chronic treatment with ang-(1-7) reverses abnormal reactivity in the corpus cavernosum and...

Research ArticleChronic Treatment with Ang-(1-7) Reverses Abnormal Reactivityin the Corpus Cavernosum and Normalizes Diabetes-InducedChanges in the Protein Levels of ACE ACE2 ROCK1 ROCK2and Omega-Hydroxylase in a Rat Model of Type 1 Diabetes

Mariam H M Yousif1 Batoul Makki1 Ahmed Z El-Hashim2

Saghir Akhtar1 and Ibrahim F Benter1

1 Department of Pharmacology and Toxicology Faculty of Medicine Kuwait University PO Box 24923 13110 Safat Kuwait2 Department of Pharmacology andTherapeutics Faculty of Pharmacy Kuwait University PO Box 24923 13110 Safat Kuwait

Correspondence should be addressed to Saghir Akhtar saghirhscedukw

Received 29 May 2014 Revised 31 July 2014 Accepted 1 August 2014 Published 16 September 2014

Academic Editor Mark A Yorek

Copyright copy 2014 Mariam H M Yousif et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Angiotensin-(1-7) [Ang-(1-7)] may have beneficial effects in diabetes mellitus-induced erectile dysfunction (DMIED) but itsmolecular actions in the diabetic corpus cavernosum (CC) are not known We characterized the effects of diabetes andor chronicin vivo administration of Ang-(1-7) on vascular reactivity in the rat corpus cavernosum (CC) and on protein expression levelsof potential downstream effectors of the renin-angiotensin-aldosterone system (RAAS) such as angiotensin-converting enzyme(ACE) ACE2 Rho kinases 1 and 2 (ROCK1 and ROCK2) and omega-hydroxylase the cytochrome-P450 enzyme that metabolizesarachidonic acid to form the vasoconstrictor 20-hydroxyeicosatetraenoic acid Streptozotocin-treated rats were chronicicallyadministered Ang-(1-7) with or without A779 a Mas receptor antagonist during weeks 4 to 6 of diabetes Ang-(1-7) reverseddiabetes-induced abnormal reactivity to vasoactive agents (endothelin-1 phenylepherine and carbachol) in the CC withoutcorrecting hyperglycemia Six weeks of diabetes led to elevated ACE ROCK1 ROCK 2 and omega-hydroxylase and a concomitantdecrease in ACE2 protein expression levels that were normalized by Ang-(1-7) treatment but not upon coadministration of A779These data are supportive of the notion that the beneficial effects of Ang-(1-7) in DMIED involve counterregulation of diabetes-induced changes in ACE ACE2 Rho kinases and omega-hydroxylase proteins in the diabetic CC via a Mas receptor-dependentmechanism

1 Introduction

Erectile dysfunction (ED) a measure of sexual dysfunctionor impotency in males is defined as the inability to achieveandor maintain an erection sufficient to permit satisfactorysexual intercourse It is commonly associated with diabetesmellitus (DM)with up to 75ofmenwith diabetes exhibitingsomedegree of erectile dysfunction (ED) [1ndash4]The incidenceof ED in large part also due to the predicted increase in DM[5 6] will rise to about 300 million sufferers worldwide by2025 [7 8] and represents a significant health burden

DM-induced ED (DMIED) is multifactorial in aetiol-ogy comprising both central (neurogenic) and peripheral

(vasculogenic) components and appears more severe andmore resistant to treatment compared with nondiabetic ED[9 10] For example treatmentwith phosphodiesterase (PDE)inhibitors such as sildenafil (Viagra) is not always effective inDMIED for reasons that are not entirely clear [3 7] Thusthere is a need for newer more effective therapies based onan increased understanding of the underlyingmechanisms ofDMIED

The exact molecular mechanisms by which DM inducesED are not fully known but chronic hyperglycemia likelydegrades both neural and vascular endothelium penile con-trol systems that eventually leads to a failure in the neuronalresponse andor increase in tone andor contractility of the

Hindawi Publishing CorporationJournal of Diabetes ResearchVolume 2014 Article ID 142154 10 pageshttpdxdoiorg1011552014142154

2 Journal of Diabetes Research

smooth muscle within the corpus cavernosum (CC) andpenile arteries [7 9] Experimental evidence suggests that thismay occur via hyperglycemia-induced modulation of nitricoxide (NO) signaling andor proinflammatory cell signalingpathways andor elevation in oxidative stress via severalpathways including increased glycolysis polyol pathway fluxformation of advanced glycation and lipoxygenation end-products [4 11 12] Additionally there is now a growingbody of evidence from our laboratory and others [7 12ndash14] on the existence of a local renin-angiotensin-aldosteronesystem (RAAS) in the penis that plays a critical role in erectilefunction

Angiotensin II (Ang II) a major effector of the RAAS isformed from the actions of angiotensin-converting enzyme(ACE) on Angiotensin 1 It is expressed in the corpuscavernosum and via its AT

1receptor activates signaling

pathways leading to vasoconstriction proliferation fibrosisand oxidative stress that are thought to play a detrimentalrole in the progression of DMIED [7 11ndash13 15] For examplewe recently reported in a rat model of type 1 diabetes thatAng II-mediated elevation in oxidative stress along witha concomitant decrease in antioxidant levels and increasedDNA damage resulted in major cellular degeneration withthe diabetic CC that could be blocked either by preventingthe formation of Ang II with an ACE inhibitor or by blockingits effects with an AT

1receptor antagonist [12]

In contrast to the detrimental ACEAng IIAT1recep-

tor ldquobranchrdquo of the RAAS there also now appears to bea counterregulating or opposing beneficial ldquobranchrdquo thatcomprises the angiotensin-converting enzyme 2 (ACE2) thatcan form the heptapeptide angiotensin-(1-7) (Ang-(1-7))from Ang II which mediates its effects via the G-proteincoupled receptor known as Mas The ACE2Ang-(1-7)Masreceptor pathway is known to oppose the detrimental effectsof ACEAng IIAT

1receptor in diabetes-induced cardio-

vascular complications [16ndash18] and recent evidence suggestsit may also be involved in DMIED [7 12ndash14 19] Severalstudies have now suggested that Ang-(1-7) has proerectilefunctions that include enhancing NO-mediated vasodilationinhibiting penile fibrosis and attenuating oxidative-stressmediated tissue degeneration [7 12ndash14 20 21] For examplewe have shown that Ang-(1-7) treatment opposed Ang II-induced oxidative stress and DNA damage that led to peniletissue degeneration in a rat model of type 1 diabetes [12]whereas acute ex vivo administration of Ang-(1-7) to diabeticrabbit CC segments was effective in attenuating diabetes orAng II-induced hyperreactivity suggesting a possible role forAng-(1-7) in the treatment of DMIED [14] Although thesestudies suggested that alterations in the fine balance betweenthe ACEAng IIAT

1receptor and the ACE2Ang-(1-7)Mas

receptor pathwayswill likely be important in the developmentof DMIED the exact molecular mechanisms underlying thedisease and how Ang-(1-7) treatment may impact on specificeffector molecules is not known in detail

2 Aims

Theaimof this studywas to characterize the effects of diabetesandor chronic administration of Ang-(1-7) on vascular

reactivity in the rat CC and in the presence or absenceof a MAS receptor antagonist on protein expression levelsin the rat CC of (a) ACE1 and ACE2 major componentsof the two counterregulatory branches of the RAAS (b)Rho kinases (ROCK) 1 and 2 important contractile reg-ulatory proteins known to be important mediators of ED[22 23] and (c) the cytochrome P450 4A1 enzyme omega-hydroxylase that metabolizes arachidonic acid (AA) to 20-hydroxyeicosatetraenoic acid (20-HETE) a potent vasocon-strictor [24ndash26] that we previously identified as a potentialtarget in the treatment of DMIED [19] Both ROCKs andomega-hydroxylase are known downstream effectors of AngII in the cardiovascular system [24 25 27 28] but the effectof Ang-(1-7) treatment on the expression of these proteins inthe diabetic CC is not known and therefore a subject of thisstudy

