(1-7) In Diabetes-Induced Erectile Dysfunctio

Research ArticleAngiotensin-(1-7) Downregulates Diabetes-Induced cGMPPhosphodiesterase Activation in Rat Corpus Cavernosum

Gursev S Dhaunsi1 Mariam Yousif2 Batoul Makki2

Saghir Akhtar2 and Ibrahim F Benter2

1Department of Pediatrics Faculty of Medicine Kuwait University Jabriya Kuwait2Department of Pharmacology and Toxicology Faculty of Medicine Kuwait University Jabriya Kuwait

Correspondence should be addressed to Gursev S Dhaunsi dhaunsighscedukw

Received 29 September 2016 Revised 24 January 2017 Accepted 30 January 2017 Published 19 February 2017

Academic Editor Fabrizio Montecucco

Copyright copy 2017 Gursev S Dhaunsi 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

Molecular mechanisms of the beneficial effects of angiotensin-(1-7) Ang-(1-7) in diabetes-related complications including erectiledysfunction remain unclear We examined the effect of diabetes andor Ang-(1-7) treatment on vascular reactivity and cyclicguanosine monophosphate (cGMP) phosphodiesterase (PDE) in corpus cavernosum Male Wistar rats were grouped as (1)control (2) diabetic (streptozotocin STZ treated) (3) control + Ang-(1-7) and (4) diabetic + Ang-(1-7) Following 3 weeks ofAng-(1-7) treatment subsequent to induction of diabetes rats were sacrificed Penile cavernosal tissue was isolated to measurevascular reactivity PDE gene expression and activity and levels of p38MAP kinase nitrites and cGMP Carbachol-inducedvasorelaxant response after preincubation of corpus cavernosumwith PEwas significantly attenuated in diabetic rats andAng-(1-7)markedly corrected the diabetes-induced impairment Gene expression and activity of PDE and p38MAP kinase were significantlyincreased in cavernosal tissue of diabetic rats and Ang-(1-7) markedly attenuated STZ-induced effects Ang-(1-7) significantlyincreased the levels of nitrite and cGMP in cavernosal tissue of control and diabetic rats Cavernosal tissue of diabetic rats hadsignificantly reduced cGMP levels and Ang-(1-7) markedly prevented the STZ-induced cGMP depletion This study demonstratesthat attenuation of diabetes-induced PDE activity might be one of the key mechanisms in the beneficial effects of Ang-(1-7)

1 Introduction

Diabetes is a major risk factor for erectile dysfunction (ED)Diabetic ED is multifactorial in etiology and is more severeand more resistant to treatment compared with nondiabeticED [1ndash3] The prevalence of ED in diabetic men is estimatedto be threefold higher than in the general population Dia-betes degrades both neural and vascular endothelium penilecontrol systems [2]

Experimental evidence suggests that hyperglycemiacauses elevated oxidative stress and the formation ofadvanced glycation and lipoxygenation end products thatlead to functional loss and degenerative changes Recentwork has implicated both the renin-angiotensin-aldosteronesystem (RAAS) and reactive oxygen species (ROS) inthe pathogenesis and vascular complications of diabetes[4 5] These mechanisms have effects on the regulation of

nitric oxide (NO) synthase expression and activity and onproinflammatory cell signaling pathways [6] Penile erectionresults from the relaxation of smooth muscle mediated bya spinal reflex to the sensory and mental stimuli In theL-argininendashNOndashguanylyl cyclasendashcyclic guanosine mono-phosphate (cGMP) pathway cGMP is the intracellulartrigger that activates cGMP-dependent protein kinase tophosphorylate several proteins that bring about erectionthrough relaxation of arterial and trabecular smooth musclein corpus cavernosum due to reduced intracellular calciumlevels Since cGMP plays a key role in this process potentialinterventions for inadequate smooth muscle relaxationand erectile dysfunction include elevating the intracellularcGMP levels through inhibition of phosphodiesterase (PDE)activity PDE normally inhibits penile erection by depletingcGMP At least more than 10 PDE families have beenidentified but PDE5 is the predominant phosphodiesterase

HindawiBioMed Research InternationalVolume 2017 Article ID 5084961 7 pageshttpsdoiorg10115520175084961

