Activation of the ET-1/ETA Pathway Contributes to ErectileDysfunction Associated with Mineralocorticoid Hypertension

Fernando S. Carneiro, MSc,*† Kênia P. Nunes, PhD,*‡ Fernanda R.C. Giachini, MSc,*†

Victor V. Lima, BSc,* Zidonia N. Carneiro,* Edson F. Nogueira, MD,* Romulo Leite, PhD,*§

Adviye Ergul, MD, PhD,* William E. Rainey, PhD,* R. Clinton Webb, PhD,* and Rita C. Tostes, PhD*†

*Medical College of Georgia, Department of Physiology, Augusta, GA, USA; †Institute of Biomedical Sciences, Universityof Sao Paulo, Pharmacology, Sao Paulo, Brazil; ‡Federal University of Minas Gerais, Department of Physiology, BeloHorizonte, Brazil; §Federal University of Ouro Preto, School of Pharmacy, Ouro Preto, Brazil

DOI: 10.1111/j.1743-6109.2008.01009.x

A B S T R A C T

Introduction. The cavernosal tissue is highly responsive to endothelin-1 (ET-1), and penile smooth muscle cells notonly respond to but also synthesize ET-1.Aim. Considering that ET-1 is directly involved in end-organ damage in salt-sensitive forms of hypertension, wehypothesized that activation of the ET-1/ETA receptor pathway contributes to erectile dysfunction (ED) associatedwith mineralocorticoid hypertension.Methods. Wistar rats were uninephrectomized and submitted to deoxycorticosterone acetate (DOCA)-salt treat-ment for 5 weeks. Control (Uni [uninephrectomized control]) animals were uninephrectomized and given tap water.Uni and DOCA-salt rats were simultaneously treated with vehicle or atrasentan (ETA receptor antagonist, 5 mg/Kg/day). Cavernosal reactivity to ET-1, phenylephrine (PE), ETB receptor agonist (IRL-1620) and electric fieldstimulation (EFS) were evaluated in vitro. Expression of ROCKa, ROCKb, myosin phosphatase target subunit 1(MYPT-1), and extracellular signal-regulated kinase 1/2 (ERK 1/2) were evaluated by western blot analysis. ET-1and ETA receptor mRNA expression was evaluated by real-time reverse-transcriptase polymerase chain reaction.Voltage-dependent increase in intracavernosal pressure/mean arterial pressure (ICP/MAP) was used to evaluateerectile function in vivo.Main Outcome Measure. ETA receptor blockade prevents DOCA-salt-associated ED.Results. Cavernosal strips from DOCA-salt rats displayed augmented preproET-1 expression, increased contractileresponses to ET-1 and decreased relaxation to IRL-1620. Contractile responses induced by EFS and PE wereenhanced in cavernosal tissues from DOCA-salt hypertensive rats. These functional changes were associated withincreased activation of the RhoA/Rho-kinase and ERK 1/2 pathways. Treatment of rats with atrasentan completelyprevented changes in cavernosal reactivity in DOCA-salt rats and restored the decreased ICP/MAP, completelypreventing ED in DOCA-salt rats.Conclusion. Activation of the ET-1/ETA pathway contributes to mineralocorticoid hypertension-associated ED.ETA receptor blockade may represent an alternative therapeutic approach for ED associated with salt-sensitivehypertension and in pathological conditions where increased levels of ET-1 are present. Carneiro FS, Nunes KP,Giachini FRC, Lima VV, Carneiro ZN, Nogueira EF, Leite R, Ergul A, Rainey WE, Webb RC, and TostesRC. Activation of the ET-1/ETA pathway contributes to erectile dysfunction associated with mineralocor-ticoid hypertension. J Sex Med 2008;5:2793–2807.

Key Words. Endothelin-1; ETA Receptor; Rho-kinase; DOCA-salt Hypertension; Erectile Dysfunction; CorpusCavernosum; Atrasentan

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Introduction

The balance between contracting and relaxingfactors controls smooth muscle tone of both

the penile vasculature and corpora cavernosa, andthereby, determines the flaccid or erectile state ofthe penis [1–3]. Neurogenic nitric oxide (NO) isconsidered the most important factor for relax-ation of penile vessels and corpus cavernosum andconsequently, penile erection. On the other hand,it is now generally accepted that the penis is keptin the flaccid state mainly via a tonic activity ofendothelins and noradrenaline, derived from thesympathetic nervous system, whereas other trans-mitters or vasoactive molecules, such as neurop-eptide Y, angiotensin II (AngII) and contractileprostanoids would play only a minor role [1–3].Numerous etiological studies have established a

positive clinical association between hypertensionand erectile dysfunction (ED) [4–6]. A high preva-lence of ED is observed among hypertensive menand antihypertensive treatment can also contributeto ED [7]. We previously reported that deoxycor-ticosterone and salt (DOCA-salt) hypertensive ratsdisplay ED that was characterized by decreasederectile response or decreased intracavernosalpressure/mean arterial pressure (ICP/MAP) ratiofollowing electrical stimulation of the major pelvicganglion [8].Granchi and colleagues made the intriguing

observation that penile smooth muscle cells notonly respond to, but also synthesize endothelin-1(ET-1) [9]. The authors reported that both endot-helial and smooth muscle cells from adult and fetalhuman penis express ET-1, endothelin convertingenzyme 1 and predominantly the endothelinreceptor A subtype (ETA) [9]. In addition, ET-1expression in human fetal penile smooth musclecells is increased by several stimuli, such as trans-forming growth factor-b1, interleukin-1a, ET-1itself, and prolonged hypoxia [9–11].ET-1 receptors, both ETA and endothelin

receptor B subtype (ETB), have been reported tobe found in the cavernosal tissue of humans[9,12,13] and in several animal species (rabbit[13–15], rat [16,17], and bovine [18]). In the rat,ET-1 injection into the penile vasculature inducesboth vasoconstriction and vasodilation [19]. ET-1-induced cavernous vasoconstriction in vivo seemsto be predominantly mediated by the ETA recep-tor, since ETA, but not ETB receptor antagonists,prevent ET-1-induced decrease of intracavernosalpressure, both in basal conditions or upon sub-maximal ganglionic stimulation [17]. In the penis,

ET-1/ETA receptor-mediated biological effectsinvolve activation of the inositol trisphosphate/calcium (Ca2+) and RhoA/Rho-kinase signalingpathways [20,21]. Alternatively, ET-1/ETBreceptor-mediated vasodilation occurs via releaseof NO from cavernous endothelial cells [19].ET-1 not only induces vasoconstriction, but it

also stimulates the production of growth factors,deposition of extracellular matrix components,induction of reactive oxygen species, and expres-sion of adhesion molecules by endothelial cells.In addition, ET-1 activates transcriptional factorsresponsible for the coordinated increase in manycytokines and enzymes, which enhance inflamma-tion and tissue damage [22,23]. ET-1 is importantin salt-sensitive forms of experimental hyperten-sion, such as DOCA-salt hypertensive rats [22,23].In these animals, ET-1 receptor antagonists havemoderate blood pressure lowering efficacy and arenot effective antihypertensive agents. However,treatment of these animals with either selectiveETA or dual ETA/ETB receptor antagonists resultsin regression of vascular damage, endothelial dys-function, cardiac and vascular hypertrophy, as wellas a delay in the progression of renal injury, thusimproving survival [22,23].Considering that ET-1 is important in salt-

sensitive forms of experimental hypertension, wehypothesized that ED in DOCA-salt hyperten-sion is associated with increased activation of theET-1/ETA receptor pathway in penises fromhypertensive rats, and that blockade of ET-1actions ameliorates ED in this salt-sensitivemodel of hypertension. To test our hypothesis,we have evaluated changes in corpora cavernosareactivity to ET-1 and an ETB receptor agonist(IRL-1620), as well as responses to other con-tractile and relaxant stimuli. In addition, theeffects of chronic treatment with an ETA receptorantagonist (atrasentan) on both cavernosal reac-tivity (in vitro) and erectile responses (in vivo)were determined.