3 Methods

31 Induction of Diabetes Experiments were performed onmale Wistar rats weighing approximately 300 g The ratshad free access to food and water throughout the studyDiabetes was induced by a single ip injection (55mgkgbody weight) of streptozotocin (STZ) Rats with fasting bloodglucose levels above 250mgdL were considered diabetic andincluded in the study The animals were sacrificed six weeksafter inducing diabetes Drug treatment was administered forthe last 3 weeks prior to sacrificing the animals at 6 weeks

32 Animal Groups All studies involving animals were con-ducted in accordance with the National Institutes of HealthGuide for the Care and Use of Laboratory Animals (NIHPublication number 85-23 Revised in 1985) as approvedby Kuwait University Research Administration Animalsincluded in this study were divided into the following groups(119873 = 12group) Group 1 nondiabetic (control) animalsGroup 2 STZ-treated diabetic animals Group 3 STZ + Ang-(1-7) and Group 4 STZ + Ang-(1-7) + A779 (a Mas receptorantagonist) Ang-(1-7) was given at a dose of 576 120583gkgdayintraperitoneally (ip) and A779 at a dose of 744 120583gkgday ipbased on our previous studies in animal models [12 18 29ndash33]

33 Vascular Reactivity Experiments Rats were anesthetizedwith ketamine and then sacrificed and the penis was removeden bloc A ventral incisionwasmade and the cavernosal tissueexposedThe corpus cavernosum was cleaned of the adjacenttissue and cut into longitudinal strips of 2 times 10mm Stripsof the corpus cavernosum were suspended longitudinallyin organ-bath chambers containing 25mL Krebs Henseleit(KH) solution at pH 74 for measurement of isometric ten-sion [14 19] The composition of KH-solution was as follows(mM) NaCl (1183) KCl (47) CaCl

2(25) MgSO

4(12)

NaHCO3(25) KH

2PO4(12) and glucose (112) The tissue

bath solution was maintained at 37∘C and was aerated with95 oxygen and 5 carbon dioxidemixture Reactivity of theisolated strips of the corpus cavernosum was determined bymeasurement of changes in the isometric tension to vasoac-tive agonists using computerized automatic organ bath LSI

Journal of Diabetes Research 3

Letica Scientific Instruments (Powerlab8sp ADInstrumentsPanlab Spain) [14 19] The preparations were left for 45minwith changing KH solution at 15min intervals A pretensionof 10 gm was applied and the preparations were allowed tostabilize (45min) until a stable baseline tone was obtained

34 Effect of Vasoactive Agonists Cumulative concentrationresponse curveswere established to investigate the contractileresponses of the corpus cavernosum to the constrictor ago-nists phenylephrine (PE) and endothelin-1 (ET-1) Ascendingconcentrations of the agonists PE (10minus9ndash5 times 10minus3M) andET-1 (10minus10ndash10minus6M) were applied to establish a cumulativedose response curve using corpus cavernosal strips isolatedfrom the different animal groups The response to any givenconcentration of the agonists was left to stabilize beforeadding the next drug concentration In another set of exper-iments the relaxant responses to carbachol were investigatedin the cavernosal strips from the different animal groupsThe relaxant response to the agonists were determined afterprecontracting the tissues with submaximal dose of PE (3 times10minus7M) added to the organ baths After obtaining a steady

level of precontraction the relaxant effects to carbachol(10minus9ndash10minus5M) were tested on different preparations of thecorpus cavernosum The response to each concentration ofthe agonists was left to stabilize before adding the next drugconcentrationThe relaxant responses were expressed as per-centage reduction of the tension induced by precontractionwith PE

35Western Blotting Studies Western blotting forACE (banddetected at molecular weight of approximately 195 kDa)ACE2 (90ndash100 kDa) ROCK1 and ROCK2 (both at around160 kDa) and omega-hydroxylase (58 kDa) was performedsimilar to that described by us previously [32 34] Strips of thecorpus cavernosum were dissected from the different animalgroups cut into small pieces and homogenized in appro-priate amounts of lysis buffer (pH 76) containing 50mMTris-base 5mM EGTA 150mM NaCl 1 Triton 100 2mMNa3VO4 50mM NAF 1mM PMSF 20120583M phenylarsine

10mM sodium molybdate 10 120583gmL leupeptin and 8 120583gmLaprotinin The tissue was homogenized at half maximumspeed for 1 minute and then left to lyse completely byincubation on shaking ice-cold shaker for 1 hour Lysates werecentrifuged at 12000 rpm at 4∘C for 15 minutes Supernatantswere then collected and protein concentration was estimatedusing Lowryrsquos method Then the samples were mixed withsample loading buffer and lysis buffer to make all the samplesof the same protein concentration The samples were thenboiled for 5min and subjected to SDS polyacrylamide gelelectrophoresisThe separated proteins were then transferredto PVDF membrane for 1 hour at 95V After blockingmembranes were incubated with the primary antibodiesspecific for each protein overnight and subsequently withappropriate secondary antibodies conjugated to horseradishperoxidase (Amersham UK) The following antibodies wereused in this study ACE (H-170) sc-20791 ACE2 (H-175)sc-20998 ROCK1 (H-85) sc-5560 ROCK2 (H-85) sc-5561(Santa Cruz USA) and omega-hydroxylase (cytochrome

P450 4A1) ab22615 (Abcam USA) In addition the anti-120573-actin rabbit polyclonal IgG (1 120583L10mL) Cat number A-2066was obtained from Sigma Chemical Co USA Immunore-active bands were detected with SuperSignal Chemilumines-cent Substrate (Pierce UK) using Kodak autoradiographyfilm (GRI Rayne UK) To ensure equal loading of proteins120573-actin levels were detected using primary rabbit anti-human120573-actin antibody followed by the secondary anti-rabbit IgGhorseradish peroxidase conjugated antibody (Cell SignalingUSA) Images were finally analysed and quantified by densit-ometry and all data normalized to 120573-actin levels as describedby us previously [32 34]

36 Statistical Analysis Results were analyzed using Graph-Pad Prism software Data are presented as Mean plusmn SE ofldquo119899rdquo number of experiments Mean values were comparedusing either 2-way analysis of variance or analysis of variancefollowed by post hoc test (Bonferroni) as appropriate Thedifference was considered to be significant when 119875 value wasless than 005

361 Main Outcome Measures Vascular responsivenessof isolated CC strips to vasoactive agents (endothelin-1phenylephrine and carbachol) and protein levels were deter-mined by western blotting

4 Results

41 Blood Glucose Levels and Body Weights STZ-induceddiabetes led to a significant elevation in blood glucose con-centration Hyperglycemia persisted in the diabetic animalsand was 312 plusmn 17mmolsdotLminus1 after 6 weeks of diabetes ascompared with 47plusmn 07mmolsdotLminus1 in the nondiabetic controlanimals but 3-week chronic treatment with Ang-(1-7) (308 plusmn14mmolsdotLminus1) did not significantly reduce blood glucoselevels There was a significant reduction in the weights ofSTZ-diabetic rats (145 plusmn 8 g) compared with the nondiabeticcontrol animals (219 plusmn 5 g) after 6 weeks of diabetes whereasAng-(1-7) treatment significantly improved the weight ofdiabetic rats to 188 plusmn 11 g

42 Chronic Treatment with Ang-(1-7) Reverses Diabetes-Induced Abnormal Vascular Reactivity in the Rat CC Fig-ures 1 and 2 show that PE and ET-1 respectively pro-duced a concentration-dependent contraction while carba-chol (Figure 3) produced relaxation in the isolated CC fromboth nondiabetic control and diabetic rats In the CC tissuestrips from diabetic animals the constrictor responses to ET-1 and PE were significantly potentiated whereas the relaxantresponses to carbachol were significantly attenuated (119875 lt005) However diabetes-induced abnormal reactivity to thevasoactive agonists was significantly corrected in cavernosaltissues isolated fromdiabetic animals chronically treatedwithAng-(1-7) (119875 lt 005) (Figures 1ndash3)

