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Circulation Research. 2004;94:1436-1442
Published online before print April 29, 2004, doi: 10.1161/01.RES.0000129578.76799.75
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(Circulation Research. 2004;94:1436.)
© 2004 American Heart Association, Inc.

Integrative Physiology

Enhanced Contractility of Renal Afferent Arterioles From Angiotensin-Infused Rabbits

Roles of Oxidative Stress, Thromboxane Prostanoid Receptors, and Endothelium

Dan Wang, Tina Chabrashvili, Christopher S. Wilcox

From Division of Nephrology and Hypertension and the Cardiovascular-Kidney Institute, Georgetown University, Washington, DC.

Correspondence to Dr Christopher S. Wilcox, Chief, Division of Nephrology and Hypertension, Georgetown University Medical Center, 3800 Reservoir Road, NW, PHC Suite F6003, Washington, DC 20007-2197. E-mail wilcoxch{at}georgetown.edu


*    Abstract
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 *Abstract
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 down arrowResults
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We tested the hypothesis that cyclooxygenase (COX), thromboxane A2 synthase (TxA2-S), thromboxane prostanoid receptors (TP-Rs), or superoxide anion (O2–) mediates enhanced contractions of renal afferent arterioles (Aff) of angiotensin II (Ang II)-infused rabbits. Rabbits were infused with vehicle (sham), Ang II 60 ng·kg–1·min–1 (Ang II 60) or 200 ng·kg–1·min–1 (Ang II 200). There was a selective enhanced vasoconstriction of Affs from Ang II 60 rabbits to Ang II ({Delta}diameter–78±8% versus –43±9%; P<0.01) that was normalized by a TP-R antagonist but not by a superoxide dismutase (SOD) mimetic. Affs from Ang II 200 rabbits had increased (P<0.01) mRNA for COX-2 and enhanced vasoconstriction to Ang II, U-46 619 (TP-R mimetic), and endothelin-1 that was normalized by ifetroban plus tempol together. Endothelium removal enhanced Ang II responses of Affs from sham rabbits but blunted responses from Ang II 200 rabbits and abolished responses to ifetroban. Affs from Ang II 200 rabbits had an endothelium-dependent contraction factor (EDCF) response to that was blunted (P<0.001) by a SOD mimetic or antagonists of COX-1 or TxA2-S but normalized by antagonists of COX-2 or TP-R. Thus, enhanced Ang II responses in Affs from rabbits infused with slow pressor Ang II are mediated independently by O2– in the vascular smooth muscle cells and by an EDCF that is principally a vasoconstrictor prostaglandin generated by COX-2 >–1 activating TP-Rs, whereas enhanced responses in rabbits infused with a lower Ang II dose are dependent on TP-R but not O2–.


Key Words: thromboxane A2 • nitric oxide • cyclooxygenase • superoxide anion • tempol • reactive oxygen species • endothelium-derived contraction factor • endothelium-derived relaxation factor


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Prolonged infusion of angiotensin II (Ang II) at an initially subpressor rate into mice, rats, rabbits, or humans gradually increases blood pressure.1–4 This "slow pressor" response increases renal vascular resistance before hypertension and increases reactivity of the afferent arterioles (Affs) to Ang II in vitro and in vivo.3,5,6

The slow pressor response activates nicotine adenine dinucleotide phosphate (NADPH) oxidase.7 In contrast, rats infused with norepinephrine (NE) do not have oxidative stress or enhanced NADPH oxidase.8 The hypertension and renal vasoconstriction depend on superoxide anion (O2–) because they are prevented by tempol (4-hydroxy-2, 2,3,6-tetramethyl piperidinoxyl), which is a permeant nitroxide superoxide dismutase (SOD) mimetic.3,9,10 There is increased expression in Affs of the p22phox11and increased renal cortical NADPH oxidase activity.6,11

The slow pressor response is accompanied by an increased excretion of prostaglandins (PGs) and thromboxane A2 (TxA2) metabolites.12–14 It is not seen in thromboxane prostanoid receptor (TP-R) knockout mice and is blunted by an antagonist of TP-R or thromboxane A2 synthase (TxA2-S).13,15–19