Methods

AnimalsMale Wistar rats (8 weeks-old, 230–250 g; Harlan,Indianapolis, IN) were used in all studies. Allprocedures were performed in accordance withthe Guiding Principles in the Care and Use ofAnimals, approved by the Medical College ofGeorgia Committee on the Use of Animals inResearch and Education. The animals were

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housed four per cage on a 12-hour light/dark cycleand fed a standard chow diet with water ad libitum.

DOCA-Salt Hypertension and Arterial BloodPressure MeasurementsDOCA-salt hypertension was induced as previ-ously described [8]. Briefly, rats were unilaterallynephrectomyzed and deoxycorticosterone-acetate(DOCA; 200 mg/Kg) silastic pellets were im-planted subcutaneously in the scapular region.DOCA rats received water containing 1% NaCland 0.2% KCl, for 5 weeks. Control (Uni [unine-phrectomized control]) rats were also uninephre-ctomized, received silastic pellets without DOCAand tap water. Animals simultaneously receivedeither the ETA antagonist atrasentan (5 mg/day/kgof body weight, p.o. per gavage) or vehicle for 5weeks. Systolic blood pressure (SBP) was mea-sured in unanaesthetized animals by tail cuff usinga RTBP1001 rat-tail blood pressure system (KentScientific Corporation, CT). At the end of 5 weeksof treatment, animals were anaesthetized with ket-amine (100 mg/Kg) and xylazine (2.9 mg/Kg) andMAP was determined by a direct method. Bothnormotensive and DOCA-salt hypertensive rats,treated with vehicle or atrasentan, were submittedto the experimental procedures described below.

Functional Studies in Cavernosal StripsAfter carbon dioxide (CO2) euthanasia, peniseswere excised, transferred into ice-cold Krebs buffer(130 mM NaCl, 14.9 mM NaHCO3, 5.5 mMdextrose, 4.7 mM KCl, 1.18 mM KH2PO4,1.17 mM MgSO4·7H2O, 1.6 mM CaCl2·2H2O,and 0.026 mM ethylenediaminetetraacetic acid(EDTA)), and dissected to remove the tunica albug-inea, as previously described [24]. One crural strippreparation (1 ¥ 1 ¥ 10 mm) was obtained fromeach corpus cavernosum (two crural strips fromeach penis). Cavernosal strips were mounted in4-mL myograph chambers (Danish Myo Technol-ogy, Aarhus, Denmark) containing buffer at 37°Ccontinuously bubbled with a mixture of 95% O2and 5% CO2. The tissues were stretched to aresting force of 3.0 millinewtons (mN) and allowedto equilibrate for 60 minutes. Changes in isometricforce were recorded using a PowerLab/8SP dataacquisition system (Chart software, version 5.0;ADInstruments, Colorado Springs, CO, USA). Toverify the contractile ability of the preparations, ahigh potassium chloride (KCl) solution (120 mM)was added to the organ baths at the end of theequilibration period. Cumulative concentration-response curves to sodium nitroprusside (SNP)

(10-8 M to 3 ¥ 10-3 M; NO donor andendothelium-independent vasodilator) and IRL-1620 (10-10 M to 10-6 M; selective ETB agonist)were performed in cavernosal strips contractedwith phenylephrine (PE; 10-5 M; a1-adrenergicreceptor agonist). Cumulative concentration-response curves to ET-1 (10-10 M to 10-6 M),IRL-1620 (10-10 M to 10-6 M), and PE (10-9 M to3 ¥ 10-4 M) were also performed with strips underbasal tone. Electrical field stimulation (EFS) wasapplied to strips placed between platinum pin elec-trodes attached to a stimulus splitter unit (Stimu-Splitter II), which was connected to a Grass S88stimulator (Astro-Med West Warwick, RI). EFSwas conducted at 20 V, 1-ms pulse width and trainsof stimuli lasting 10 s at varying frequencies(1–64 Hz). To evaluate adrenergic nerve-mediatedresponses, strips were incubated with Nw-nitro-L-arginine methyl ester (L-NAME) (10-4 M)plus atropine (10-6 M) before EFS was per-formed. When atropine and L-NAME were used,drugs were administered 30–45 minutes beforeconcentration-response or frequency-responsecurves were performed. Time control experimentswere performed to determine force developmentof cavernosal strips not related to the effects ofeach antagonist/inhibitor. Control solutions con-taining vehicle levels of ethanol and dimethyl sul-foxide were also used throughout the experimentalprotocols.

RNA Extraction, cDNA Synthesis, and QuantitativeReal-Time Reverse-Transcriptase PolymeraseChain ReactionTotal RNA was extracted from cavernosal stripsusing the RNeasy kit (Qiagen Sciences, MD,USA). The quantity, purity and integrity of allRNA samples were determined by NanoDropspectrophotometers (NanoDrop Technologies,Wilmington, DE, USA). One microgram oftotal RNA was reverse transcribed in a finalvolume of 50 mL using the high-capacity cDNAarchive kit (Applied Biosystems, Foster City,CA, USA), and single-strand cDNA was storedat -20°C. Primers for preproendothelin-1(preproET-1) (N° Rn00561129_m1), ETAreceptor (N° Rn00561137_m1) and ETB(N° Rn00569139_m1) mRNA were obtainedfrom Applied Biosystems. Real-time reverse-transcriptase polymerase chain reaction (qPCR)reactions were performed using the 7,500 fastReal-Time PCR system (Applied Biosystems) in atotal volume of 20 mL reaction mixture followingthe manufacturer’s protocol, using the TaqMan®

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fast universal PCR master mix (2¥) (Applied Bio-systems), and 0.1 mM of each primer. Negativecontrols contained water instead of first-strandcDNA. Each sample was normalized on the basisof its 18S ribosomal RNA content. The 18S quan-tification was performed using a TaqMan riboso-mal RNA reagent kit (Applied Biosystems)following the manufacturer’s protocol. Relativegene expression for preproET-1 mRNA was nor-malized to a calibrator that was chosen to be thebasal condition (samples from Uni) for each timepoint. Results were calculated with the DDCtmethod and expressed as n-fold differences inpreproET-1 gene expression relative to 18S rRNAand to the calibrator and were determined asfollows: n-fold = 2-(DCt sample - DCt calibrator), where theparameter Ct (threshold cycle) is defined as thefractional cycle number at which the PCR reactionreporter signal passes a fixed threshold. DCt valuesof the sample and the calibrator were determinedby subtracting the average Ct value of the tran-script under investigation from the average Ctvalue of the 18S rRNA gene for each sample.

Protein ExpressionCavernosal tissues were dissected and homog-enized in protease inhibitor cocktail ([4-{2-aminoethyl} benzenesulfonyl fluoride, pepstatin A,E-64, bestatin, leupeptin, and aprotinin]; SigmaAldrich) using a small tissue homogenizer. TheBradford assay was used to measure protein con-centration. A total of 40 mg of protein was loadedonto an SDS-PAGE 10% gel and transferred to anitrocellulose membrane. Nonspecific bindingsites were blocked with 5% nonfat milk in Tris-buffered solution with 1% Tween for 1 hour atroom temperature. Membranes were then probedwith rabbit anti-ETA receptor antibody (1:400;Alamone), rabbit anti-endothelial nitric oxidesynthase (anti-eNOS) (1:500; Cell Signaling),mouse anti-ROCKa (1:1,000; BD TransductionLaboratories), mouse anti-ROCKb (1:250; BDTransduction Laboratories), rabbit anti-phospho-MYPT-1 (Thr 696) (1:1,000; Upstate); mouseanti-MYPT-1 (1:1,000, BD Transduction Labora-tories), rabbit anti-phospho-ERK 1/2 (Thr 202/Tyr 204) (1:1,000, Cell Signaling), rabbit anti-ERK 1/2 (1:1,000, Cell Signaling), and mouseanti-b-actin (1:20,000; Sigma Aldrich). All primaryantibodies were incubated overnight at 4°C.Immunostaining was detected using horseradishperoxidase-conjugated anti-rabbit IgG (1:2,000;GE Healthcare) and anti-mouse IgG (1:2,000; GEHealthcare) for 1 hour at room temperature. For

b-actin detection, the secondary antibody was usedin a higher dilution (1:5,000). Immunoblots wererevealed by the SuperSignal Substrate (Pierce Bio-technology, Rockford, IL, USA) and quantitatedby densitometry using a Scion Image analyzer soft-ware (Scion Image Corporation, Frederick, MD,USA).