43 Chronic Treatment with Ang-(1-7) Attenuates Diabetes-Induced Changes in ACE and ACE2 Protein Expressionvia Its Mas Receptor Figure 4 shows that 6 weeks of dia-betes led to significantly increased expression of ACE and


0

05

1

15

2

Con

trac

tions

(gm

gm

)

minus100 minus90 minus80 minus75

lowast

lowastlowast

ControlDiabetesDiabetes + Ang-(1-7)

Log[ET-1] (M)

Figure 1 Endothelin-1-induced vasoconstriction in the corpuscavernosum segments from nondiabetic (control) diabetic anddiabetic rats treated with Ang-(1-7) (MeanplusmnSEM 119899 = 6ndash8) Asterisk(lowast) indicates significantly different mean values as compared tocontrol and hash () indicates significantly different mean valuesas compared to diabetes 119875 lt 005 Tissues isolated from 6-weekdiabetic rats treated with Ang-(1-7) for the last 3 weeks of the study

0

1

2

3

4

5

Con

trac

tions

(gm

gm

)

minus70 minus60 minus50minus65 minus55

lowast

lowast

lowast

lowast


Log[PE] (M)

Figure 2 Phenylephrine-induced vasoconstriction in the corpuscavernosum segments from nondiabetic (control) diabetic anddiabetic rats treated with Ang-(1-7) (MeanplusmnSEM 119899 = 6ndash8) Asterisk(lowast) indicates significantly different mean values as compared tocontrol and hash () indicates significantly different mean valuesas compared to diabetes 119875 lt 005 Tissues isolated from 6-weekdiabetic rats treated with Ang-(1-7) for the last three weeks of thestudy

decreased expression ofACE2 proteins in the rat CC Chronictreatment with Ang-(1-7) significantly attenuated diabetes-induced changes in both proteins (119875 lt 005) Chroniccoadministration of the Mas receptor antagonist A779 withAng-(1-7) reversed the effects of Ang-(1-7) on ACE and ACEprotein expression in the diabetic rat CC (119875 lt 005)

0

20

40

60

80

Rela

xatio

n

minus80 minus70 minus60 minus50 minus40


Log[carbachol] (M)

lowast

lowast

lowast

lowast

Figure 3 Carbachol-induced relaxation (expressed as ) in thecorpus cavernosum segments from nondiabetic (control) diabeticand diabetic rats treated with Ang-(1-7) (Mean plusmn SEM 119899 = 6ndash8) Asterisk (lowast) indicates significantly different mean values ascompared to control and hash () indicates significantly differentmean values as compared to diabetes 119875 lt 005 Tissues isolatedfrom 6-week diabetic rats treated with Ang-(1-7) for the last threeweeks of the study

44 Chronic Ang-(1-7) Treatment Corrects Diabetes-InducedElevation in ROCK1 and ROCK2 Protein Expression via ItsMas Receptor Figure 5 shows that diabetes led to signifi-cantly increased protein expression of ROCK1 and ROCK2in the rat CC (119875 lt 005) Chronic treatment with Ang-(1-7) significantly attenuated diabetes-induced changes in bothROCK proteins (119875 lt 005) Chronic coadministration ofA779 with Ang-(1-7) reversed the effects of Ang-(1-7) onROCK1 and ROCK2 protein expression in the diabetic ratCC (119875 lt 005) to a level that was not significantly differentto nondiabetic controls (Figure 5)

45 Chronic Treatment with Ang-(1-7) Attenuates Diabetes-Induced Increase in Omega-Hydroxylase Protein Expressionvia Its Mas Receptor Figure 6 shows that diabetes led to sig-nificantly increased expression of omega-hydroxylase proteinin the rat CC (119875 lt 005) Chronic treatment with Ang-(1-7) significantly attenuated diabetes-induced changes inomega-hydroxylase protein expression (119875 lt 005) Chroniccoadministration of A779 with Ang-(1-7) significantly atten-uated the effects of Ang-(1-7) on omega-hydroxylase proteinexpression in the diabetic rat CC (119875 lt 005) (Figure 5)

5 Discussion

The major findings of this study were that diabetes ledto an upregulation in ACE ROCK1 ROCK2 and omega-hydroxylase proteins and a downregulation in ACE2 proteinthat was accompanied by abnormal vascular reactivity in the


ACE

ACE2

Actin

STZ

+A779

C +Ang-(1-7) +Ang-(1-7)

(a)

lowast

lowast

C

STZ

0

10

20

30

Rela

tive i

nten

sity

of

ACE

actin

+A779

+Ang-(1-7) +Ang-(1-7)

(b)

0

04

08

12

ACE2

act

inRe

lativ

e int

ensit

y of

lowast

lowast

C

STZ+A779

+Ang-(1-7) +Ang-(1-7)

(c)

Figure 4The levels of ACE and ACE2 protein expression in corpus cavernosum of nondiabetic (C) diabetic (STZ) and STZ-rats chronicallytreated either with Ang-(1-7) alone (STZ + Ang-(1-7)) or in combination with A779 (STZ + Ang-(1-7) + A779) Panel (a) is a representativewestern blot showing the levels of ACE ACE2 and the protein loading control 120573-actin Panels (b) and (c) are densitometry histogramsshowing levels of ACE and ACE2 normalized to actin respectively119873 = 4 MeanplusmnSD Asterisk (lowast) indicates significantly different (119875 lt 005)mean values from normal nondiabetic rats (C) whereas hash () indicates significantly different mean values (119875 lt 005) from diabetic rats(STZ)

rat CC Importantly chronic treatment with exogenous Ang-(1-7) was able to reverse diabetes-induced abnormal CC vas-cular reactivity without correcting hyperglycemia and via itsMas receptor normalized diabetes-induced changes in ACEACE2 ROCK1 ROCK2 and omega-hydroxylase proteins inthe rat CC Our study therefore showed for the first time thatan imbalance in the ACE-ACE2 enzymes is associated withdevelopment of DMIED and further highlights ROCKs andomega-hydroxylase as novel protein effectors by which Ang-(1-7) mediates its beneficial effects in DMIED Furthermoresince only a relatively short term 3-week chronic treatmentwith Ang-(1-7) in animals with preestablished diabetes was

effective in reversing DM-induced protein and vascularreactivity changes in the diabetic CC our study has clinicalrelevance in suggesting that therapeutic strategies aimedat activating the endogenous ACE2Ang-(1-7)Mas receptorsignaling pathway for even a short time course may bebeneficial in DMIED

The fact that in our study we were able to measuredetectable levels of ACE andACE2 in normal and diabetic CCprovided further evidence for the existence of a local renin-angiotensin system (RAS) within the CC [3 7 11] Moreoversince diabetes led to a significant upregulation of ACE anddownregulation of ACE2 in the rat CC (Figure 4) our data


ROCK1

ROCK2

Actin

STZ

+A779

C +Ang-(1-7) +Ang-(1-7)

(a)

0

8

16

24Re

lativ

e int

ensit

y of

RO

CK1

act

inlowast lowast

C

STZ+A779

+Ang-(1-7) +Ang-(1-7)

(b)

0

8

16

24

Rela

tive i

nten

sity

of

ROCK

2a

ctin

lowast

lowast

C

STZ+A779

+Ang-(1-7) +Ang-(1-7)

(c)

Figure 5 The levels of ROCK1 and ROCK2 protein expression in corpus cavernosum of nondiabetic (C) diabetic (STZ) and STZ-ratschronically treated either with Ang-(1-7) alone (STZ + Ang-(1-7)) or in combination with A779 (STZ + Ang-(1-7) + A779) Panel (a) is arepresentativewestern blot showing the levels of ROCK1 ROCK2 and the protein loading control120573-actin Panels (b) and (c) are densitometryhistograms showing levels of ROCK1 and ROCK2 normalized to actin respectively 119873 = 4 Mean plusmn SD Asterisk (lowast) indicates significantlydifferent (119875 lt 005) mean values from normal nondiabetic rats (C) whereas hash () indicates significantly different mean values (119875 lt 005)from diabetic rats (STZ)

implies that disruption in the ACE-ACE2 balance with aresulting hyperactivity of the detrimental ACEAng IIAT