2 BioMed Research International

in the corpus cavernosum [7] It has been demonstrated thattreatment with PDE inhibitors such as sildenafil (Viagra)is less effective in diabetic patients for unknown reasonsand gene therapy with endothelial NO synthase fails tototally restore erectile function in diabetes [8] Furthermorethese therapies do not address the underlying causes ofdiabetes-induced ED

We have previously reported that older and diabeticrabbits showed impaired Ang-(1-7)-mediated relaxation sug-gesting that aging and diabetes-related erectile dysfunction(ED) may be partly due to decreased Ang-(1-7)-mediatedrelaxation of the corpus cavernosum [9] In addition wehave also found that acute preincubation with Ang-(1-7) waseffective in attenuating Ang II-induced contraction of rabbitcorpus cavernosum suggesting that the possible role of Ang-(1-7) in treatment of ED should be investigated In this studypresented in part at the 3rdWorldCongress onPharmacology[10] we investigated the effect of Ang-(1-7) on PDE at themolecular level in corpus cavernosum of diabetic rats

2 Materials and Methods

21 Materials Wistar rats (male weighing approximately300 g) used in this study were supplied by the Animal HouseHealth Sciences Center Kuwait University Angiotensin-(1-7) norepinephrine bitartrate and carbachol were all pur-chased from Sigma Chemical Co (St Louis USA)

22 Methods

221 Animal Groups Animal studies were conducted inaccordance with the National Institutes of Health Guide forthe Care and Use of Laboratory Animals (NIH Publicationnumber 85-23 Revised 1985) as approved by Kuwait Univer-sity Research Administration

Animals included in this study were divided into the fol-lowing groups (119873 = 8group) Group 1 nondiabetic (control)animals Group 2 diabetic animals Group 3 control + Ang-(1-7) Group 4 diabetic + Ang-(1-7) Diabetes was induced bya single intraperitoneal injection (55mgkg body weight) ofstreptozotocin (STZ) Rats with fasting blood glucose levelsabove 250mgdL were declared diabetic and included in thestudy Ang-(1-7) was given at a dose of 576 120583gkgday afterthree weeks of diabetes induction and continued for the last 3weeks prior to sacrificing the animalsDoses of the drugs usedin this study were based on our previous findings in modelsof hypertension and diabetes [11 12] The rats had free accessto food and water throughout the study

The animals were sacrificed after six weeks of inducingdiabetes Rats were anesthetized with ketamine and the peniswas removed en bloc after sacrificing the animals A ventralincisionwasmade to expose the cavernosal tissueThe corpuscavernosum was cleaned of the adjacent tissue and cut intolongitudinal strips of 2 times 10mm

222 Vascular Reactivity Studies Strips of the corpus caver-nosum were suspended longitudinally in organ bath cham-bers [13] containing 25mL Krebs Henseleit (KH) solution

at pH 74 The composition of KH solution was as follows(mM) NaCl (1183) KCl (47) CaCl2 (25) MgSO4 (12)NaHCO3 (25) KH2PO4 (12) and glucose (112) The tissuebath solution was maintained at 37∘C and was aerated with95 oxygen and 5 carbon dioxide mixture Reactivity ofthe isolated strips of the corpus cavernosum was determinedby measurement of changes in the isometric tension to Ang-(1-7) and carbachol using computerized automatic organbath LSI (Power Lab8sp AD Instruments Pan Lab Spain)Letica Scientific Instruments [9 14] The preparations wereleft to stabilize for 45min with changing KH solution at15min intervals A pretension of 10 gm was applied andthe preparations were left until a stable baseline tone wasobtained The relaxant effect to the vasodilator agonist Ang-(1-7) was determined after precontracting the tissues withphenylephrine (PE) (3 times 10minus7M) added to the organ bathsAfter obtaining a steady level of precontraction the vasodila-tor effect to Ang-(1-7) (10minus12ndash10minus5M) was tested on differentpreparations of the corpus cavernosumThe response to eachconcentration of the agonist was left to stabilize before addingthe next drug concentration The relaxant responses wereexpressed as percentage reduction of the tension inducedby precontraction with PE Based on the constructed dose-response curves Emax (themaximal percentage of relaxationattained) and ED50 (the concentration of Ang-(1-7) requiredto reduce the induced tone by 50) were calculated usingGraph Pad Prism software In another group of experimentsthe effect of incubation with Ang-(1-7) on the responsivenessof the corpus cavernosum to carbachol was investigated Acumulative concentration response curve was established forcarbachol as described earlier [13] The corpus cavernosalstrips were then incubated with KH solution containing Ang-(1-7) (3 times 10minus6) for 20min Following the incubation periodcumulative concentration response curves for carbachol wereestablished in the presence of Ang-(1-7)