In Vivo Measurements of ICP/MAPAfter animals were anaesthetized with ketamine(100 mg/Kg) and xylazine (2.9 mg/Kg), the leftfemoral artery and right corpus cavernosum of therat penis were cannulated for continuous monitor-ing of MAP and ICP, respectively (as previouslydescribed) [8]. For experiments measuringvoltage-stimulated changes in ICP and MAP,stainless steel bipolar electrodes were positionedon the right major pelvic ganglion. In order toexamine the effects of ganglionic stimulation onICP/MAP, the major pelvic ganglion was stimu-lated with various voltages (1–5 V, 5 ms pulses at afrequency of 12 Hz).

Drugs and SolutionsPhysiological salt solution of the following com-position was used: 130 mM NaCl, 14.9 mMNaHCO3, 5.5 mM dextrose, 4.7 mM KCl,1.18 mM KH2PO4, 1.17 mM MgSO4·7H2O,1.6 mM CaCl2·2H2O, and 0.026 mM EDTA.Atropine, L-NAME, PE, sodium nitroprusside,and bretylium tosylate ([o-bromobenzyl] eth-yldimethylammonium p-toluenesulfonate) werepurchased from Sigma Chemical Co. (St. Louis,MO, USA). ET1 and IRL-1620 (Succinyl-[Glu9,Ala11,15]-endothelin-1[8-21]) were fromTocris (Ellisville, MO, USA). Atrasentan waskindly provided by Abbott Laboratories (AbbottPark, IL, USA). All reagents used were of analyti-cal grade. Stock solutions were prepared in deion-ized water, and stored in aliquots at -20°C;dilutions were prepared immediately before use.

Statistical AnalysisContractions were recorded as changes in the dis-placement from baseline and are represented asmN for n experiments. Relaxation is expressed aspercentage change from the PE-contracted levels.Agonist concentration–response curves were fittedusing a nonlinear interactive fitting program(Graph Pad Prism 4.0; GraphPad Software Inc.,San Diego, CA, USA). Agonist potencies areexpressed as pD2 (negative logarithm of the molarconcentration of agonist producing 50% of themaximum response). Maximum effect elicited by

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the agonist (Emax) values are represented as mN forcontractile responses or as a percentage changefrom the PE-contracted levels for relaxationresponses. Constraining curve-fit parameters wereused to fit a sigmoidal curve and to determine pD2values for IRL-1620 and sodium nitroprusside.Statistical analyses of Emax and pD2 values wereperformed using one-way anova or student’st-test. Western blot data were analyzed by one-sample t-test and the P value was computed fromthe t-ratio and the numbers of degrees of freedom.Values of P < 0.05 were considered statisticallysignificant.

Results

At 3 weeks of treatment, SBP of awake DOCAanimals was higher than the Uni rats(Uni: 125 � 3.6 mm Hg vs. DOCA: 164 �6.4 mm Hg). Atrasentan treatment improved thehypertension; however it did not return to basalvalues (Uni+atrasentan: 111 � 7.7 mm Hg vs.DOCA+atrasentan: 138 � 5.7 mm Hg) (Table 1).At 5 weeks of treatment, MAP measured directlyin anesthetized animals was higher in DOCA-saltin comparison to Uni rats (Uni: 96 � 4.3 mm Hgvs. DOCA: 144 � 10.2 mm Hg). Treatmentwith the ETA antagonist did not change MAP inUni or in DOCA-salt rats (Uni+atrasentan: 91 �1.2 mm Hg vs. DOCA+atrasentan: 150 �5.65 mm Hg) (Table 1). DOCA-salt rats exhibiteddecreased body weight in comparison to their nor-motensive littermates and ETA receptor blockadedid not change body weight in Uni or DOCA-saltanimals (Table 1).

Effects of DOCA-salt Hypertension onCavernosal ReactivityStimulation with 120 mM KCl induced a contrac-tile response (mN) of 1.76 � 0.36 (N = 5) and2.26 � 0.49 (N = 5) in strips from Uni andDOCA-salt rats, respectively. Treatment of ratswith atrasentan decreased contractile responses to

KCl in tissues from Uni (1.07 � 0.05 [N = 5]) andDOCA-salt rats (1.35 � 0.27 [N = 5]) (P < 0.05).In the first set of experiments, we evaluated

responses induced by ET-1, IRL-1620, EFS, andPE. Contractile responses to ET-1, which werenearly abolished by the addition of ETA antagonistin all groups (data not shown), were enhanced instrips from DOCA-salt rats (P < 0.05) (Figure 1A).Activation of smooth muscle cell ETB receptorsinduces contraction. In contrast, endothelial cellETB receptors activation stimulates the release ofNO and prostacyclin, and consequently, inducesvasodilation. Considering that, responses to theETB agonist IRL-1620 were tested under bothbasal tone and after cavernosal strips were stimu-lated with PE (10-5 M). IRL-1620 did not inducecontractile responses when tested in tissues at basaltone, either in the absence or presence of 10-4 ML-NAME (data not shown), but produced relax-ation of cavernosal strips contracted with PE10-5 M (Figure 1B). IRL-1620-induced relaxationwas impaired in cavernosum fromDOCA-salt rats.EFS (1–64 Hz), after 45 minutes of incubationwith atropine (muscarinic receptor antagonist;10-6 M) plus L-NAME (nonselective inhibitor ofNO synthase, 10-4 M) to avoid responses due tothe stimulation of cholinergic and nonadrenergic-noncholinergic (NANC) nerves, produced fre-quency dependent contractions in cavernosalsmooth muscle strips (Figure 1C). EFS-dependentcontractions were virtually abolished by the sym-pathetic nerve blocking agent bretylium tosylate(3 ¥ 10-5 M) and by the alpha-adrenergic antago-nist terazosin (10-6 M), confirming that theseresponses are neuronal in origin and adrenergic innature (data not shown). Contractile responsesinduced by EFS (adrenergic nerve stimulation) athigher frequencies, were enhanced in cavernosalstrips from DOCA-salt rats in comparison toUni tissues (Figure 1C). Similarly, contractile res-ponses to the alpha-adrenergic receptor agonistPE were enhanced in cavernosal tissues fromDOCA-salt hypertensive rats (Figure 1D). The

Table 1 SBP at 3 weeks of treatment (awake animals), MAP at 5 weeks of treatment (anaesthetized animals), and bodyweight of Uni and DOCA-salt hypertensive rats

Uni DOCA Uni+atrasentan DOCA+atrasentan

SBP (mm Hg) 125 � 3.6 164 � 6.4* 111 � 7.7 138 � 5.7*†

MAP (mm Hg) 96 � 4.3 144 � 10.2* 91 � 1.2 150 � 5.65*Body weight (g) 390 � 14 319 � 7* 374 � 17 308 � 13*

*P < 0.05 vs. respective control; †P < 0.05 vs. vehicle-treated.Values are means � SEM for N = 6 in each group.SBP = systolic blood pressure; MAP = mean arterial pressure; Uni = uninephrectomized control; DOCA = deoxycorticosterone-salt hypertensive rats;Uni+atrasentan = uninephrectomized control rats treated with atrasentan; DOCA+atrasentan = DOCA-salt hypertensive rats treated with atrasentan.