Affs of rabbits infused with Ang II at a slow pressor rate have a decreased endothelium-dependent relaxation factor (EDRF) response to acetylcholine (ACh) and an increased contractile response to Ang II, but a normal response to NE.6 Tempol corrects the defective relaxation to ACh and moderates the enhanced response to Ang II. One aim of the present study was to compare vasoconstrictor responsiveness of Affs of Ang II-infused rabbits with that to U-46 619 (TP-R agonist) and endothelin-1 (ET-1) and to determine the roles of O2– and the endothelium. A second aim was to investigate the role of TP-Rs. We compared responses from rabbits infused with Ang II at 200 and at 60 ng·kg–1·min–1 (Ang II 200 and 60) because Ang II 60 rabbits do not have hypertension, oxidative stress, or enhanced expression of mRNA for p22phox in their Affs.6

O2– can impair ACh-induced relaxation by bioinactivation of nitric oxide (NO) and generation of an endothelium-dependent contraction factor (EDCF) that depends on TP-R.20–28 Ligands for the TP-R include prostaglandin endoperoxides (PGH2) generated by cyclooxygenase (COX) 1 and 2, TxA2, isoprostanes (Iso) generated nonenzymatically by interaction of O2– with arachidonate, and COX metabolites of hydroxyeicosatetraenoic acids.29 We tested the hypothesis that endothelium-dependent generation of O2– and/or products activating TP-Rs mediate the EDCF response in Affs from rabbits infused with Ang II. We tested the role of O2– and Isos from the effects of tempol, of TP-R from ifetroban,17 and COX-1, COX-2, and TxA2-S from the effects of paracoxib (P), SC-560 (SC), and OKY-046 (OKY), respectively.30–33


*    Methods
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 *Methods
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Animal Protocols
The study was approved by the institutional Animal Care and Use Committee. Male New Zealand White rabbits (1.6 to 1.8 kg; n=104; Covance Inc., Denver, Pa) were maintained on standard chow (Na+ content 0.4 g·100 g–1). Rabbits (n=6 per group) were implanted subcutaneously with sterile osmotic minipumps (Alzet; DURET Corporation, Cupertino, Calif) to infusion vehicle (0.154 mol/L NaCl; Sham) or human Ang II (Peninsula Laboratories, San Carlos, Calif). All studies were performed under a protocol approved by the Georgetown University Animal Care and Use Committee.

Isolation and Microperfusion of Rabbit Afferent Arterioles
Rabbits were anesthetized with xylazine (10 mg·kg–1 intramuscularly), ketamine (50 mg·kg–1 intramuscularly), and pentobarbital sodium (10 mg·kg–1 intravenous), followed by heparin (1000 units intravenous) for anticoagulation. Microdissection and microperfusion of Aff was as described previously.6,10 Only arterioles showing basal tone and >50% contraction to NE at the beginning and end were selected. One Aff was used from each animal. Drugs were added to the bath.

Experimental Protocol
The first aim was to compare the contractile responses of Affs from Ang II 60 and Ang II 200 rabbits with sham rabbits (n=6 per group). Responses to superfusion with NE (10–7 mol/L), Ang II (10–8 mol/L), U-46 619 (10–8 M, a TP-R agonist), or endothelin 1 (ET-1, 10–8 mol/L) were related to a standard contraction with NE (10–7 mol/L) plus 40 mmol/L KCl (NAK). Responses were maintained for 3 minutes and allowed to recover for 10 minutes. Because Affs from Ang II 60 rabbits had an enhanced response to Ang II, but normal responses to U-46 619 and ET-1, subsequent protocols with Ang II 60 rabbits were confined to the Ang II response.

The second aim was to evaluate the effects of blockade of TP-R on contractile responses. Affs were incubated for 30 minutes with ifetroban, which blocks completely contractions to U-46 619.17,34,35 The protocol followed series 1 in a bath containing ifetroban (10–6 mol/L). There were 3 groups of rabbits (n=6 per group).