223 Assay of Cyclic GMP Phosphodiesterase (PDE) ActivityExperiments were carried out to determine the levels ofphosphodiesterase in the corpus cavernosum tissues from thedifferent groups in the study PDE activity was measured inpenile tissue homogenates using a luminescence-based kitfrom Promega (Madison WI USA) Briefly the PDE assaywas started by adding substrate (cGMP 10 120583M final concen-tration) to the reaction mixture containing PDE buffer and aknown amount of tissue homogenate PDE-Glo terminationbuffer containing 3-isobutyl-1-methylxanthine (IBMX) wasadded to stop the reaction Luminescence was measuredusing a plate-reading luminometer following addition ofPDE-Glo detection solution provided in the kit Specificactivity PDE was calculated as Relative Light Units (RLU)120583gof tissue protein

224 Western Blotting Studies Western blotting for phos-phorylated forms of p38MAP kinase (p-p38Map kinase) wasperformed as described previously [15] Strips of the corpuscavernosum were cut into small pieces and homogenizedin appropriate amounts of lysis buffer protease inhibitorand phosphatase inhibitor cocktails A and B Lysates were

BioMed Research International 3

centrifuged 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 loaded in the 12 SDS poly-acrylamide gel for electrophoresis Separated proteins werethen transferred to PVDF membrane for detection of p-p38 MAP kinase After blocking with buffer Blotto B mem-branes were incubated with the specific primary antibodiesovernight then washed with TBST and incubated with thehorseradish peroxidase-conjugated secondary antibody for2 hours Protein bands were detected with Super Signalchemiluminescent substrate

225 RT-PCR Detection of cGMP-PDE Total RNA was iso-lated from corpus cavernosum using Qiagen kit including anRNase-free DNase treatment to prevent coamplification ofgenomic DNA The concentration of the purified RNAwas determined spectrophotometrically at 260 nm and theintegrity was checked by agarose gel electrophoresis indenaturing condition The reverse transcription was per-formed using iScriptH cDNA synthesis kit from Bio-RadGAPDH house-keeping gene was used for normalizationSpecific primers for PDE-5 and GAPDH used in this studyhad the following sequences PDE-5 forward 51015840-TTGGAG-AGCCCTTGAACATCA-31015840 reverse 51015840-GTAGCCTGT-AATTTGGTCAACTTCTG-31015840 GAPDH forward 51015840-AAG-GTCGGAGTCAACGGATTT-31015840 reverse 51015840-ACCAGA-GTTAAAAGCAGCCCTG-31015840The transcript levels of PDE-5and GAPDH were quantified by PCR using iTaq DNA poly-merase under the following amplification conditions 95∘Cfor 30 sec 58∘C or 62∘C for 30 sec 68∘C for 45 sec and 40cycles after denaturing at 95∘C for 1min Specificity of ampli-fication products was assessed by their size using agarose gelelectrophoresis

226 Measurement of Nitrites and cGMP Levels of nitritesand index ofNOgeneration weremeasured in serum and cellculture supernatants using Greiss reaction assay according tothe kit purchased from Calbiochem (CA) Levels of cGMPwere determined using the cGMP Immunoassay Kit (RampDSystems IncMinneapolisMN) following themanufacturerrsquosinstructions The levels were normalized by total protein ineach sample

227 Statistical Analysis Sigmoidal dose-response curveswere established using nonlinear regression The concen-tration of the drug is shown on the 119909-axis while the 119910-axis plots response The 119883 values are logarithm of drugsrsquoconcentrations Data were analyzed using Graph Pad Prismsoftware and presented as mean plusmn SEM of ldquo119899rdquo numberof experiments as described previously The difference wasconsidered to be significant when 119901 value was less than 005

3 Results

31 Ex Vivo Relaxation Effect of Ang-(1-7) on Corpus Ca-vernosum Figure 1 shows that Ang-(1-7) (10minus12ndash10minus5M)produced concentration-dependent relaxation of the corpus