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cumulative addition of SNP (10-8 to 3 ¥ 10-3 M)produced concentration-dependent relaxations ofPE-contracted tissues. Relaxation responses toSNP were reduced in cavernosal tissues fromDOCA-salt (Table 2).

Effects of DOCA-salt Hypertension on Componentsof the ET-1 System and RhoA/Rho-kinase PathwayIn the second set of experiments, we evaluatedpreproET-1, ETA, and ETB mRNA levels in cav-ernosal tissue from Uni and DOCA rats. Cavern-

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Figure 1 Responses of cavernosal strips from (A) Uni (�) and deoxycorticosterone and salt (�) rats to endothelin-1, (B)IRL-1620 (an endothelin receptor B subtype agonist), (C) stimulation of adrenergic nerves by EFS, and (D) the alpha1-adrenergic receptor agonist PE. Frequency response curves elicited by EFS (1–64 Hz) were performed in the presence ofL-NAME 10-4 M plus atropine 10-6 M (N = 5). PE concentration-response curves were performed in the absence of L-NAME(N = 5 in all groups). Relaxations induced by IRL-1620 (N = 5) were calculated relative to the maximal changes from thecontraction produced by PE in each tissue, which was taken as 100%. Data represent the mean � SEM of N experiments.*P < 0.05 compared with values of cavernosal strips from normotensive rats. EFS = electrical field stimulation;L-NAME = Nw-nitro-L-arginine methyl ester; PE = phenylephrine.

Table 2 Emax and pD2 values for agonists-induced responses in cavernosal strips from Uni and DOCA-salt rats, treatedwith vehicle or atrasentan

Agonist

Uni DOCA Uni+atrasentan DOCA+atrasentan

Emax pD2 Emax pD2 Emax pD2 Emax pD2

PE (N = 5) 2.6 � 0.1 5.4 � 0.1 3.6 � 0.3* 5.4 � 0.2 1.3 � 0.1† 5.5 � 0.2 1.5 � 0.1† 5.5 � 0.2ET-1 (N = 4–6) 0.8 � 0.1 6.9 � 0.1 1.3 � 0.1* 7.0 � 0.1 0.3 � 0.1† 6.1 � 0.1† 0.9 � 0.04*† 6.7 � 0.3*IRL-1620 (N = 4–5) 68 � 1.7 8.8 � 0.1 47 � 3.6* 7.6 � 0.2* n.d. n.d. 66 � 4.5† 7.5 � 0.1*SNP (N = 4–5) 72 � 13 4.0 � 0.3 45 � 21* 3.7 � 0.6 n.d. n.d. 88 � 19† 3.4 � 0.3

*P < 0.05 vs. respective control; †P < 0.05 vs. vehicle treated.Values are means � SEM for n experiments in each group. Emax values for PE and ET-1 are represented as mN. Relaxation induced by IRL-1620 and SNP wascalculated relative to the maximal changes from the contraction produced by PE and Emax values are represented as % of relaxation.PE = phenylephrine; ET-1 = endothelin-1; SNP = sodium nitroprusside; Emax = maximum effect elicited by the agonist; pD2 = negative logarithm of EC50;Uni = uninephrectomized control; DOCA = DOCA-salt hypertensive rats; Uni+atrasentan = uninephrectomized control rats treated with atrasentan;DOCA+atrasentan = DOCA-salt hypertensive rats treated with atrasentan; n.d. = not determined.

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osal strips from DOCA animals displayedincreased preproET-1 expression (Figure 2A) incomparison to Uni group. Although, the levels ofETA receptor mRNA expression were not altered(Figure 2B), cavernosal tissue from DOCAanimals displayed decreased ETB receptor mRNAexpression (Figure 2C). Additional support tothese data was the fact that ETA receptor protein

expression was similar between Uni and DOCA(Figure 2D). ETB receptor-mediated vasodilationoccurs via release of NO from cavernous endothe-lial cells. Considering that cavernosal strips fromDOCA-salt rats displayed decreased IRL-1620-induced relaxation and ETB receptor mRNAexpression, we further determined eNOS expres-sion levels in the cavernosal tissue from these

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Figure 2 Messenger RNA expression of (A) preproET-1 and (B) ETA and (C) ETB determined by real-time reverse-transcriptase polymerase chain reaction using total RNA extracted from cavernosal tissue of Uni (open bar) and DOCA-salt(filled bar) rats. Bar graphs show fold of change in preproET-1 and ETA mRNA expression. Values were normalized by thecorrespondent 18S rRNA of each sample. Results are mean � SEM (N = 5–6). Representative immunoblots for ETA

receptors (D) and eNOS (E) proteins (upper panel). Corresponding bar graphs demonstrating ETA receptors and eNOSexpression in the corporal tissue from Uni (open bar) and DOCA-salt (filled bar) rats (bottom panel). Results aremean � SEM of N = 5–6 experiments. *P < 0.05, DOCA-salt vs. Uni. ETA = endothelin receptor A subtype; ETB = endothelinreceptor B subtype; eNOS = endothelial nitric oxide synthase; DOCA-salt = deoxycorticosterone and salt; Uni =uninephrectomized control.

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animals. Decreased relaxation to IRL-1620 wasalso associated with decreased eNOS proteinexpression (Figure 2E).Considering that ET-1 stimulates the RhoA/

Rho-kinase pathway in the penis and that DOCA-salt rats displayed increased cavernosal reactivityto PE and adrenergic nerve-mediated responses,we also determined whether DOCA-salt hyper-tension alters the RhoA/Rho-kinase pathway.Although protein expression levels of ROCKaand ROCKb (Rho-kinase isozymes) were similarbetween corpora cavernosa from Uni and DOCA(Figure 3A,B, respectively), the phosphorylatedform of myosin phosphatase regulatory subunit(MYPT-1), a downstream effector of Rho-kinase,

was increased in cavernosal tissue from DOCAanimals (Figure 3C). In addition, because ET-1can also regulate contractile response via phos-phorylation of p44/42 isoforms of the extracel-lular signal-regulated kinase (ERK 1/2), weinvestigated the phosphorylation state of ERK1/2. Cavernosal tissue from DOCA rats displayedan increased ERK 1/2 phosphorylation level(Figure 3D).

Effects of ETA Antagonist Treatment on CavernosalReactivity and ET-1/RhoA/Rho-kinase PathwayIn the third set of experiments we determined iftreatment of animals with atrasentan would at-tenuate changes in cavernosal reactivity induced

A B

C D

b-actin

ROCKa ROCKb

b-actin

tERK 1/2tMYPT-1

2/1 KREp1-TPYMp

Uni DOCA Uni DOCA

Uni DOCA Uni DOCA

Uni DOCA0.0

0.5

1.0

1.5

RO

CK

α/ β

-ac

tin

Uni DOCA0.0

0.5

1.0

1.5

RO

CK

β/β-

ac

tin

Uni DOCA0

1

2

3

4

*

pM

YP

T/t

MY

PT

Uni DOCA0

1

2

3

4

5

*

pE

RK

1/2

/to

tal

ER

K 1

/2

Figure 3 Western immunoblots for (A) ROCKa, (B) ROCKb, (C) pMYPT-1, and (D) pERK 1/2. Representative immunoblotsare presented in the upper panels. Corresponding bar graphs comparing ROCKa and ROCKb expression, and the levelof pMYPT-1 and perk 1/2 in the corporal tissue from Uni (open bar) and DOCA-salt (filled bar) rats are given in the bottompanel. Activation is represented by the ratio between the phosphorylation and total forms of the protein. Results are mean �SEM of N = 5 experiments. *P < 0.05, DOCA-salt rats vs. Uni. DOCA-salt = deoxycorticosterone and salt; Uni =uninephrectomized control.