The third aim was to evaluate the effects of metabolism of O2– on contractile responses. Affs were incubated for 20 minutes with tempol, which, at 10–4 M, blocks fully the contractile responses to O2– generated by a quinolone or by prolonged administration of U-46 619.31,34 The protocol followed series 1 in a bath containing tempol (10–4 mol/L). There were 3 groups of rabbits (n=6 per group).

The fourth aim was to evaluate the effects of TP-R blockade on contractile responses in Affs incubated with tempol. The protocol followed series 1 in arterioles preincubated for 20 to 30 minutes in a bath containing 10–4 mol/L tempol and 10–6 mol/L ifetroban. There were 3 groups of rabbits (n=6 per group).

The fifth aim was to contrast the effect of removal of the endothelium (Endo–) on responses to NE and Ang II of arterioles from sham and Ang II 200-infused rabbits. The endothelium was removed by luminal perfusion with compliment and an antibody to factor VIII, which prevents relaxation to acetylcholine (ACh) but not sodium nitroprusside.10,34

The sixth aim was to evaluate the effects of an SOD mimetic or an antagonist to COX-1 or COX-2 and TxA2-S or TP-R on EDCF responses to ACh of Affs from Ang II 200 rabbits. ACh given to preconstricted Affs from Ang II 200 rabbits caused constriction at >10–5 mol/L. This indicated an EDCF. The EDRF response of Affs from normal rabbits is blocked by superfusion with a combination of NG-Nitro-L-arginine (LNNA) (10–5 mol/L; NO synthase inhibitor) and 14,15 epoxyeicoa-5(Z)-enoic acid (EEZE) (10–5 mol/L; epoxyeicosatrienoic acid antagonist).35 Therefore, these EDCF studies (ACh 10–6 to 10–3 mol/L responses) were undertaken without preconstriction in vessels superfused with LNNA and EEZE. Subgroups were superfused with SC-560 (10–7 mol/L) or paracoxib (10–6 mol/L) to assess the contributions of COX-1 and COX-2, or with ifetroban (10–6 mol/L) or OKY-046 (10–5 mol/L) to assess the roles of TP-R and TxA2 or with tempol (10–4 mol/L) to assess the role of O2– and Isos.

Analysis of mRNA in Individual Isolated Afferent Arterioles
The methods for microdissection, mRNA extraction, and real-time polymerase chain reaction analysis have been published.6,36 The primers used for amplification of COX-1, COX-2, TP-R, and TxA2-S are shown in online Table I (available at http://circres.ahajournals.org). The amplified products were sequenced. The real-time polymerase chain reaction primers and probes were designed from the sequenced regions.

Drugs and Solutions
Solutions were prepared fresh daily. U-46 619 (Cayman Chemical, Ann Arbor, Mich) was evaporated under N2 and reconstituted using 97% ethanol, and 55 mmol/L Tris purchased from Sigma (St. Louis, Mo). Earle deficient basal medium solution was used for dissections. It contains 8.89g·L–1 BME powder, 26 mol/L NaHCO3, 2 mmol/L L-glutamine, and 5% bovine serum albumin (5%; pH 7.40 to 7.45). MEM solution (10.1g MEM powder, 5 mol/L NaHCO3, 10 mmol/L HEPES, 14 mol/L NaOH, and 1 g glucose/L) containing 5% bovine serum albumin was used for perfusion and with 0.15% bovine serum albumin for superfusion.6,22 Tempol (4-hydroxy TEMPO), ACh, NE, LNNA, goat–antihuman F8-Rag antibody, guinea pig complement, and SC-560 were purchased from Sigma (St. Louis, Mo). Ifetroban (BMS-180291) was gift from Dr Martin Ogletree (Bristol Myers Squib, Princeton, NJ), paracoxib was from Pfizer-Pharmacia Pharmaceuticals, OKY-056 was from ONO Pharmaceutical, Osaka, Japan, and 14, 15-epoxyeicosa-5(Z)-enoic acid was from John R. Falck (University of Texas).