1

receptor ldquobranchrdquo of the RAAS together with a concomi-tant attenuation of ACE2Ang-(1-7)Mas receptor signalingpathway is associated with the development of DMIEDOur finding in the diabteic rat CC is consistent with thedevelopment of other cardiovascular and renal pathologiesthat are also thought to involve an imbalance in ACE-ACE2enzymes representing the two opposing ldquobranchesrdquo of theRAAS [16 17 35]

The observed upregulation of ACE in the diabetic CC(Figure 4) which converts the inactive decapeptide Ang Iinto the octapeptide Ang II the major effector of the RAASis consistent with previous reports showing higher levels of

Ang II in the diabetic CC [36ndash38] that lead to deleteriouseffects such as vasoconstriction proliferation fibrosis andoxidative stress [7 12 15] Pharmacological blockage of AngIIAT

1actions using ACE inhibitors andor AT

1receptor

blockers (ARBs) has beneficial effects on erectile functionin animal models and humans [1 3 11ndash13] Since both ACEinhibitors and ARBs can increase Ang-(1-7) levels in plasmaand tissue [39] it is possible that the beneficial effects ofthe Ang II blockade on erectile function might at least inpart bemediated byAng-(1-7) Indeed we previously showedthat not only modulation of Ang II signaling by captopriland losartan but also administration of Ang-(1-7) restoredthe diabetes-induced structural changes and oxidative DNAdamage in the diabetic CC [12]


Actin

120596-Hydroxylase

STZ

+A779

C +Ang-(1-7) +Ang-(1-7)

(a)

0

4

8

12

16lowast

lowast

C

STZ

Rela

tive i

nten

sity

of

120596-h

ydro

xyla

sea

ctin

+A779

+Ang-(1-7) +Ang-(1-7)

(b)

Figure 6 The level of omega-hydroxylase protein expression in the corpus cavernosum of nondiabetic (C) diabetic (STZ) and STZ-ratschronically treated either with Ang-(1-7) alone (STZ + Ang-(1-7)) or in combination with A779 (STZ + Ang-(1-7) + A779) Panel (a) isa representative western blot showing the level of omega(120596)-hydroxylase protein and 120573-actin as a loading control Panels (b) and (C) aredensitometry histograms showing levels of omega(120596)-hydroxylase protein normalized to actin 119873 = 4 Mean plusmn SD Asterisk (lowast) indicatessignificantly different (119875 lt 005) mean values from normal nondiabetic rats (C) whereas hash () indicates significantly differentmean values(119875 lt 005) from diabetic rats (STZ)

Our observation that ACE2 is downregulated in diabetessuggests reduced formation of endogenous Ang-(1-7) in thediabetic rat CC though its levels were not measured directlyin this study Importantly our results showed that chronic3-week daily treatment with exogenous Ang-(1-7) was ableto reverse diabetes-induced changes in ACE1 and ACE2 andvascular reactivity in the rat CC presumably by activationof the endogenous ACE2Ang-(1-7)Mas receptor pathwayand readdressing of the proposed imbalances in the twocounterregulatory branches of the RAAS in the diabetic CC

The fact that administration of Ang-(1-7) also reverseddiabetes-induced upregulation of Rho kinases ROCK1 andROCK2 suggests a novelmechanismbywhichAng-(1-7)mayexerts its beneficial effects on DMIED Upregulation of theRhoROCK pathway has been implicated in a variety of car-diovascular complications including ED [13 15 22] and mayrepresent a downstream effector of Ang II ACEAng IIAT

1

receptor signaling is known to exert its vasoconstrictor effectson smooth muscle via its downstream effects on RhoROCKpathway [15 40 41] Thus ROCK upregulation might becontributing to the observed exaggerated responsiveness ofthe diabetic rat CC to vasoconstrictor agents (Figures 1 and2) and attenuated response to relaxant effects of carbachol(Figure 3) in a manner similar to that observed by us inthe diabetic mesenteric bed where diabetes-induced alteredvascular reactivity was corrected upon ROCK inhibition[34] Inhibition of ROCKs is also known to improve erectilefunction by a variety of mechanisms including increased

NO signaling and suppressing penile apoptosis and corporalfibrosis [11 42ndash44] As such ROCK inhibitors are increas-ingly being considered for the treatment of ED [1 11] Rho-kinase inhibitors improve erectile function in hypertensivediabetic and aged rats as well as in cavernous nerve injuryinduced rats [11 42] These reports clearly illustrate thatdirect inhibition of RhoARho-kinase pathwaymay representa suitable therapeutic approach in the treatment of EDHowever our data presented here suggests for the firsttime that chronic treatment with Ang-(1-7) can also reverseupregulation of ROCKs in the diabetic CC andmay representa novel alternative to conventional ROCK inhibitors for thetreatment of ED especially in DMIED Whether attenua-tion of ROCK protein expression in diabetic CC occursvia a direct effect of Ang-(1-7) or by counterregulation ofACEAng IIAT

1receptor pathway is not entirely clear and

requires further studyThe fact that diabetes led to an elevation in omega-

hydroxylase protein expression implies that DMIED is asso-ciated with increased production of 20-HETE-a poten-tial downstream effector of the ACEAng IIAT

1receptor

ldquobranchrdquo of the RAAS [24] though its levels were not mea-sured here and is a potential limitation of this study Howeverwe previously showed that Ang II via its AT

1receptors

increases 20-hydroxyeicosatetraenoic acid (20-HETE) pro-duction in vascular smooth muscle cells [28] It is now wellestablished that an imbalance in the metabolism of AA and20-HETE levels contributes to development of cardiovascular


dysfunction and end-organ damage [20 24 25 45ndash48] butthis is the first report in the CC showing an imbalance inomega-hydroxylase being associated with DMIED 20-HETEis a potent vasoconstrictor that has important roles in theregulation of vascular tone in several different tissues [24] Itsupregulation in the diabetic CC implies that it may have a sig-nificant role in contributing to the exaggerated vasoconstric-tor response to PE and ET-1 observed in the diabetic CC inthis study (Figures 1 and 2) Elevated 20-HETE production isalso known to occur in other vasculatures as well as the heartliver and kidney of diabetic animals [24 47 48] Indeed wewere the first to show that selective inhibition of 20-HETEproduction with N-hydroxy-N1015840-(4-butyl-2-methyl-phenyl)-formamidine (HET0016 an inhibitor of CYP450 4A1 omega-hydroxylase) can prevent diabetes-induced hyperreactivity tovasoconstrictors in the rat carotid artery [47] and can furtherattenuate cardiovascular end-organ damage in animalmodelsof diabetes andor hypertension [20 45 48] Interestinglyinhibition of 20-HETE also improved vasodilator responsesin several vascular beds [20 45 48] Thus removal of thevasoconstrictor signaling mediated via 20-HETE leads tocorrection of diabetes-induced abnormal responsiveness tovasoconstrictors and vasodilatorsThismay also be occurringin our present study in the CC where we observed exag-gerated CC vascular reactivity to PE and ET-1 (Figures 1and 2) and an attenuated responsiveness to the vasorelaxantcarbachol (Figure 3) after 6 weeks of diabetes Althoughnot directly studied here we previously showed that acuteex vivo inhibition of omega-hydroxylase with HET0016 inCC segments resulted in correction of abnormal reactivityto phenylephrine and carbachol in diabetic and aged rats[19] The fact that in the present study we observed anormalization of the abnormal CC reactivity upon chronictreatment with Ang-(1-7) suggests that Ang-(1-7) can alsocounterregulate omega-hydroxylase upregulation and thelikely increased 20-HETE production in the diabetic CCTaken together our data are therefore consistent with thehypothesis that ACEAng IIAT

1receptor arm of the RAAS

and likely downstream effectors omega-hydroxylase20-HETE and RhoROCK pathways are driving the vasocon-striction and other deleterious antierectile actions inDMIEDwhereas ACE2Ang-(1-7)Mas receptor signaling pathwayleads to vasorelaxation and generally beneficial or proerectileactions in DMIED