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by DOCA-salt hypertension. Contractileresponses induced by EFS and to the alpha-adrenergic receptor agonist PE were enhanced incavernosal strips from DOCA rats in comparisonto Uni tissues (Figure 4A,B, respectively). Treat-ment of animals with atrasentan decreased con-tractile responses to both EFS and PE in tissues

from Uni and DOCA rats (P < 0.05). At these con-ditions, no differences were observed betweenresponses in tissues from normotensive and hyper-tensive animals (Figure 4A, B). SNP and IRL-1620 responses were restored by treatment ofanimals with the ETA antagonist. Emax and pD2values are listed in Table 2.

A

C

0.5 1 2 4 8 16 32 64 128

0

1

2

3UniUni+atrasentanDOCADOCA+atrasentan

**

Frequency [Hz]

Contr

acti

on (

mN

)

-10 -9 -8 -7 -6 -5 -4 -3

0

1

2

3

4

5 UniDOCADOCA+atrasentanUni+atrasentan

*

* †

phenylephrine log [M]C

ontr

acti

on (

mN

)

0.0

0.5

1.0

1.5

Uni + atrasentan DOCA + atrasentan

ET

A r

ecep

tor

mR

NA

(fo

ld o

f in

cre

ase)

0.0

0.5

1.0

1.5

2.0

2.5

*

Uni + atrasentan DOCA + atrasentan

pre

pro

-en

do

theli

n-1

mR

NA

(fo

ld o

f in

cre

ase)

0.0

0.5

1.0

Uni+atrasentan DOCA+atrasentan

ET

B r

ece

pto

r m

RN

A

(Fo

ld o

f ch

an

ge)

E

B

D

Figure 4 Effects of the ETA antagonist, atrasentan, on contractile responses of cavernosal strips from Uni and DOCA-saltrats upon stimulation of adrenergic nerves by (A) EFS, (B) PE, and (C) mRNA expression of preproET-1, (D) ETA, and (E)ETB receptor. Frequency-response curves elicited by EFS (1–64 Hz) were performed in the presence of L-NAME 10-4 M plusatropine 10-6 M (N = 5). PE concentration-response curves were performed in the absence of L-NAME (N = 5 in all groups).PreproET-1 and ETA receptor mRNA expression were determined by qPCR using total RNA extracted from cavernosal tissueof uninephrectomized control (open bar) and DOCA-salt (filled bar) rats treated with atrasentan. Bar graphs show fold ofchange in preproET-1 and ETA mRNA expression. Values were normalized by the correspondent 18S rRNA of each sample.Results are mean � SEM (N = 5–6). ETA = endothelin receptor A subtype; ETB = endothelin receptor B subtype; DOCA-salt = deoxycorticosterone and salt; EFS = electric field stimulation; PE = phenylephrine; L-NAME = Nw-nitro-L-argininemethyl ester.

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Considering that the ETA receptor antagonisttreatment prevented changes in cavernosal reactiv-ity induced by DOCA-salt hypertension, we per-formed experiments to verify if these effects wereassociated with changes in the signaling pathwaysaddressed in the second set of experiments. Treat-ment of DOCA rats for 5 weeks with atrasentandid not prevent increased preproET-1 mRNAexpression (Figure 4C) or change ETA receptormRNA levels (Figure 4D). However, it did abolishdecreased ETB receptor expression (Figure 4E).Moreover, atrasentan treatment reverted increasedphosphorylation of MYPT-1 (Figure 5A) andERK 1/2 (Figure 5B) and restored eNOS expres-sion levels (Figure 5C).

Effects of ETA Antagonist Treatment on ErectileResponses in vivoTo confirm that DOCA-salt rats display ED andto determine whether ETA receptor antagonistimproves ED in mineralocorticoid hypertensiveanimals, the major pelvic ganglion was stimulatedover a range of voltages (1–5 V) and the effectsof ganglionic-mediated increases in ICP/MAPwere evaluated. Stimulation resulted in a voltage-dependent increase in ICP/MAP, which wasdecreased in DOCA-salt rats (Figure 6). Erectilefunction in atrasentan-treated DOCA-salt rats wassimilar to that observed in Uni animals. Treatmentof Uni animals with atrasentan did not changeganglionic-mediated increases in ICP/MAP.

Discussion

The findings of this study demonstrate that ED inDOCA-salt hypertension is prevented by treat-ment of hypertensive rats with an ETA receptorantagonist. In addition, it shows that DOCA-saltrats exhibit altered cavernosal reactivity to ET-1and to other contractile and relaxant stimuli andthat treatment with atrasentan prevents changesin cavernosal reactivity induced by DOCA-salthypertension.A high prevalence of ED is observed among

hypertensive men [4,5] and increased levels ofET-1 is considered one of the best independentpredictors for ED [25]. In addition, greater plasmalevels of ET-1 were reported in patients withorganic and psychogenic ED in comparison tohealthy controls. Interestingly, patients with or-ganic ED have significantly higher ET-1 levels inboth venous and cavernosal blood than patientswith psychogenic ED [26–28].

A previous report showed that DOCA-salthypertensive rats display decreased erectile func-tion, represented by a significant suppression ofganglionic-induced increases in ICP/MAP ratioupon electrical stimulation of the major pelvicganglion. This study also shows that intracavern-osal injection of a selective Rho-kinase inhibitoraugments ICP/MAP and that this effect is signi-ficantly suppressed in DOCA-salt rats, indicat-ing that increased Rho-kinase activity may be themechanism underlying the decreased erectilefunction in hypertensive subjects [8].The importance of Rho-kinase in Ca2+ sensiti-

zation of corpus cavernosum smooth muscle cellsand maintenance of penile flaccidity [3,29,30] aswell as increased Rho-kinase activity in the vascu-lature of various hypertensive animal models hasbeen extensively demonstrated [31–33]. Rho-kinase inhibition increases corpus cavernosumpressure in an in vivo rat model [29] and allthe components of RhoA/Rho-kinase signalingpathway (RhoA, phosphatase inhibitor CPI-17,MYPT-1 and catalytic (PP1d) subunits, two iso-forms of Rho-kinase [ROCKa and ROCKb])are present in corpora cavernosa from differentspecies [30].ET-1, which potently induces slow developing,

long-lasting contractions in the corporal smoothmuscle cells and penile vessels, has been suggestedto contribute to the maintenance of corpus caver-nosal smooth muscle tone [1–3,19–21]. In addi-tion, it is well established that ET-1 increasesRho-kinase activity in the cavernous circulation[20] and directly induces Ca2+ sensitization in cav-ernosal smooth muscle cells [29,30]. Therefore,considering that ET-1 may be the link betweenincreased Rho-kinase activity and ED in DOCA-salt hypertension, we hypothesized that ET-1acting through ETA receptors contributes to EDin DOCA-salt hypertensive rats.Herein we present the novel finding that erec-

tile tissue from DOCA-salt rats has enhancedET-1 contractile responses and impaired IRL-1620-induced relaxation, indicating that ET-1may contribute to ED in this salt-sensitive modelof hypertension. Increased contraction as well asdecreased relaxation of cavernosal smooth musclecells would favor penile flaccidity in vivo. Our dataare in agreement with previous reports showingthat contractile responses to ET-1 in isolated cav-ernosal strips are abolished by ETA receptor block-ade [21] and that administration of an ETBantagonist does not produce any significant effecton the vasoconstrictor action of ET-1 in vivo [17].