ControlSTZ

lowast lowast

lowast

lowast

lowast

lowast

lowast

0

20

40

60

80

100

re

laxa

tion

minus12 minus10 minus8 minus6 minus4minus14log [Ang-(1-7)] (M)

Figure 1 Ang-(1-7) induced relaxation (10minus12ndash3times 10minus5M) in the iso-lated corpus cavernosal strips from (◼) control and (998771) STZ-induceddiabetic rats Tissues were precontracted with phenylephrine (3 times10minus8M) Mean plusmn SEM (119899 = 5) (lowastsignificantly different compared tocontrol 119901 lt 005)

Control STZ

minus9 minus8 minus7 minus6 minus5

Log [carbachol] (M)

0

20

40

60

80

100

re

laxa

tion

lowastlowast

lowast

lowast lowast

STZ + Ang-(1-7) 10minus5 MControl + Ang-(1-7) 10minus5 M

Figure 2 Effect of incubationwithAng-(1-7) (10minus6M)on carbachol-induced relaxation in the isolated corpus cavernosal strips fromdiabetic ratsMeanplusmn SEM (119899 = 5) (lowastsignificantly different comparedto control 119901 lt 005 significantly different compared to STZ 119901 lt005)

cavernosum isolated from control and STZ-diabetic ratsTherelaxant response to Ang-(1-7) was significantly impairedin diabetic corpus cavernosum strips compared to controlnondiabetic rats (Figure 1) The maximal relaxation to Ang-(1-7) was significantly reduced in the corpus cavernosumisolated from diabetic rats The maximal percentagerelaxation (Emax) induced by Ang-(1-7) was 91 plusmn 2 and51 plusmn 4 in the control and diabetic tissues respectively TheED50 values for Ang-(1-7) were significantly not different inthe corpus cavernosum strips of control and diabetic rats79 plusmn 01 and 75 plusmn 01 respectively The difference betweenthe ED50 values in the two groups was not significantCarbachol-induced relaxation of the corpus cavernosumwas significantly reduced in the diabetic groups compared


Control STZ Ang-(1-7) STZ + Ang-(1-7)0

25

50

75

100

125

150

Nitr

ite le

vels

(as

of c

ontro

l)

lowastlowast

Figure 3 Nitrite levels in corpus cavernosum tissues from controland diabetic rats withwithout Ang-(1-7) treatment Values aremeanplusmn SEM of six measurements and lowast119901 lt 005 when compared withcontrol

Control STZ Ang-(1-7) STZ + Ang-(1-7)

lowast

lowast

0

25

50

75

100

125

150

cGM

P le

vels

(as

of c

ontro

l)

lowast

Figure 4 Levels of cGMP in corpus cavernosum tissues fromcontrol and diabetic rats withwithout Ang-(1-7) treatment Valuesare mean plusmn SEM of six measurements lowast119901 lt 005 when comparedwith control and 119901 lt 001when compared with STZ-treated group

with the controls (Figure 2) Preincubation of the corpuscavernosum tissues with Ang-(1-7) (10minus5M) resulted insignificant enhancement in carbachol-induced relaxantresponses in the tissues isolated from diabetic rats

32 Effect of Ang-(1-7) on Levels of Nitrites and cGMP inCorpus Cavernosum of Control and Diabetic Rats In view ofthe observed relaxation effects of Ang-(1-7) levels of NO andcGMPweremeasured in corpus cavernosum tissue of controland diabetic rats (Figures 3 and 4) Nitrite levels in tissuesfrom diabetic rats were not markedly different from controlshowever treatment with Ang-(1-7) significantly (119901 lt 005)increased the nitrite content in both diabetic and controltissues (Figure 3) Figure 4 shows that cGMP levels weresignificantly (119901 lt 001) decreased in corpus cavernosum ofdiabetic rats when compared with control tissues and Ang-(1-7) treatment significantly restored the diabetes-induceddepletion of cGMP Ang-(1-7) treatment markedly increasedthe cGMP levels in controls tissues also


100

200

300

400

500

PDE

activ

ity (a

s o

f con

trol)

lowast

lowast

Figure 5 PDE levels (shown as percent of control) in corpuscavernosal tissues from control STZ-diabetic and Ang-(1-7) treateddiabetic Values are mean plusmn SEM of six measurements lowast119901 lt 005when compared with control and 119901 lt 001 when compared withSTZ-treated group