Calculations of Results and Statistics
The change of luminal diameter to an agonist (in series 1 to 5) was expressed as percent change compared with NAK (series 1 to 5), given by: Down


The change of luminal diameter to ACh (10–6 to 10–3 M, in series 6) was expressed as percent change compared with NE (10–7M), given by: Down


where C0 is the basal diameter, C is the minimal diameter with NAK (series 1 to 5) or NE (aim 6), C1 is the luminal diameter to agonist (series 1 to 5), and C2 is the luminal diameter to ACh (series 6).

Statistical tests used 2-factor repeated-measures analysis of variance (ANOVA). When appropriate, post hoc comparisons between groups were made with Student t tests. Statistical significance with Bonferroni correction for 4 genes was P<0.05/4=0.0125.36 Data are presented as mean±SD.


*    Results
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 *Results
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The diameters of Affs were similar in the basal state (sham: 17.3±1.2 µm versus 17.2±1.1 µm [Ang II 60] versus 16.9±1.2 µm [Ang II 200]; NS) and during NAK (sham: 6.5±0.5 versus 6.6±0.8 [Ang II 60] versus 6.3±0.6 µm [Ang II 200]; NS). Ang II 60 does not change MAP, oxidative stress, renal cortical NADPH oxidase activity, or mRNA for p22phox in Affs, whereas Ang II 200 increases all these parameters significantly.37

In series 1, Affs from Ang II 60 rabbits had a significantly enhanced contraction to Ang II (change in luminal diameter, –78±8% versus –43±9%; P<0.001) but a normal response to NE, U-46 619, and ET-1 (Figure 1). Affs from Ang II 200 rabbits had a normal response to NE but an enhanced response to Ang II (–110±10% versus –43±9%; P<0.001), U-46 619 (–89±8% versus –55±4%; P<0.01), and ET-1 (–110±10% versus –63±6%; P<0.001). Responses to Ang II, U-46 619, and ET-1 were significantly (P<0.001) greater than those from Ang II 60 rabbits.



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  		Figure 1. Mean±SD values for contractile responses of afferent arterioles in sham, Ang II 60, and Ang II 200 rabbits. The percentage changes in luminal diameter are compared with NAK during bath addition of norepinephrine (10–7 mol/L), Ang II (10–8 mol/L), U-46 619 (10–6 mol/L), and endothelin I (10–8 mol/L). Compared with sham rabbits: **P<0.01; ***P<0.001. Compared with Ang II 60 rabbits: {dagger}P<0.05; {dagger}{dagger}P<0.01.

In series 2, ifetroban did not affect the basal luminal diameter (17±1 µm [vehicle] versus 18±2 µm [ifetroban]; NS) or the change with NAK (–66±6% [vehicle] versus –61±6% [ifetroban]; NS) but abolished the response to U-46 619. Ifetroban did not modify contractile responses in sham rabbits (Figure 2) or NE responses in Ang II 60 rabbits (Figure 3) but normalized responses to Ang II in Ang II 60 Affs (sham: –43±9% versus –78±8% [Ang II 60 + vehicle] versus 47±9% [Ang II 60 + ifetroban]) (Figure 3). Ifetroban did not modify responses to NE in Ang II 200 rabbits but blunted responses to Ang II (–85±8% versus –110.0±10%; P<0.01) and normalized responses to ET-1 (–80±8% versus –110±10%; P<0.001) (Figure 4).



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  		Figure 2. Mean±SD values for contractile responses of afferent arterioles from sham rabbits during bath addition of vehicle, ifetroban (10–6 mol/L), and/or tempol (10–4M). Compared with vehicle: ***P<0.001.



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  		Figure 3. Mean±SD values for contractile responses of afferent arterioles from Ang II 60 rabbits during bath addition of vehicle, ifetroban, and/or tempol, and responses in sham rabbits (S). Compared with vehicle: **P<0.01. Compared with sham rabbits: {dagger}{dagger}P<0.01.



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  		Figure 4. Mean±SD values of contractile responses of afferent arterioles from Ang II-infused 200 rabbits during bath addition of vehicle, ifetroban, and/or tempol, and responses in sham rabbits (S). Compared with vehicle: *P<0.05; **P<0.01; ***P<0.001. Compared with sham rabbits: {dagger}P<0.05; {dagger}{dagger}P<0.01; {dagger}{dagger}{dagger}P<0.001.