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Our results, showing that IRL-1620 induces relax-ation, but not contraction, of cavernosal stripssuggest a role for endothelial cell ETB receptorsand are supported by a previous report showingthat ET-1 injection into rat penile vasculatureinduces both vasoconstriction and vasodilation[19].ET-1 activates the RhoA/Rho-kinase pathway

[21,30] and in vivo responses to Rho-kinase inhibi-tion are decreased in DOCA-salt hypertensive rats[8]. Considering that ET-1 is increased in DOCA-salt hypertensive model [22,23], we verified theexpression of components of the RhoA/Rho-kinasepathway in cavernosal tissues. In recent years,studies have revealed that the small GTPase Rhoand its downstream target Rho-kinase mediate theCa+2 sensitization of smooth muscle contraction[34]. The exchange of bound guanosine diphos-phate (GDP) for guanosine triphosphate (GTP)activates Rho and stimulates its translocation fromthe cytosol to the plasma membrane. Rho-GTPphosphorylates Rho-kinase, which inhibits myosinlight chain (MLC) phosphatase activity by phos-phorylation of the MLC phosphatase targetsubunit (MYPT1). A decrease inMLCphosphataseactivity increases phosphorylation of myosin andtherefore contributes to smooth muscle contrac-tion at low levels of intracellular (Ca+2). Strongevidence suggests that increasedRhoA/Rho kinase-dependent Ca+2 sensitization contributes to hyper-tension and inhibition of RhoA/Rho kinase reducesblood pressure [31,35]. Some heterotrimetric G

A

B

C

tMYPT-1

pMYPT-1

tERK 1/2

pERK 1/2

eNOS

b-actin

Uni +

atrasentan

DOCA +

atrasentan

Uni +

atrasentan

Uni +

atrasentan

DOCA +

atrasentan

DOCA +

atrasentan

0.0

0.5

1.0

1.5

2.0

Uni + atrasentan DOCA + atrasentan

pM

YP

T/t

MY

PT

0.0

0.5

1.0

1.5

Uni + atrasentan DOCA + atrasentan

pE

RK

1/2

/to

tal

ER

K 1

/2

0.0

0.5

1.0

1.5

2.0

Uni + atrasentan DOCA + atrasentan

eN

OS

/B-a

cti

n

Figure 5 Effects of the endothelin receptor A subtypeantagonist atrasentan on (A) pMYPT-1 (B) and perk 1/2activation and (C) eNOS expression. Representative immu-noblots are presented in the upper panels. Correspondingbar graphs demonstrating activation of pMYPT-1 and perk1/2, represented by the ratio between the phosphorylationand total forms of the protein, and eNOS expression in thecorporal tissue from Uni (open bar) and DOCA-salt (filled bar)rats treated with atrasentan (bottom panels). Results aremean � SEM of N = 5 experiments. *P < 0.05, DOCA-saltrats vs. Uni. eNOS = endothelial nitric oxide synthase;DOCA-salt = deoxycorticosterone and salt; Uni =uninephrectomized control.

0 1 2 3 4 5 60.0

0.2

0.4

0.6

0.8

1.0

Control (n=5)

DOCA (n=4)

DOCA+atrasentan (n=5)

** * *#

## #

Voltage [volts]

ICP

/MA

P

Figure 6 Effect of ETA antagonist treatment on voltage-mediated increases in ICP/MAP. ICP/MAP response to gan-glionic stimulation was assessed over a range of voltages(1–5 v). The voltage-stimulated increase in ICP/MAP fol-lowing the administration of atrasentan (5 mg/day/kg ofbody weight, p.o. per gavage for 5 weeks) was significantlyincreased in DOCA-salt rats, as compared to nontreated(vehicle) animals. Data represent mean � SEM of N = 4–5per group. *P < 0.05 compared with values from normoten-sive rats; #P < 0.05 compared with values from DOCA-salt rats treated with vehicle (ANOVA). ICP = intracavernosalpressure; MAP = mean arterial pressure; DOCA-salt =deoxycorticosterone and salt.

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protein-coupled receptor agonists, includingAngIIand ET-1, induce smooth muscle contractionthrough both intracellular Ca+2-dependent andRhoA/Rho kinase-mediated Ca+2-independent sig-naling pathways [20,36].An inverse functional relationship between the

NO and RhoA/Rho-kinase signaling pathwaysis well established [3]. Accordingly, Rho-kinaseactivation suppresses eNOS expression in thecavernosal tissue [37] and decreased sensitivityof contractile proteins to Ca2+ is considered a keymechanism in NO-induced relaxation of cavern-osal smooth muscle [3]. This is further corrobo-rated by our results demonstrating increasedRhoA/Rho kinase signaling and decreased eNOSexpression in cavernosal strips from DOCA-saltrats. In addition, ETA blockade reversed bothdecreased eNOS expression and increased phos-phorylation of MYPT-1. Although atrasentan nor-malized MYPT-1 phosphorylation in DOCA-saltcavernosal tissue, our study presents the limitationthat functional in vivo studies with a Rho-kinaseinhibitor were not performed in animals treatedwith the ETA antagonist. However, chronicadministration of Rho-kinase inhibitor has beenshown to prevent the development of vasculogenicED in a rat model [38].On the other hand, ERK 1/2 has been suggested

to play a role in vascular reactivity. In vivo studieshave shown that ERK 1/2 inhibition attenuatescerebral blood flow reduction and vasoconstrictionafter subarachnoid hemorrhage in rats and alsoprevents the up-regulation of vascular endothelinETB receptors [39]. In addition, ERK 1/2 areinvolved in the up-regulation of endothelin ETAandETB receptors in porcine coronary arteries [40]and ETB receptors in rat cerebral arteries [41].Furthermore, Somer and collaborators [42] havedemonstrated that patients with ED exhibited agreater amount of active ERK 1/2 in the cavernosalsmooth muscle than patients with normal erectilefunction. Taken together, these results suggest thatERK 1/2 play a role in ED and that ET-1 may be afactor that triggers its activation, especially in salt-sensitive forms of experimental hypertension, suchas mineralocorticoid hypertension. Our datashowing increased activity of MYPT-1 and ERK1/2 in cavernosal tissues in the DOCA-salt hyper-tensive group reinforce both this suggestion andour previous in vivo observations.The suggestion that ET-1/ETA receptors may

contribute to ED in DOCA-salt hypertensive ratsis reinforced by the set of experiments showingthat cavernosal strips from hypertensive animals

display abnormal responses to adrenergic nervestimulation, PE (alpha1 adrenergic receptoragonist) and sodium nitroprusside (NO donor)and that treatment of DOCA-salt hypertensiverats with the ETA antagonist atrasentan preventschanges in cavernosal reactivity.Since ET-1 increases contractile responses to

other vasoactive agents, it is possible that increasedcontractile responses to EFS and PE are dueto increased ET-1 expression in penises fromDOCA-salt rats. In the rat penis, ET-1 increasescontractile responses to PE via activation of ETAreceptors and this synergistic effect involves acti-vation of the RhoA/Rho-kinase pathway [21]. Thesynergism between ET-1 and PE is associated withincreased expression of RhoA in the membranefraction of penis homogenates and the Rho-kinaseinhibitor, Y-27632, relaxes the contraction inducedby ET-1, PE, or its combination [21]. ET-1 alsoenhances contractile responses elicited by PEon isolated human corporal strips, independentof age- or diabetes-related alterations [43,44]. Inaccordance with these reports, our study showsthat both PE and EFS-mediated contractions weredecreased in cavernosal tissues from animalstreated with the ETA antagonist. In rat fibroblasts,activation of ETA receptors was found to induce(1-adrenergic receptor phosphorylation, possiblyinvolving the protein kinase (PKC) and mitogen-activated protein kinases (MAPK) signaling [45]. Acrosstalk between ET-1 and a1-adrenergic signal-ing pathways process may also account for thissynergistic interaction.It is assumed that Rho-kinase and ET-1-

regulated vasoconstriction occurs in the smoothmuscle of the cavernosal arterioles and the walls ofthe cavernosal sinuses. However, the possibilitythat they act on the penile helicine arteries and notdirectly on the cavernosal sinuses and arteriolesshould be considered. Binding sites for ET-1 weredemonstrated, using autoradiography in the deeppenile artery, circumflex veins and through thecorpus cavernosum tissue [13]. In addition, ET-1induces contractions in penile smooth muscle cav-ernosal tissue, cavernous artery and deep dorsal andpenile circumflex veins [13,18]. Finally, the mostconvincing evidence for the contribution of ET-1to ED in DOCA-salt hypertension was obtainedusing in vivo experiments, showing that treatmentof DOCA-salt hypertensive rats with atrasentancompletely prevents ED, evidenced by decreasedganglionic-mediated increases in ICP/MAP.A pilot study for the treatment of mild-to-