33 Effect of Ang-(1-7) on cGMP Phosphodiesterase in CorpusCavernosum of Control and Diabetic Rats Enzymatic activityof PDE was significantly (119901 lt 0001) higher in corpus caver-nosum of diabetic rats when compared with control tissues(Figure 5) Ang-(1-7) treatment did not have any markedeffect on the PDE activity in control tissues but significantly(119901 lt 001) reduced the STZ-induced increase in PDE activityRT-PCR studies revealed that diabetes-induced enhancementof PDE activity was due to an induction of the PDE-5 geneexpression (Figure 6) STZ treatment significantly increasedthe PDE mRNA content in corpus cavernosum and thediabetes-mediated increase in gene expression of PDE wasdownregulated by Ang-(1-7) treatment as evidenced by asignificant decrease (119901 lt 005) in PDE mRNA levels Ang-(1-7) however did not influence the gene expression of PDEin control tissues

34 Effect of Ang-(1-7) on p38MAP Kinase in Corpus Caver-nosum of ControlDiabetic Rats Western blot studies (Fig-ure 7) revealed that protein expression of p38MAP kinasewas significantly (119901 lt 001) higher in corpus cavernosum ofdiabetic rats than the control tissue suggesting an associationof p38MAP kinase with observed diabetes-induced increasein PDE activity Ang-(1-7) treatment produced slight reduc-tion in p38MAP kinase protein content of control corpuscavernosum but significantly (119901 lt 005) corrected thediabetes-induced increase in protein expression of p38MAPkinase

4 Discussion

Diabetes is a multifaceted metabolic disorder with anexpanding list of clinical complications Diabetes-relatederectile dysfunction is a well-known effect of long termhyperglycemia and associated metabolic disturbances how-ever its pathogenic mechanisms have remained unclear Wereported earlier that Ang-(1-7) has beneficial effects againstdiabetes-induced erectile dysfunction and this study now


Control STZ

(a)

(b)Ang-(1-7) STZ + Ang-(1-7)

Ratio

of P

DE

GA

PDH

mRN

A

0

100

200

300

(as

of c

ontro

l) lowast

PDE-5GAPDH

Figure 6 Gene expression of PDE-5 in corpus cavernosum tissues of control and diabetic rats withwithout Ang-(1-7) treatment Values(mean plusmn SEM of six measurements) presented as percent of control are the ratio of PDE and GAPDH mRNA lowast119901 lt 005 when comparedwith control and 119901 lt 001 when compared with STZ-treated group


lowast

0

1

2

3

4

5

6

Nor

mal

ized

leve

l of p

-p38

p-p38

Actin

(a)

(b)

Figure 7 Western blot analysis of p38MAP kinase from control control-Ang-(1-7) diabetic and diabetic-Ang-(1-7) treated animals (meanplusmn SEM 119899 = 4) (lowast119901 lt 005 compared to control 119901 lt 005 compared to diabetic)

demonstrates that Ang-(1-7) downregulates the gene expres-sion of cGMP-PDE in corpus cavernosum of diabetic ratsthereby suggesting a new molecular mechanism related todiabetes-induced erectile dysfunction and the protective roleof Ang-(1-7)

Our findings in this study show that acute ex vivoadministration of Ang-(1-7) produces significant relaxingresponses in corpus cavernosum of diabetic rats and alsocorrects the diabetes-induced impairment of carbachol relax-ant responses These results further support our previousin vivo studies on chronic treatment with Ang-(1-7) and

strengthening the notion that Ang-(1-7) is a potent thera-peutic tool to correct erectile dysfunction [13] The fact thatAng-(1-7) treatment significantly increased the levels of NO amajor vasodilator known to play a key role in normal erectilefunction through activation of soluble guanylyl cyclase [1617] and cGMP in corpus cavernosum of control and STZ-treated rats in this study suggests that the observed relaxationeffects of Ang-(1-7) are mediated via NOcGMP Howevera marked decrease in the cGMP levels with unaltered NOlevels in corpus cavernosum of diabetic rats suggests thatprotective effect of Ang-(1-7) against erectile dysfunction