In series 3, tempol did not modify basal luminal diameter (18±1 µm) or diameter during NAK (6±1 µm). Tempol did not modify responses from sham rabbits (Figure 2) or responses to NE or Ang II in Ang II 60 rabbits (Figure 3). However, tempol blunted responses to Ang II in Ang II 200 Affs (–62±8 versus –110±10; P<0.001) and normalized responses to U-46 619 (–60±6% versus –89±9%; P<0.001) and ET-1 (–59±7% versus –110±10%; P<0.001). During tempol, the luminal diameter responses to NE, U-46 619, and ET-1 did not differ significantly among the 3 groups of rabbits, but the response to Ang II in arterioles from Ang II 60 (–67±13%; Figure 3) and Ang II 200 rabbits (–62±8%; Figure 4) remained significantly greater than in arterioles from sham rabbits (–43±9%; P<0.01 versus Ang II rabbits).

In series 4, ifetroban plus tempol did not modify the basal luminal diameters (18±1 µm; NS) or the responses from sham-infused rabbits except to abolish the response to the U-46 619 (Figure 2). Tempol plus ifetroban added to arterioles from Ang II 60 rabbits, like ifetroban alone, normalized the response to Ang II (Figure 3). When added to arterioles from Ang II 200 rabbits, the combination reduced the response to Ang II significantly more than tempol alone (–47±6% versus –62±8%; P<0.05) to values of sham-infused rabbits (–43±9%; NS). Ifetroban plus tempol, like tempol alone, normalized the response to ET-1.

In series 5, endothelium removal of arterioles from sham rabbits did not affect the basal diameter (18±1 µm versus 17±1 µm; P=NS) or the response to NE but enhanced the response to Ang II (Endo+: –43±9% versus Endo: –69±8%; P<0.001) (Figure 5). Addition of ifetroban or tempol to these Endo– arterioles did not modify the response to Ang II (Figure 5). Endothelium removal of arterioles from Ang II 200 rabbits likewise did not modify the basal luminal diameter (17±1 µm versus 17±2 µm; P=NS) or the response to NE but diminished the response to Ang II (Endo+: –110±10% versus Endo–: –96±3%; P<0.05) (Figure 6). This was unaffected by ifetroban but was reduced further (–75±9%; P<0.05) by tempol to values not significantly different from the response to Ang II of de-endothelialized vessels of sham-infused rabbits (–69±10%; NS).



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  		Figure 5. Mean±SD values for contractile responses of afferent arterioles from sham rabbits comparing endothelium intact (Endo+) with endothelium removal (Endo–) during bath addition of vehicle, ifetroban, or tempol. Compared with Endo+: **P<0.01.



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  		Figure 6. Mean±SD values for contractile responses of afferent arterioles from Ang II 200 rabbits comparing Endo+ with Endo– during bath addition of vehicle, ifetroban, or tempol and for responses of Endo– vessels in sham rabbits (S). Compared with arterioles from Ang II 200 rabbits with Endo(+): *P<0.05; **P<0.01. Compared with afferent arterioles from sham rabbits with Endo–: {dagger}{dagger}{dagger}P<0.001.

In series 6, the abundance of mRNA in individual afferent arterioles from Ang II 200 rabbits for COX-1 and TP-R was unchanged but was increased 2.6- fold for COX-2 (P<0.01; online Table II). The mRNA for TxA2-S was less than the limit for quantification.

Affs from Ang II 200 rabbits had an impaired EDRF response and a contractile response to higher concentration of ACh that was abolished by endothelium removal (Figure 7A). This EDCF response was analyzed further in vessels without preconstriction and with EDRF/NO and endothelium-derived hyperpolarizing factor responses blocked with LNNA plus EEZE.10 These vessels demonstrated a graded contractile response to ACh that was not present in vessels from Sham rabbits (–42±4% versus –5±3% in sham, P<0.001). This was abolished by removing the endothelium (Figure 7B). The contractile response was reduced by bath addition of tempol (Figure 7B), SC-560 (Figure 7C), or OKY-046 (Figure 7D) and was abolished by bath addition of paracoxib (Figure 7C) or ifetroban (Figure 7D).