moderate ED, showed that intravenous admin-

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istration of BMS-193884, an ETA receptorantagonist, in male rabbits increases the durationof pelvic nerve-stimulated penile erection [46].However, in males diagnosed with mild-to-moderate ED, administration of BMS-193884 didnot significantly improve erectile function [46]. Itis possible that ET-1 is involved in ED associatedwith specific conditions (diabetes- or salt-sensitivehypertension-associated ED) and that ETA or dualETA/ETB antagonists may be beneficial only inthese subsets of patients. It is noteworthy that theETA receptor antagonist had a blood pressure-lowering effect in awake, but not in anaesthetized,animals. Anesthesia effects on blood pressure mayaccount for such differences. Although possible,it is unlikely that the beneficial effects of ETAantagonismonEDare related to its blood pressure-lowering effects. Accordingly, Fibbi and coworkers[47] have recently demonstrated that atorvastatintreatment improves sildenafil-induced erections inspontaneously hypertensive rats. The effects ofatorvastatin were associated with a decrease inRhoA/Rho-kinase pathway activity, but were bloodpressure-independent. In addition, Jin and cowork-ers have recently shown that ED in AngII-infusedrats is reverted by inhibition of reactive oxygenspecies (ROS) production with only mild bloodpressure-lowering effects [48]. Finally, althoughantihypertensive therapies effectively lower bloodpressure, they often fail to ameliorate erectile func-tion in hypertensive patients [7].In summary, our study shows that DOCA-salt

rats exhibit altered cavernosal reactivity to ET-1and to the ETB agonist IRL-1620, as well as toother contractile and relaxant stimuli. These func-tional changes were associated with increased acti-vation of the RhoA/Rho-kinase and ERK 1/2pathways. Treatment of DOCA-salt rats with theETA antagonist atrasentan prevented changesin cavernosal reactivity induced by DOCA-salthypertension and, very importantly, its associatedED. ETA antagonists may represent an alternativetherapeutic approach for ED associated with salt-sensitive hypertension or other pathological con-ditions where increased levels of ET-1 are present.

Acknowledgement

This study was supported by grants from the NationalInstitutes of Health (HL71138 and HL74167) andFundacao de Amparo a Pesquisa do Estado de SaoPaulo—FAPESP, Brazil.

Corresponding Author: Fernando S. Carneiro, MSc,Medical College of Georgia, Department of Physiology,

1120 15th St., Augusta, GA, USA. Tel: 706-721-3547;Fax: 706-721-7299; E-mail: fcarneiro@mcg.edu.

Conflict of Interest: None declared.

Statement of Authorship

Category 1(a) Conception and Design

Fernando S. Carneiro; Rita C. Tostes(b) Acquisition of Data

Fernando S. Carneiro; Kênia Pedrosa Nunes;Romulo Leite; Edson F. Nogueira; Fernanda R.C.Giachini; Zidonia N. Carneiro; Victor V. Lima

(c) Analysis and Interpretation of DataFernando S. Carneiro; Kênia Pedrosa Nunes;Edson F. Nogueira

Category 2(a) Drafting the Article

Fernando S. Carneiro; Rita C. Tostes(b) Revising It for Intellectual Content

Rita C. Tostes; R. Clinton Webb; William E.Rainey; Adviye Ergul

Category 3(a) Final Approval of the Completed Article

Rita C. Tostes; R. Clinton Webb

References

1 Andersson KE. Pharmacology of penile erection.Pharmacol Rev 2001;53:417–50.

2 Andersson KE. Erectile physiological and patho-physiological pathways involved in erectile dysfunc-tion. J Urol 2003;170:S6–13; discussion S13–4.

3 Leite R, Giachini FR, Carneiro FS, Nunes KP,Tostes RC, Webb RC. Targets for the treatment oferectile dysfunction: is NO/cGMP still the answer?Recent Patents Cardiovasc Drug Discov 2007;2:119–32.

4 Muller A, Mulhall JP. Cardiovascular disease, meta-bolic syndrome and erectile dysfunction. Curr OpinUrol 2006;16:435–43.

5 Lewis RW. Sexual Medicine: Sexual Dysfunction inMen and Women. Paris: Health Publications; 2004.

6 Shabsigh R, Shah M, Sand M. Erectile dysfunctionand men’s health: developing a comorbidity risk cal-culator. J Sex Med 2008;5:1237–43.

7 Keene LC, Davies PH. Drug-related erectile dys-function. Adverse Drug React Toxicol Rev 1999;18:5–24.

8 Chitaley K, Webb RC, Dorrance AM, Mills TM.Decreased penile erection in DOCA-salt and strokeprone-spontaneously hypertensive rats. Int J ImpotRes 2001;13(Suppl 5):S16–20.

9 Granchi S, Vannelli GB, Vignozzi L, Crescioli C,Ferruzzi P, Mancina R, Vinci MC, Forti G, Filippi

Endothelin-1 and Erectile Dysfunction in Hypertension 2805

J Sex Med 2008;5:2793–2807

S, Luconi M, Ledda F, Maggi M. Expression andregulation of endothelin-1 and its receptors inhuman penile smooth muscle cells. Mol HumReprod 2002;8:1053–64.

10 Vignozzi L, Filippi S, Morelli A, Ambrosini S,Luconi M, Vannelli GB, Donati S, Crescioli C,Zhang XH, Mirone V, Forti G, Maggi M. Effect ofchronic tadalafil administration on penile hypoxiainduced by cavernous neurotomy in the rat. J SexMed 2006;3:419–31.

11 Filippi S, Marini M, Vannelli GB, Crescioli C,Granchi S, Vignozzi L, Luconi M, Ferruzzi P,Morelli A, Forti G, Ledda F, Maggi M. Effects ofhypoxia on endothelin-1 sensitivity in the corpuscavernosum. Mol Hum Reprod 2003;9:765–74.

12 Saenz de Tejada I, Carson MP, de las Morenas A,Goldstein I, Traish AM. Endothelin: localization,synthesis, activity, and receptor types in humanpenile corpus cavernosum. Am J Physiol 1991;261:H1078–85.

13 Holmquist F, Kirkeby HJ, Larsson B, Forman A,Alm P, Andersson KE. Functional effects, bindingsites and immunolocalization of endothelin-1 in iso-lated penile tissues from man and rabbit. J Pharma-col Exp Ther 1992;261:795–802.

14 Sullivan ME, Dashwood MR, Thompson CS,Muddle JR, Mikhailidis DP, Morgan RJ. Alterationsin endothelin B receptor sites in cavernosal tissue ofdiabetic rabbits: potential relevance to the patho-genesis of erectile dysfunction. J Urol 1997;158:1966–72.

15 KhanMA,ThompsonCS,AngeliniGD,MorganRJ,MikhailidisDP, Jeremy JY. Prostaglandins and cyclicnucleotides in the urinary bladder of a rabbit modelof partial bladder outlet obstruction. ProstaglandinsLeukot Essent Fatty Acids 1999;61:307–14.

16 Bell CR, Sullivan ME, Dashwood MR, Muddle JR,Morgan RJ. The density and distribution of endot-helin 1 and endothelin receptor subtypes in normaland diabetic rat corpus cavernosum. Br J Urol1995;76:203–7.

17 Dai Y, Pollock DM, Lewis RL, Wingard CJ,Stopper VS, Mills TM. Receptor-specific influenceof endothelin-1 in the erectile response of the rat.Am J Physiol Regul Integr Comp Physiol 2000;279:R25–30.