might not be solely NO-mediated but likely occurs throughregulation of PDE A remarkable increase in PDE enzymeactivity in corpus cavernosum of diabetic rats justifies atleast in part the depletion of diabetes-induced depletionof cGMP in this study and indicates that activation ofPDE under hyperglycemic conditions may be a contributingfactor for erectile dysfunction Though Ang-(1-7) did notinfluence the PDE activity in control tissues it markedlyabated the diabetes-induced activation of PDE suggestingthat Ang-(1-7) triggers compensatory responses to fallingintracellular cGMP levels possibly through downregulationof PDE Our findings that gene expression of PDE-5 wassignificantly enhanced in corpus cavernosum of diabeticrats and Ang-(1-7) markedly mitigated the STZ-inducedoverexpression further indicating that Ang-(1-7)-mediatedrelaxation response might occur by increasing cGMP levelsthrough transcriptional regulation of PDE geneThe fact thatAng-(1-7) treatment does not affect PDE gene expressionin control tissue reaffirms our notion that cGMP depletionpossibly triggers the Ang-(1-7)-mediated response of PDEactivation

Diabetes being a multifactorial disease involves a widerange of pathophysiological events that include developmentof stress due to overproduction of cytokines and reactiveoxygen species [18ndash20] It remains to be investigated if cellularoxidative stress is at all associated with findings of this studyhowever we have reported earlier that Ang-(1-7) attenuatesdiabetes-induced activation of NADPH oxidase in kidneys[21] There are a number of reports that strongly link acti-vation of p38MAP kinase with increased cellular oxidativestress [22 23] and indication of an increase in p38MAPkinaseprotein expression in corpus cavernosum of diabetic rats aswas observed in this study suggesting involvement of cel-lular oxidative stress in diabetes-related erectile dysfunctionand supportive of earlier reports regarding association ofhyperglycemia with cellular redox [24] Observed effect ofAng-(1-7) on p38 kinase in control and STZ-treated tissueswarrants further studies to examine possible association ofp38MAP kinase with PDE regulation in corpus cavernosumThe findings reported herein provide a new insight into thepossible molecular mechanisms of diabetes-induced erectiledysfunction and the beneficial effects of Ang-(1-7)

Competing Interests

There is no conflict of interests financial or any other in thisstudy

Authorsrsquo Contributions

Gursev S Dhaunsi planned and conducted the studyanalyzed biochemical data and wrote major part of themanuscriptMariamYousif planned and conducted the studyanalyzed data from vascular reactivity analyzed the resultsand contributed significantly in writing the manuscriptBatoul Makki carried out the experiments and collected dataSaghir Akhtar analyzed data and contributed in manuscriptwriting Ibrahim F Benter planned the study analyzed dataand contributed in manuscript writing

Acknowledgments

Authors are thankful to Mrs Bindu Chandrasekhar MrsNini Mathews and Mrs Heba Dalvi for their technicalassistance

References

[1] V Phe and M Roupret ldquoErectile dysfunction and diabetes areview of the current evidence-based medicine and a synthesisof the main available therapiesrdquo Diabetes and Metabolism vol38 no 1 pp 1ndash13 2012

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

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

[4] B Musicki and A L Burnett ldquoEndothelial dysfunction in dia-betic erectile dysfunctionrdquo International Journal of ImpotenceResearch vol 19 no 2 pp 129ndash138 2007

[5] 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

[6] N F Gonzalez-Cadavid ldquoMechanisms of penile fibrosisrdquo Jour-nal of Sexual Medicine vol 6 S3 pp 353ndash362 2009

[7] J D Corbin ldquoMechanisms of action of PDE5 inhibition in erec-tile dysfunctionrdquo International Journal of Impotence Researchvol 16 no 1 pp S4ndashS7 2004

[8] T J Bivalacqua M F Usta H C Champion P J Kadowitzand W J G Hellstrom ldquoEndothelial dysfunction in erectiledysfunction role of the endothelium in erectile physiology anddiseaserdquo Journal of Andrology vol 24 no 6 pp S17ndashS37 2003

[9] M H M Yousif E O Kehinde and I F Benter ldquoDifferentresponses to angiotensin-(1-7) in young aged and diabeticrabbit corpus cavernosumrdquo Pharmacological Research vol 56no 3 pp 209ndash216 2007

