallskog
Moderate hypothermia induces a preferential increase in pancreatic islet blood flow in anesthetized rats
Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2007; 293(3): R1438 - R1443.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
ASN.2006030212v1
17/12/3347    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wang, D.
Right arrow Articles by Wilcox, C. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wang, D.
Right arrow Articles by Wilcox, C. S.

HOME CURRENT ISSUE ARCHIVES JASN Express ONLINE SUBMISSION AUTHOR INFO
EDITORIAL BOARD SUBSCRIBE FEEDBACK ALERTS HELP