18 Parkkisenniemi UM, Klinge E. Functional charac-terization of endothelin receptors in the bovineretractor penis muscle and penile artery. PharmacolToxicol 1996;79:73–9.

19 Ari G, Vardi Y, Hoffman A, Finberg JP. Possible rolefor endothelins in penile erection. Eur J Pharmacol1996;307:69–74.

20 Mills TM, Chitaley K, Wingard CJ, Lewis RW,Webb RC. Effect of Rho-kinase inhibition on vaso-constriction in the penile circulation. J Appl Physiol2001;91:1269–73.

21 Wingard CJ, Husain S, Williams J, James S. RhoA-Rho kinase mediates synergistic ET-1 and phenyle-

phrine contraction of rat corpus cavernosum. AmJ Physiol Regul Integr Comp Physiol 2003;285:R1145–52.

22 Schiffrin EL. Vascular endothelin in hypertension.Vascul Pharmacol 2005;43:19–29.

23 Tostes RC, Muscara MN. Endothelin receptorantagonists: another potential alternative for cardio-vascular diseases. Curr Drug Targets CardiovascHaematol Disord 2005;5:287–301.

24 Tostes RC, Giachini FR, Carneiro FS, Leite R,Inscho EW,Webb RC. Determination of adenosineeffects and adenosine receptors in murine corpuscavernosum. J Pharmacol Exp Ther 2007;322:678–85.

25 Bocchio M, Desideri G, Scarpelli P, Necozione S,Properzi G, Spartera C, Francavilla F, Ferri C,Francavilla S. Endothelial cell activation in menwith erectile dysfunction without cardiovascular riskfactors and overt vascular damage. J Urol 2004;171:1601–4.

26 Francavilla S, Properzi G, Bellini C, Marino G,Ferri C, Santucci A. Endothelin-1 in diabetic andnondiabetic men with erectile dysfunction. J Urol1997;158:1770–4.

27 El Melegy NT, Ali ME, Awad EM. Plasma levels ofendothelin-1, angiotensin II, nitric oxide and pros-taglandin E in the venous and cavernosal blood ofpatients with erectile dysfunction. BJU Int 2005;96:1079–86.

28 Hamed EA, Meki AR, Gaafar AA, Hamed SA. Roleof some vasoactivemediators in patients with erectiledysfunction: their relationship with angiotensin-converting enzyme and growth hormone. Int J ImpotRes 2003;15:418–25.

29 Chitaley K, Wingard CJ, Webb RC, Branam H,Stopper VS, Lewis RW, Mills TM. Antagonismof Rho-kinase stimulates rat penile erection via anitric oxide-independent pathway. Nat Med 2001;7:119–22.

30 Wang H, Eto M, Steers WD, Somlyo AP, SomlyoAV. RhoA-mediated Ca2+ sensitization in erectilefunction. J Biol Chem 2002;277:30614–21.

31 Uehata M, Ishizaki T, Satoh H, Ono T, KawaharaT, Morishita T, Tamakawa H, Yamagami K, Inui J,Maekawa M, Narumiya S. Calcium sensitization ofsmooth muscle mediated by a Rho-associatedprotein kinase in hypertension. Nature 1997;389:990–4.

32 Weber DS, Webb RC. Enhanced relaxation to therho-kinase inhibitor Y-27632 in mesenteric arteriesfrom mineralocorticoid hypertensive rats. Pharma-cology 2001;63:129–33.

33 Mukai Y, Shimokawa H, Matoba T, Kandabashi T,Satoh S, Hiroki J, Kaibuchi K, Takeshita A. Involve-ment of Rho-kinase in hypertensive vascular disease:a novel therapeutic target in hypertension. FASEB J2001;15:1062–4.

34 Somlyo AP, Somlyo AV. Signal transduction byG-proteins, rho-kinase and protein phosphatase to

2806 Carneiro et al.

J Sex Med 2008;5:2793–2807

smooth muscle and non-muscle myosin II. J Physiol2000;522 Pt 2:177–85.

35 Seko T, Ito M, Kureishi Y, Okamoto R, Moriki N,Onishi K, Isaka N, Hartshorne DJ, Nakano T. Acti-vation of RhoA and inhibition ofmyosin phosphataseas important components in hypertension in vascularsmooth muscle. Circ Res 2003;92:411–8.

36 Matrougui K, Tanko LB, Loufrani L, Gorny D,Levy BI, Tedgui A, Henrion D. Involvement ofRho-kinase and the actin filament network in angio-tensin II-induced contraction and extracellularsignal-regulated kinase activity in intact rat mesen-teric resistance arteries. Arterioscler Thromb VascBiol 2001;21:1288–93.

37 Bivalacqua TJ, Champion HC, Usta MF, Cellek S,Chitaley K, Webb RC, Lewis RL, Mills TM,Hellstrom WJ, Kadowitz PJ. RhoA/Rho-kinasesuppresses endothelial nitric oxide synthase in thepenis: a mechanism for diabetes-associated erectiledysfunction. Proc Natl Acad Sci U S A 2004;101:9121–6.

38 Park K, Kim SW, RhuKS, Paick JS. Chronic admin-istration of an oral Rho kinase inhibitor prevents thedevelopment of vasculogenic erectile dysfunction ina rat model. J Sex Med 2006;3:996–1003.

39 Beg SA, Hansen-Schwartz JA, Vikman PJ, Xu CB,Edvinsson LI. ERK1/2 inhibition attenuates cere-bral blood flow reduction and abolishes ET(B) and5-HT(1B) receptor upregulation after subarachnoidhemorrhage in rat. J Cereb Blood Flow Metab2006;26:846–56.

40 Nilsson D, Wackenfors A, Gustafsson L, UganderM, Paulsson P, Ingemansson R, Edvinsson L,Malmsjo M. Endothelin receptor-mediated vasodi-latation: effects of organ culture. Eur J Pharmacol2008;579:233–40.

41 Henriksson M, Xu CB, Edvinsson L. Importanceof ERK1/2 in upregulation of endothelin type B

receptors in cerebral arteries. Br J Pharmacol 2004;142:1155–61.

42 Sommer F, Klotz T, Steinritz D, Schmidt A,Addicks K, Engelmann U, Bloch W. MAP kinase1/2 (Erk 1/2) and serine/threonine specific proteinkinase Akt/PKB expression and activity in thehuman corpus cavernosum. Int J Impot Res 2002;14:217–25.

43 Kim DC, Gondre CM, Christ GJ. Endothelin-1-induced modulation of contractile responses elicitedby an alpha 1-adrenergic agonist on human corpuscavernosum smooth muscle. Int J Impot Res 1996;8:17–24.

44 Christ GJ, Lerner SE, Kim DC, Melman A.Endothelin-1 as a putative modulator of erectiledysfunction: I. Characteristics of contraction of iso-lated corporal tissue strips. J Urol 1995;153:1998–2003.

45 Vazquez-Prado J, Medina LC, Garcia-Sainz JA.Activation of endothelin ETA receptors inducesphosphorylation of alpha1b-adrenoreceptors inRat-1 fibroblasts. J Biol Chem 1997;272:27330–7.

46 Kim NN, Dhir V, Azadzoi KM, Traish AM,Flaherty E, Goldstein I. Pilot study of theendothelin-A receptor selective antagonist BMS-193884 for the treatment of erectile dysfunction.J Androl 2002;23:76–83.

47 Fibbi B, Morelli A, Marini M, Zhang XH, MancinaR, Vignozzi L, Filippi S, Chavalmane A, SilvestriniE, Colli E, Adorini L, Vannelli GB, Maggi M. Ator-vastatin but not elocalcitol increases sildenafilresponsiveness in spontaneously hypertensive ratsby regulating the RhoA/ROCK pathway. J Androl2008;29:70–84.

48 Jin L, Lagoda G, Leite R, Webb RC, Burnett AL.NADPH oxidase activation: a mechanism ofhypertension-associated erectile dysfunction. J SexMed 2008;5:544–51.

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