The data presented here is also consistent with previousreports on the beneficial actions of Ang-(1-7) in erectilefunction Both Ang-(1-7) and its Mas receptor are known tobe present in the human CC [11 21] Our group previouslyreported that Ang-(1-7) via Mas receptor can produce nitricoxide-dependent relaxation of the rabbit CC [14] We furthershowed that acute ex vivo preincubation of the diabeticrabbit CC with Ang-(1-7) corrected the diabetes-inducedhyperresponsiveness to Ang II [14] Ang-(1-7) has also beenshown to normalize the severely depressed erectile functionobserved in DOCA-salt hypertensive rats by activation ofMas receptor and subsequent NO release [21] and oraldelivery of Ang-(1-7) in a cyclodextrin formulation reducedpenile fibrosis and improved cavernosal endothelial functionin mice with hypercholesterolemia [7] Additionally also

the synthetic Mas receptor agonist AVE 0991 potentiatedrat penile erectile response [49] These studies suggest thatproerectile vasculogenic actions of the ACE2Ang-(1-7)Masreceptor axis involve increased cavernosal vasodilation byincreasing NO productionbioavailability [7 14 20 21 49]reduced oxidative-stress mediated damage to DNA andpenile tissue [12] and blocking development of penile fibrosis[7 13] Although the above studies and our data presentedhere suggest that Ang-(1-7) counteracts the ACEAng IIAT

1

receptor armof theRAAS in theCCvia itsMas receptor thereis a possibility that it may also block AT

1andor stimulate

AT2receptors in certain cell types and tissues [7] However

whether Ang-(1-7) in addition to its effects via Mas receptoraffects AT

1or AT

2receptors in the penis remains to be

studiedIn conclusion in this study we showed that diabetes-

induced abnormal cavernosal vascular reactivity a markerfor DMIED was accompanied by imbalances in ACE-ACE2ROCK1 ROCK2 and omega-hydroxylase proteins whichcould be reversed by 3-week chronic treatment with Ang-(1-7) via activation of its Mas receptor Whether more longer-term treatments with Ang-(1-7) would have any additionalbenefit remains to be determinedHowever despite the caveatthat only protein expression and not activity was measuredour study provides a novel insight into the mechanismof action of ACE2Ang-(1-7)Mas receptor pathway in thediabetic CC whereby we show for the first time that thebeneficial effects of Ang-(1-7) in DMIED are at least partiallydue to inhibition of omega-hydroxylase ROCKs and ACElevels through activation of its Mas receptorThus we furthersuggest that activating the endogenous ACE2Ang-(1-7)Masreceptor pathway of the RAAS system may represent anattractive strategy for the treatment of ED associated withdiabetes

Conflict of Interests

The authors report no conflict of interests

Authorsrsquo Contribution

Mariam HM Yousif Ahmed Z El-Hashim Saghir Akhtarand Ibrahim F Benter were responsible for conception anddesign of the paper and revising it for intellectual contentBatoul Makki and Mariam HM Yousif were responsibleforacquisition of data Mariam HM Yousif Batoul MakkiSaghir Akhtar and Ibrahim F Benter drafted the paperMariam HM Yousif Batoul Makki Ahmed Z El-HashimSaghir Akhtar and Ibrahim F Benter were responsible foranalysis and interpretation of data and the final approval ofthe completed paper

Acknowledgments

This study was funded by a grant from Kuwait Univer-sity Research Administration Project no MR0409 Theauthors also acknowledge support from the OMICS ResearchUnitRCF and the General Facility Grant (SRUL0213)


References

[1] S Gur T C Peak P J Kadowitz S C Sikka and W J GHellstrom ldquoReview of erectile dysfunction in diabetic animalmodelsrdquoCurrent Diabetes Reviews vol 10 no 1 pp 61ndash73 2014

[2] H Zheng X Liu and K P Patel ldquoCentrally mediated erectiledysfunction in rats with type 1 diabetes role of angiotensin IIand superoxiderdquo Journal of Sexual Medicine vol 10 no 9 pp2165ndash2176 2013

[3] K-E Andersson ldquoMechanisms of penile erection and basis forpharmacological treatment of erectile dysfunctionrdquo Pharmaco-logical Reviews vol 63 no 4 pp 811ndash859 2011

[4] V S Thorve A D Kshirsagar N S Vyawahare V S Joshi KG Ingale and R J Mohite ldquoDiabetes-induced erectile dysfunc-tion epidemiology pathophysiology andmanagementrdquo JournalofDiabetes and its Complications vol 25 no 2 pp 129ndash136 2011

[5] T Scully ldquoDiabetes in numbersrdquo Nature vol 485 pp S2ndashS32012

[6] D RWhiting LGuariguata CWeil and J Shaw ldquoIDFdiabetesAtlas global estimates of the prevalence of diabetes for 2011 and2030rdquo Diabetes Research and Clinical Practice vol 94 no 3 pp311ndash321 2011

[7] RA Fraga-Silva FMontecucco FMach RA S Santos andNStergiopulos ldquoPathophysiological role of the renin-angiotensinsystem on erectile dysfunctionrdquo European Journal of ClinicalInvestigation vol 43 no 9 pp 978ndash985 2013

[8] I AAytac J BMcKinlay andR J Krane ldquoThe likelyworldwideincrease in erectile dysfunction between 1995 and 2025 andsome possible policy consequencesrdquo BJU International vol 84no 1 pp 50ndash56 1999

[9] L S Malavige and J C Levy ldquoErectile dysfunction in diabetesmellitusrdquo Journal of Sexual Medicine vol 6 no 5 pp 1232ndash12472009

[10] D Price and G Hackett ldquoManagement of erectile dysfunctionin diabetes an update for 2008rdquo Current Diabetes Reports vol8 no 6 pp 437ndash443 2008

[11] K Decaluwe B Pauwels C Boydens and J van de VoordeldquoTreatment of erectile dysfunction new targets and strategiesfrom recent researchrdquo Pharmacology Biochemistry and Behav-ior vol 121 pp 146ndash157 2014

[12] R A Fraga-Silva F P Costa-Fraga S Q Savergnini et al ldquoAnoral formulation of Angiotensin-(1-7) reverses corpus caver-nosum damages induced by Hypercholesterolemiardquo Journal ofSexual Medicine vol 10 no 10 pp 2430ndash2442 2013

[13] N Kilarkaje M H M Yousif A Z El-Hashim B Makki SAkhtar and I F Benter ldquoRole of angiotensin II and angiotensin-(1-7) in diabetes-induced oxidative DNA damage in the corpuscavernosumrdquo Fertility and Sterility vol 100 no 1 pp 226ndash2332013

[14] M H Yousif E O Kehinde and I F Benter ldquoDifferentresponses to angiotensin-(1ndash7) in young aged and diabeticrabbit corpus cavernosumrdquo Pharmacological Research vol 56no 3 pp 209ndash216 2007

[15] L-M Jin ldquoAngiotensin II signaling and its implication inerectile dysfunctionrdquo Journal of Sexual Medicine vol 6 no 3pp 302ndash310 2009

[16] V B Patel N Parajuli and G Y Oudit ldquoRole of angiotensin-converting enzyme 2 (ACE2) in diabetic cardiovascular com-plicationsrdquo Clinical Science vol 126 no 7 pp 471ndash482 2014

[17] R A S Santos A J Ferreira T Verano-Braga and MBader ldquoAngiotensin-converting enzyme 2 angiotensin-(1-7)

and Mas new players of the renin-angiotensin systemrdquo Journalof Endocrinology vol 216 no 2 pp R1ndashR17 2013

[18] I F Benter M H M Yousif C Cojocel M Al-Maghrebiand D I Diz ldquoAngiotensin-(1-7) prevents diabetes-inducedcardiovascular dysfunctionrdquo American Journal of PhysiologyHeart and Circulatory Physiology vol 292 no 1 pp H666ndashH672 2007