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  		Figure 7. Mean±SD values for acetylcholine responses of afferent arterioles. A, Vasodilation responses in preconstricted vessels, whereas other panels depict vasoconstriction responses. A, Compared with sham rabbits: {dagger}{dagger}P<0.01; {dagger}{dagger}{dagger}P<0.005. Compared with Ang II 200 rabbits with LNNA plus EEZE in Endo+ vessels: *P<0.05; **P<0.01; ***P<0.005. B, Before addition of ACh.


*    Discussion
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The main new findings of this study are that Affs from rabbits infused at a nonpressor rate of Ang II that does not cause oxidative stress or endothelial dysfunction have a selective increase in the response to Ang II. This is normalized by a TP-R antagonist. Affs from rabbits infused at a rate that increases MAP by 38 mm Hg over 12 days and causes oxidative stress and enhanced expression of p22phox have enhanced responses to Ang II, U-46 619, and ET-1 but not to NE. A TP-R antagonist and a SOD mimetic together are required to normalize the Ang II response in Ang II 200 arterioles.6 Endothelium removal enhanced Ang II responses of Affs from sham rabbits but blunted it in those from Ang II 200 rabbits. Ifetroban was ineffective after endothelium removal. Affs from Ang II 200 rabbits have a 2.6-fold increase in the mRNA for COX-2 and an EDCF response to ACh that is reduced by an antagonist to COX-1 or TxA2-S or by an SOD mimetic, but is prevented fully by an antagonist to COX-2 or TP-R.

U-46 619 superfused over Affs from normal rabbits for a longer period of 15 to 20 minutes causes contractions that are moderated by NO and are promoted by O2–.34 Therefore, the duration of agonist exposure was limited in this series to obviate these effects in normal vessels.

The present study confirms that infusion of Ang II at a slow pressor rate enhances responses of Affs to Ang II and shows further that there is an enhanced response to U-46 619 and ET-1, but not to NE alone or NE plus high [K+].6 The preservation of responses to NE is unexplained but suggests that the enhanced vascular reactivity to Ang II, U-46 619, and ET-1 is not likely to be secondary to structural vascular amplification.38

Role of O2–
Oxidative stress in large vessels activates phospholipase A2.11 and TxA2-S and enhances the responses to TP-R activation,20,21,25,27,39 likely by stabilizing the beta isoform of the TP-R.40 Furthermore, oxidative stress generates ET-1 in vascular smooth muscle cells (VSMCs).41 The present study shows that oxidative stress enhances the contractile responses of the major resistance vessel of the kidney not only to a TP-R agonist but also to Ang II and ET-1.

Antioxidants prevent enhanced endothelin generation in rats infused with slow pressor Ang II.9 Activation of endothelin and TP-Rs enhance myogenic constriction in VSMCs from hypertensive animals.42,43 Ang II infusion upregulates Ca++ channel activity in blood vessels, whereas antioxidants improve VSMC Ca++ uptake into the sarcoplasmic reticulum in animals with oxidative stress.43,44 The finding that Affs from Ang II 200 rabbits have exaggerated responses to Ang II, U-46 619, and ET-1 that are partially or fully corrected by tempol and that the effects of tempol survive de-endothelialization indicate that oxidative stress increases the reactivity of the renal afferent arteriole VSCMs to a number of agonists. However, there is selectivity because responses to NE and high [K+] are unaffected.