[10] M H M Yousif G S Dhaunsi S Akhtar and I F BenterldquoAngiotensin-(1ndash7) corrects diabetes-induced activation ofphosphodiesterase in rat corpus cavernosumrdquo in Proceedings ofthe 3rd World Congress on Pharmacology vol 6 no 4 pp 226ndash233 Birmingham UK August 2016

[11] M M Muthalif I F Benter M R Uddin J L Harper andK U Malik ldquoSignal transduction mechanisms involved inangiotensin-(1-7)-stimulated arachidonic acid release and pros-tanoid synthesis in rabbit aortic smoothmuscle cellsrdquo Journal ofPharmacology and ExperimentalTherapeutics vol 284 no 1 pp388ndash398 1998

[12] 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 PhysiologymdashHeart and Circulatory Physiology vol 292 no 1 pp H666ndashH672 2007

[13] M H M Yousif B Makki A Z El-Hashim S Akhtar and IF Benter ldquoChronic treatment with ang-(1-7) reverses abnormalreactivity in the corpus cavernosum and normalizes diabetes-induced changes in the protein levels of ACE ACE2 ROCK1ROCK2 and omega-hydroxylase in a rat model of type 1diabetesrdquo Journal of Diabetes Research vol 2014 Article ID142154 10 pages 2014


[14] 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

[15] S Akhtar M HM Yousif G S Dhaunsi B Chandrasekhar OAl-Farsi and I F Benter ldquoAngiotensin-(1-7) inhibits epidermalgrowth factor receptor transactivation via a Mas receptor-dependent pathwayrdquo British Journal of Pharmacology vol 165no 5 pp 1390ndash1400 2012

[16] J Rajfer W J Aronson P A Bush F J Dorey and L JIgnarro ldquoNitric oxide as a mediator of relaxation of the corpuscavernosum in response to nonadrenergic noncholinergicneurotransmissionrdquo New England Journal of Medicine vol 326no 2 pp 90ndash94 1992

[17] M Boolell S Gepi-Attee J C Gingell and M J AllenldquoSildenafil a novel effective oral therapy for male erectiledysfunctionrdquo British Journal of Urology vol 78 no 2 pp 257ndash261 1996

[18] J Donate-Correa E Martın-Nunez M Muros-de-FuentesC Mora-Fernandez and J F Navarro-Gonzalez ldquoInflamma-tory cytokines in diabetic nephropathyrdquo Journal of DiabetesResearch vol 2015 Article ID 948417 9 pages 2015

[19] V Rani G Deep R K Singh K Palle and U C S YadavldquoOxidative stress and metabolic disorders pathogenesis andtherapeutic strategiesrdquo Life Sciences vol 148 pp 183ndash193 2016

[20] Y Kayama U Raaz A Jagger et al ldquoDiabetic cardiovasculardisease induced by oxidative stressrdquo International Journal ofMolecular Sciences vol 16 no 10 pp 25234ndash25263 2015

[21] I F Benter M H M Yousif G S Dhaunsi J Kaur M CChappell and D I Diz ldquoAngiotensin-(1ndash7) prevents activationof NADPH oxidase and renal vascular dysfunction in diabetichypertensive ratsrdquo American Journal of Nephrology vol 28 no1 pp 25ndash33 2008

[22] Y Son S Kim H-T Chung and H-O Pae ldquoReactive oxygenspecies in the activation of MAP kinasesrdquoMethods in Enzymol-ogy vol 528 pp 27ndash48 2013

[23] F Jiang Y Zhang and G J Dusting ldquoNADPH oxidase-mediated redox signaling roles in cellular stress response stresstolerance and tissue repairrdquo Pharmacological Reviews vol 63no 1 pp 218ndash242 2011

[24] R F Mapanga and M F Essop ldquoDamaging effects of hyper-glycemia on cardiovascular function spotlight on glucosemetabolic pathwaysrdquo American Journal of PhysiologymdashHeartand Circulatory Physiology vol 310 no 2 pp H153ndashH173 2016

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MEDIATORSINFLAMMATION

of


in the corpus cavernosum [7] It has been demonstrated thattreatment with PDE inhibitors such as sildenafil (Viagra)is less effective in diabetic patients for unknown reasonsand gene therapy with endothelial NO synthase fails tototally restore erectile function in diabetes [8] Furthermorethese therapies do not address the underlying causes ofdiabetes-induced ED