[19] M H M Yousif and I F Benter ldquoRole of cytochrome P450metabolites of arachidonic acid in regulation of corporalsmooth muscle tone in diabetic and older ratsrdquo VascularPharmacology vol 47 no 5-6 pp 281ndash287 2007

[20] M H M Yousif I F Benter K M J Dunn A J Dahly-VernonS Akhtar andR J Roman ldquoRole of 20-hydroxyeicosatetraenoicacid in altering vascular reactivity in diabetesrdquo Autonomic andAutacoid Pharmacology vol 29 no 1-2 pp 1ndash12 2009

[21] A C Da Costa Goncalves R Leite R A Fraga-Silva et alldquoEvidence that the vasodilator angiotensin-(1-7)-Mas axis playsan important role in erectile functionrdquo The American Journalof PhysiologymdashHeart and Circulatory Physiology vol 293 no 4pp H2588ndashH2596 2007

[22] J Shi and LWei ldquoRho kinases in cardiovascular physiology andpathophysiology the effect of fasudilrdquo Journal of CardiovascularPharmacology vol 62 no 4 pp 341ndash354 2013

[23] T J Bivalacqua H C Champion M F Usta et al ldquoRhoARho-kinase suppresses endothelial nitric oxide synthase in the penisa mechanism for diabetes-associated erectile dysfunctionrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 101 no 24 pp 9121ndash9126 2004

[24] AMAlsaad BN ZordokyMMTse andAO El-Kadi ldquoRoleof cytochrome P450-mediated arachidonic acid metabolitesin the pathogenesis of cardiac hypertrophyrdquo Drug MetabolismReviews vol 45 no 2 pp 173ndash195 2013

[25] F Fan C-W Sun K G Maier et al ldquo20-Hydroxyeicos-atetraenoic acid contributes to the inhibition of K+ channelactivity and vasoconstrictor response to angiotensin II in ratrenal microvesselsrdquo PLoS ONE vol 8 no 12 Article ID e824822013

[26] D L Kroetz and F Xu ldquoRegulation and inhibition of arachi-donic acid 120596-hydroxylases and 20-HETE formationrdquo AnnualReview of Pharmacology and Toxicology vol 45 pp 413ndash4382005

[27] J F Giani M M Gironacci M C Munoz D Turyn andF P Dominici ldquoAngiotensin-(1-7) has a dual role on growth-promoting signalling pathways in rat heart in vivo by stim-ulating STAT3 and STAT5ab phosphorylation and inhibitingangiotensin II-stimulated ERK12 and Rho kinase activityrdquoExperimental Physiology vol 93 no 5 pp 570ndash578 2008

[28] M M Muthalif I F Benter M R Uddin J L Harperand K U Malik ldquoSignal transduction mechanisms involvedin angiotensin-(1-7)-stimulated arachidonic acid release andprostanoid synthesis in rabbit aortic smooth muscle cellsrdquoJournal of Pharmacology and Experimental Therapeutics vol284 no 1 pp 388ndash398 1998

[29] I F Benter M H M Yousif F M Al-Saleh R RaghupathyM C Chappell and D I Diz ldquoAngiotensin-(1-7) blockadeattenuates captopril- or hydralazine-induced cardiovascularprotection in spontaneously hypertensive rats treated withNG-nitro-l-arginine methyl esterrdquo Journal of CardiovascularPharmacology vol 57 no 5 pp 559ndash567 2011

[30] A Z El-Hashim W M Renno R Raghupathy H T AbduoS Akhtar and I F Benter ldquoAngiotensin-(1-7) inhibits allergicinflammation via the MAS1 receptor through suppression of


ERK12- and NF-120581B-dependent pathwaysrdquo British Journal ofPharmacology vol 166 no 6 pp 1964ndash1976 2012

[31] WM Renno A G Al-Banaw P George A A Abu-Ghefreh SAkhtar and I F Benter ldquoAngiotensin-(1-7) via the mas receptoralleviates the diabetes-induced decrease in GFAP and GAP-43 immunoreactivity with concomitant reduction in the COX-2 in hippocampal formation an immunohistochemical studyrdquoCellular and Molecular Neurobiology vol 32 no 8 pp 1323ndash1336 2012

[32] S Akhtar M H Yousif G S Dhaunsi B Chandrasekhar OAl-Farsi and I F Benter ldquoAngiotensin-(1ndash7) inhibits epidermalgrowth factor receptor transactivation via a Mas receptor-dependent pathwayrdquo British Journal of Pharmacology vol 165no 5 pp 1390ndash1400 2012

[33] M H M Yousif G S Dhaunsi B M Makki B A Qabazard SAkhtar and I F Benter ldquoCharacterization of Angiotensin-(1-7)effects on the cardiovascular system in an experimental modelof Type-1 diabetesrdquo Pharmacological Research vol 66 no 3 pp269ndash275 2012

[34] S Akhtar M H M Yousif G S Dhaunsi et al ldquoActivation ofErbB2 and downstream signalling via Rho kinases and ERK12contributes to diabetes-induced vascular dysfunctionrdquo PLoSONE vol 8 no 6 Article ID e67813 2013

[35] M Bader ldquoACE2 angiotensin-(1-7) and Mas The other side ofthe coinrdquo Pflugers Archiv European Journal of Physiology vol465 no 1 pp 79ndash85 2013

[36] Y Chen S X Li L S Yao R Wang and Y T Dai ldquoValsartantreatment reverses erectile dysfunction in diabetic ratsrdquo Inter-national Journal of Impotence Research vol 19 no 4 pp 366ndash370 2007

[37] M T Abdel Aziz M F El Asmer T Mostafa et al ldquoEffectsof Losartan HO-1 Inducers or HO-1 inhibitors on erectilesignaling in diabetic ratsrdquo Journal of Sexual Medicine vol 6 no12 pp 3254ndash3264 2009

[38] I Kifor G HWilliamsM A VickersM P Sullivan P Jodbertand R G Dluhy ldquoTissue angiotensin II as a modulator oferectile function I Angiotensin peptide content secretion andeffects in the corpus cavernosumrdquo Journal of Urology vol 157no 5 pp 1920ndash1925 1997

[39] D Iusuf R H Henning W H van Gilst and A J M RoksldquoAngiotensin-(1-7) pharmacological properties and pharma-cotherapeutic perspectivesrdquo European Journal of Pharmacologyvol 585 no 2-3 pp 303ndash312 2008

[40] Z Ying L Jin T Palmer and R C Webb ldquoAngiotensin IIup-regulates the leukemia-associated Rho guanine nucleotideexchange factor (RhoGEF) a regulator of G protein signalingdomain-containing RhoGEF in vascular smooth muscle cellsrdquoMolecular Pharmacology vol 69 no 3 pp 932ndash940 2006

[41] A V Schofield and O Bernard ldquoRho-associated coiled-coilkinase (ROCK) signaling and diseaserdquo Critical Reviews inBiochemistry and Molecular Biology vol 48 no 4 pp 301ndash3162013

[42] J L Hannan M Albersen O Kutlu et al ldquoInhibition of Rho-kinase improves erectile function increases nitric oxide signal-ing and decreases penile apoptosis in a rat model of cavernousnerve injuryrdquo Journal of Urology vol 189 no 3 pp 1155ndash11612013

[43] MCChoK Park J S Chai SH Lee SWKim and J S PaickldquoInvolvement of sphingosine-1-PhosphateRhoARho-Kinasesignaling pathway in corporal fibrosis following cavernousnerve injury in male ratsrdquo Journal of Sexual Medicine vol 8 no3 pp 712ndash721 2011

[44] H A Toque K P Nunes L Yao et al ldquoActivated rho kinasemediates diabetes-induced elevation of vascular arginase acti-vation and contributes to impaired corpora cavernosa relax-ation possible involvement of p38 MAPK activationrdquo Journalof Sexual Medicine vol 10 no 6 pp 1502ndash1515 2013

[45] M H M Yousif I F Benter and R J Roman ldquoCytochromeP450metabolites of arachidonic acid play a role in the enhancedcardiac dysfunction in diabetic rats following ischaemic reper-fusion injuryrdquo Autonomic and Autacoid Pharmacology vol 29no 1-2 pp 33ndash41 2009