Role of TP Receptors
The identified ligands for TP-Rs are PGH2, TxA2, Isos such as 8-Iso-PGF2{alpha}, COX metabolites of HETEs, and the stable agonist U-46 619.1,29 Ang II infusion activates phospholipase A2 (PLA2) and increases expression of COX-2 in VSMCs. In contrast, COX-2 in the macula densa is suppressed.45–47 Ang II also increases the generation of TxA2 and 8-Iso PGF2{alpha}.11,12,14,48 The present study demonstrates that Ang II 200 upregulates the mRNA for COX-2 in Affs. Thus, an Ang II infusion likely increases all the endogenous ligands for the TP-R in the Aff. Moreover, oxidative stress enhances vasoconstriction to PGH2.27 The importance of these TP-R ligands in the response to Ang II is apparent from the effect of an antagonist of TxA2-S or TP-R, or deletion of the gene for TP-R, to moderate or prevent hypertension and renal vasoconstriction during a slow pressor response to Ang II.15,18,19,49

The effect of ifetroban to normalize responses to Ang II in Ang II 60 Affs can be dissociated from O2– because these rabbits do not have increased lipid peroxidation or increased NADPH oxidase activity in their kidneys or increased mRNA expression for p22phox in their Affs.6 Moreover, incubation of Affs from Ang II 60 or Ang II 200 rabbits with tempol did not moderate the effect of ifetroban to blunt contraction to Ang II. Presumably the ligand for the TP-R is not an Iso but a COX metabolite.

Ifetroban blunted the response to Ang II and to ET-1 in arterioles from Ang II 200 rabbits. ET-1 releases PGH2 and/or TxA2 from VSMCs of the rat, from damaged coronary arteries of the pig, and from postischemic rat heart.37,42,50 A component of renal microvascular contraction during prolonged Ang II-induced hypertension entails generation of ET-1 with release of PGH2 and TxA2 that activate TP-Rs on VSMCs.

Role of Endothelium
A novel finding is that the afferent arteriolar responses to Ang II depend on the endothelium. Endothelium removal increased contractile responses to Ang II by {approx}40% in normal arterioles, likely reflecting NO generation.51 In contrast, endothelium removal reduced Ang II responses in vessels from Ang II 200 rabbits and prevented the blunting of the Ang II response by ifetroban, but not tempol. Thus, Ang II releases an EDCF from the endothelium of these vessels. This EDCF is distinct from O2– but entails a COX-2 product that activates TP-Rs on VSMCs.

Perspective
Infusion of Ang II at a slow pressor rate causes preglomerular vasoconstriction.3 This may contribute to hypertension because the tone of the Aff determines the transmission of pressure to the glomeruli and tubules. Indeed, during prolonged subpressor infusion of Ang II, antioxidants, ifetroban, or deletion of the gene for TP-R all prevent preglomerular vasoconstriction and hypertension.3,9,15,18,19,49,52 The present studies have shown that the enhanced preglomerular vasoconstriction is intrinsic to the afferent arteriole where oxidative stress acts on VSMCs to promote contractile responses to a range of pathophysiologic agonists. These are reinforced by an EDCF that depends predominantly on COX-2 products that activate TP-Rs on VSMCs. Lower rates of Ang II infusion enhance Ang II responses by a mechanism that depends selectively on TP-Rs.


*    Acknowledgments
 
This work was supported by grants from the NIDDK (DK-36079 and DK-49870), the NHLBI (HL68686), by funds from George E. Schriener Chair of Nephrology, and by a grant-in-aid to Dan Wang from the NKF, Nations Capital Affiliate. We appreciate the assistance of Lillian Borrego-Conde. We are grateful to Martin Ogletree, PhD (Bristol Myers Squib, Princeton, NJ) for a generous gift of ifetroban, to Pfizer Pharmacia for a gift of paracoxib, to John R Falck (Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Tex) for a gift of EEZE, and to Kate Scata for preparation of the manuscript.


*    Footnotes
 
Original received October 30, 2003; revision received April 12, 2004; accepted April 15, 2004.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Ames RP, Borkowski AJ, Sicinski AM, Laragh JH. Prolonged infusions of angiotensin II and norepinephrine and blood pressure, electrolyte balance, aldosterone and cortisol secretion in normal man and in cirrhosis with ascites. J Clin Invest. 1965; 44: 1171–1186.[Medline] [Order article via Infotrieve]
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  3. 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. 2002; 13: 2860–2868.[Abstract/Free Full Text]
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