[46] M H M Yousif and I F Benter ldquoRole of 20-hydroxyeicosa-tetraenoic and epoxyeicosatrienoic acids in the regulation ofvascular function in a model of hypertension and endothelialdysfunctionrdquo Pharmacology vol 86 no 3 pp 149ndash156 2010

[47] I F Benter M H Yousif H Canatan and S Akhtar ldquoInhibi-tion of Ca2+calmodulin -dependent protein kinase II RAS-GTPase and 20-hydroxyeicosatetraenoic acid attenuates thedevelopment of diabetes-induced vascular dysfunction in therat carotid arteryrdquo Pharmacological Research vol 52 no 3 pp252ndash257 2005

[48] I F Benter I Francis C Cojocel J S Juggi M H M YousifandHCanatan ldquoContribution of cytochromeP450metabolitesof arachidonic acid to hypertension and end-organ damagein spontaneously hypertensive rats treated with L-NAMErdquoAutonomic and Autacoid Pharmacology vol 25 no 4 pp 143ndash154 2005

[49] A C da Costa Goncalves R A Fraga-Silva R Leite and RA S Santos ldquoAVE 0991 a non-peptide Mas-receptor agonistfacilitates penile erectionrdquo Experimental Physiology vol 98 no3 pp 850ndash855 2013


smooth muscle within the corpus cavernosum (CC) andpenile arteries [7 9] Experimental evidence suggests that thismay occur via hyperglycemia-induced modulation of nitricoxide (NO) signaling andor proinflammatory cell signalingpathways andor elevation in oxidative stress via severalpathways including increased glycolysis polyol pathway fluxformation of advanced glycation and lipoxygenation end-products [4 11 12] Additionally there is now a growingbody of evidence from our laboratory and others [7 12ndash14] on the existence of a local renin-angiotensin-aldosteronesystem (RAAS) in the penis that plays a critical role in erectilefunction

Angiotensin II (Ang II) a major effector of the RAAS isformed from the actions of angiotensin-converting enzyme(ACE) on Angiotensin 1 It is expressed in the corpuscavernosum and via its AT

1receptor activates signaling

pathways leading to vasoconstriction proliferation fibrosisand oxidative stress that are thought to play a detrimentalrole in the progression of DMIED [7 11ndash13 15] For examplewe recently reported in a rat model of type 1 diabetes thatAng II-mediated elevation in oxidative stress along witha concomitant decrease in antioxidant levels and increasedDNA damage resulted in major cellular degeneration withthe diabetic CC that could be blocked either by preventingthe formation of Ang II with an ACE inhibitor or by blockingits effects with an AT

1receptor antagonist [12]

In contrast to the detrimental ACEAng IIAT1recep-

tor ldquobranchrdquo of the RAAS there also now appears to bea counterregulating or opposing beneficial ldquobranchrdquo thatcomprises the angiotensin-converting enzyme 2 (ACE2) thatcan form the heptapeptide angiotensin-(1-7) (Ang-(1-7))from Ang II which mediates its effects via the G-proteincoupled receptor known as Mas The ACE2Ang-(1-7)Masreceptor pathway is known to oppose the detrimental effectsof ACEAng IIAT

1receptor in diabetes-induced cardio-

vascular complications [16ndash18] and recent evidence suggestsit may also be involved in DMIED [7 12ndash14 19] Severalstudies have now suggested that Ang-(1-7) has proerectilefunctions that include enhancing NO-mediated vasodilationinhibiting penile fibrosis and attenuating oxidative-stressmediated tissue degeneration [7 12ndash14 20 21] For examplewe have shown that Ang-(1-7) treatment opposed Ang II-induced oxidative stress and DNA damage that led to peniletissue degeneration in a rat model of type 1 diabetes [12]whereas acute ex vivo administration of Ang-(1-7) to diabeticrabbit CC segments was effective in attenuating diabetes orAng II-induced hyperreactivity suggesting a possible role forAng-(1-7) in the treatment of DMIED [14] Although thesestudies suggested that alterations in the fine balance betweenthe ACEAng IIAT

1receptor and the ACE2Ang-(1-7)Mas

receptor pathwayswill likely be important in the developmentof DMIED the exact molecular mechanisms underlying thedisease and how Ang-(1-7) treatment may impact on specificeffector molecules is not known in detail

2 Aims

Theaimof this studywas to characterize the effects of diabetesandor chronic administration of Ang-(1-7) on vascular

reactivity in the rat CC and in the presence or absenceof a MAS receptor antagonist on protein expression levelsin the rat CC of (a) ACE1 and ACE2 major componentsof the two counterregulatory branches of the RAAS (b)Rho kinases (ROCK) 1 and 2 important contractile reg-ulatory proteins known to be important mediators of ED[22 23] and (c) the cytochrome P450 4A1 enzyme omega-hydroxylase that metabolizes arachidonic acid (AA) to 20-hydroxyeicosatetraenoic acid (20-HETE) a potent vasocon-strictor [24ndash26] that we previously identified as a potentialtarget in the treatment of DMIED [19] Both ROCKs andomega-hydroxylase are known downstream effectors of AngII in the cardiovascular system [24 25 27 28] but the effectof Ang-(1-7) treatment on the expression of these proteins inthe diabetic CC is not known and therefore a subject of thisstudy

3 Methods

31 Induction of Diabetes Experiments were performed onmale Wistar rats weighing approximately 300 g The ratshad free access to food and water throughout the studyDiabetes was induced by a single ip injection (55mgkgbody weight) of streptozotocin (STZ) Rats with fasting bloodglucose levels above 250mgdL were considered diabetic andincluded in the study The animals were sacrificed six weeksafter inducing diabetes Drug treatment was administered forthe last 3 weeks prior to sacrificing the animals at 6 weeks

32 Animal Groups All studies involving animals were con-ducted in accordance with the National Institutes of HealthGuide for the Care and Use of Laboratory Animals (NIHPublication number 85-23 Revised in 1985) as approvedby Kuwait University Research Administration Animalsincluded in this study were divided into the following groups(119873 = 12group) Group 1 nondiabetic (control) animalsGroup 2 STZ-treated diabetic animals Group 3 STZ + Ang-(1-7) and Group 4 STZ + Ang-(1-7) + A779 (a Mas receptorantagonist) Ang-(1-7) was given at a dose of 576 120583gkgdayintraperitoneally (ip) and A779 at a dose of 744 120583gkgday ipbased on our previous studies in animal models [12 18 29ndash33]

33 Vascular Reactivity Experiments Rats were anesthetizedwith ketamine and then sacrificed and the penis was removeden bloc A ventral incisionwasmade and the cavernosal tissueexposedThe corpus cavernosum was cleaned of the adjacenttissue and cut into longitudinal strips of 2 times 10mm Stripsof the corpus cavernosum were suspended longitudinallyin organ-bath chambers containing 25mL Krebs Henseleit(KH) solution at pH 74 for measurement of isometric ten-sion [14 19] The composition of KH-solution was as follows(mM) NaCl (1183) KCl (47) CaCl

2(25) MgSO

4(12)

NaHCO3(25) KH

2PO4(12) and glucose (112) The tissue

bath solution was maintained at 37∘C and was aerated with95 oxygen and 5 carbon dioxidemixture Reactivity of theisolated strips of the corpus cavernosum was determined bymeasurement of changes in the isometric tension to vasoac-tive agonists using computerized automatic organ bath LSI










4 Results





0

05

1

15

2

Con

trac

tions

(gm

gm

)


lowast

lowastlowast


Log[ET-1] (M)


0

1

2

3

4

5

Con

trac

tions

(gm

gm

)


lowast

lowast

lowast

lowast


Log[PE] (M)



0

20

40

60

80

Rela

xatio

n



Log[carbachol] (M)

lowast

lowast

lowast

lowast




5 Discussion



ACE

ACE2

Actin

STZ

+A779

C +Ang-(1-7) +Ang-(1-7)

(a)

lowast

lowast

C

STZ

0

10

20

30

Rela

tive i

nten

sity

of

ACE

actin

+A779

+Ang-(1-7) +Ang-(1-7)

(b)

0

04

08

12

ACE2

act

inRe

lativ

e int

ensit

y of

lowast

lowast

C

STZ+A779

+Ang-(1-7) +Ang-(1-7)

(c)






ROCK1

ROCK2

Actin

STZ

+A779

C +Ang-(1-7) +Ang-(1-7)

(a)

0

8

16

24Re

lativ

e int

ensit

y of

RO

CK1

act

inlowast lowast

C

STZ+A779

+Ang-(1-7) +Ang-(1-7)

(b)

0

8

16

24

Rela

tive i

nten

sity

of

ROCK

2a

ctin

lowast

lowast

C

STZ+A779

+Ang-(1-7) +Ang-(1-7)

(c)



1





1receptor



Actin

120596-Hydroxylase

STZ

+A779

C +Ang-(1-7) +Ang-(1-7)

(a)

0

4

8

12

16lowast

lowast

C

STZ

Rela

tive i

nten

sity

of

120596-h

ydro

xyla

sea

ctin

+A779

+Ang-(1-7) +Ang-(1-7)

(b)




1






1receptor


1receptors








1


1andor stimulate



1or AT








Acknowledgments



References






























































4 Results





0

05

1

15

2

Con

trac

tions

(gm

gm

)


lowast

lowastlowast


Log[ET-1] (M)


0

1

2

3

4

5

Con

trac

tions

(gm

gm

)


lowast

lowast

lowast

lowast


Log[PE] (M)



0

20

40

60

80

Rela

xatio

n



Log[carbachol] (M)

lowast

lowast

lowast

lowast




5 Discussion



ACE

ACE2

Actin

STZ

+A779

C +Ang-(1-7) +Ang-(1-7)

(a)

lowast

lowast

C

STZ

0

10

20

30

Rela

tive i

nten

sity

of

ACE

actin

+A779

+Ang-(1-7) +Ang-(1-7)

(b)

0

04

08

12

ACE2

act

inRe

lativ

e int

ensit

y of

lowast

lowast

C

STZ+A779

+Ang-(1-7) +Ang-(1-7)

(c)






ROCK1

ROCK2

Actin

STZ

+A779

C +Ang-(1-7) +Ang-(1-7)

(a)

0

8

16

24Re

lativ

e int

ensit

y of

RO

CK1

act

inlowast lowast

C

STZ+A779

+Ang-(1-7) +Ang-(1-7)

(b)

0

8

16

24

Rela

tive i

nten

sity

of

ROCK

2a

ctin

lowast

lowast

C

STZ+A779

+Ang-(1-7) +Ang-(1-7)

(c)



1





1receptor



Actin

120596-Hydroxylase

STZ

+A779

C +Ang-(1-7) +Ang-(1-7)

(a)

0

4

8

12

16lowast

lowast

C

STZ

Rela

tive i

nten

sity

of

120596-h

ydro

xyla

sea

ctin

+A779

+Ang-(1-7) +Ang-(1-7)

(b)




1






1receptor


1receptors








1


1andor stimulate



1or AT








Acknowledgments



References






















































0

05

1

15

2

Con

trac

tions

(gm

gm

)


lowast

lowastlowast


Log[ET-1] (M)


0

1

2

3

4

5

Con

trac

tions

(gm

gm

)


lowast

lowast

lowast

lowast


Log[PE] (M)



0

20

40

60

80

Rela

xatio

n



Log[carbachol] (M)

lowast

lowast

lowast

lowast




5 Discussion



ACE

ACE2

Actin

STZ

+A779

C +Ang-(1-7) +Ang-(1-7)

(a)

lowast

lowast

C

STZ

0

10

20

30

Rela

tive i

nten

sity

of

ACE

actin

+A779

+Ang-(1-7) +Ang-(1-7)

(b)

0

04

08

12

ACE2

act

inRe

lativ

e int

ensit

y of

lowast

lowast

C

STZ+A779

+Ang-(1-7) +Ang-(1-7)

(c)






ROCK1

ROCK2

Actin

STZ

+A779

C +Ang-(1-7) +Ang-(1-7)

(a)

0

8

16

24Re

lativ

e int

ensit

y of

RO

CK1

act

inlowast lowast

C

STZ+A779

+Ang-(1-7) +Ang-(1-7)

(b)

0

8

16

24

Rela

tive i

nten

sity

of

ROCK

2a

ctin

lowast

lowast

C

STZ+A779

+Ang-(1-7) +Ang-(1-7)

(c)



1





1receptor



Actin

120596-Hydroxylase

STZ

+A779

C +Ang-(1-7) +Ang-(1-7)

(a)

0

4

8

12

16lowast

lowast

C

STZ

Rela

tive i

nten

sity

of

120596-h

ydro

xyla

sea

ctin

+A779

+Ang-(1-7) +Ang-(1-7)

(b)




1






1receptor


1receptors








1


1andor stimulate



1or AT








Acknowledgments



References






















































ACE

ACE2

Actin

STZ

+A779

C +Ang-(1-7) +Ang-(1-7)

(a)

lowast

lowast

C

STZ

0

10

20

30

Rela

tive i

nten

sity

of

ACE

actin

+A779

+Ang-(1-7) +Ang-(1-7)

(b)

0

04

08

12

ACE2

act

inRe

lativ

e int

ensit

y of

lowast

lowast

C

STZ+A779

+Ang-(1-7) +Ang-(1-7)

(c)






ROCK1

ROCK2

Actin

STZ

+A779

C +Ang-(1-7) +Ang-(1-7)

(a)

0

8

16

24Re

lativ

e int

ensit

y of

RO

CK1

act

inlowast lowast

C

STZ+A779

+Ang-(1-7) +Ang-(1-7)

(b)

0

8

16

24

Rela

tive i

nten

sity

of

ROCK

2a

ctin

lowast

lowast

C

STZ+A779

+Ang-(1-7) +Ang-(1-7)

(c)



1





1receptor



Actin

120596-Hydroxylase

STZ

+A779

C +Ang-(1-7) +Ang-(1-7)

(a)

0

4

8

12

16lowast

lowast

C

STZ

Rela

tive i

nten

sity

of

120596-h

ydro

xyla

sea

ctin

+A779

+Ang-(1-7) +Ang-(1-7)

(b)




1






1receptor


1receptors








1


1andor stimulate



1or AT








Acknowledgments



References






















































ROCK1

ROCK2

Actin

STZ

+A779

C +Ang-(1-7) +Ang-(1-7)

(a)

0

8

16

24Re

lativ

e int

ensit

y of

RO

CK1

act

inlowast lowast

C

STZ+A779

+Ang-(1-7) +Ang-(1-7)

(b)

0

8

16

24

Rela

tive i

nten

sity

of

ROCK

2a

ctin

lowast

lowast

C

STZ+A779

+Ang-(1-7) +Ang-(1-7)

(c)



1





1receptor



Actin

120596-Hydroxylase

STZ

+A779

C +Ang-(1-7) +Ang-(1-7)

(a)

0

4

8

12

16lowast

lowast

C

STZ

Rela

tive i

nten

sity

of

120596-h

ydro

xyla

sea

ctin

+A779

+Ang-(1-7) +Ang-(1-7)

(b)




1






1receptor


1receptors








1


1andor stimulate



1or AT








Acknowledgments



References






















































Actin

120596-Hydroxylase

STZ

+A779

C +Ang-(1-7) +Ang-(1-7)

(a)

0

4

8

12

16lowast

lowast

C

STZ

Rela

tive i

nten

sity

of

120596-h

ydro

xyla

sea

ctin

+A779

+Ang-(1-7) +Ang-(1-7)

(b)




1






1receptor


1receptors








1


1andor stimulate



1or AT








Acknowledgments



References



























































1


1andor stimulate



1or AT








Acknowledgments



References






















































References





















































chronic treatment with ang-(1-7) reverses abnormal reactivity in the corpus cavernosum and...

Documents