We have previously reported that older and diabeticrabbits showed impaired Ang-(1-7)-mediated relaxation sug-gesting that aging and diabetes-related erectile dysfunction(ED) may be partly due to decreased Ang-(1-7)-mediatedrelaxation of the corpus cavernosum [9] In addition wehave also found that acute preincubation with Ang-(1-7) waseffective in attenuating Ang II-induced contraction of rabbitcorpus cavernosum suggesting that the possible role of Ang-(1-7) in treatment of ED should be investigated In this studypresented in part at the 3rdWorldCongress onPharmacology[10] we investigated the effect of Ang-(1-7) on PDE at themolecular level in corpus cavernosum of diabetic rats

2 Materials and Methods

21 Materials Wistar rats (male weighing approximately300 g) used in this study were supplied by the Animal HouseHealth Sciences Center Kuwait University Angiotensin-(1-7) norepinephrine bitartrate and carbachol were all pur-chased from Sigma Chemical Co (St Louis USA)

22 Methods

221 Animal Groups Animal studies were conducted inaccordance with the National Institutes of Health Guide forthe Care and Use of Laboratory Animals (NIH Publicationnumber 85-23 Revised 1985) as approved by Kuwait Univer-sity Research Administration

Animals included in this study were divided into the fol-lowing groups (119873 = 8group) Group 1 nondiabetic (control)animals Group 2 diabetic animals Group 3 control + Ang-(1-7) Group 4 diabetic + Ang-(1-7) Diabetes was induced bya single intraperitoneal injection (55mgkg body weight) ofstreptozotocin (STZ) Rats with fasting blood glucose levelsabove 250mgdL were declared diabetic and included in thestudy Ang-(1-7) was given at a dose of 576 120583gkgday afterthree weeks of diabetes induction and continued for the last 3weeks prior to sacrificing the animalsDoses of the drugs usedin this study were based on our previous findings in modelsof hypertension and diabetes [11 12] The rats had free accessto food and water throughout the study

The animals were sacrificed after six weeks of inducingdiabetes Rats were anesthetized with ketamine and the peniswas removed en bloc after sacrificing the animals A ventralincisionwasmade to expose the cavernosal tissueThe corpuscavernosum was cleaned of the adjacent tissue and cut intolongitudinal strips of 2 times 10mm

222 Vascular Reactivity Studies Strips of the corpus caver-nosum were suspended longitudinally in organ bath cham-bers [13] containing 25mL Krebs Henseleit (KH) solution

at pH 74 The composition of KH solution was as follows(mM) NaCl (1183) KCl (47) CaCl2 (25) MgSO4 (12)NaHCO3 (25) KH2PO4 (12) and glucose (112) The tissuebath solution was maintained at 37∘C and was aerated with95 oxygen and 5 carbon dioxide mixture Reactivity ofthe isolated strips of the corpus cavernosum was determinedby measurement of changes in the isometric tension to Ang-(1-7) and carbachol using computerized automatic organbath LSI (Power Lab8sp AD Instruments Pan Lab Spain)Letica Scientific Instruments [9 14] The preparations wereleft to stabilize for 45min with changing KH solution at15min intervals A pretension of 10 gm was applied andthe preparations were left until a stable baseline tone wasobtained The relaxant effect to the vasodilator agonist Ang-(1-7) was determined after precontracting the tissues withphenylephrine (PE) (3 times 10minus7M) added to the organ bathsAfter obtaining a steady level of precontraction the vasodila-tor effect to Ang-(1-7) (10minus12ndash10minus5M) was tested on differentpreparations of the corpus cavernosumThe response to eachconcentration of the agonist was left to stabilize before addingthe next drug concentration The relaxant responses wereexpressed as percentage reduction of the tension inducedby precontraction with PE Based on the constructed dose-response curves Emax (themaximal percentage of relaxationattained) and ED50 (the concentration of Ang-(1-7) requiredto reduce the induced tone by 50) were calculated usingGraph Pad Prism software In another group of experimentsthe effect of incubation with Ang-(1-7) on the responsivenessof the corpus cavernosum to carbachol was investigated Acumulative concentration response curve was established forcarbachol as described earlier [13] The corpus cavernosalstrips were then incubated with KH solution containing Ang-(1-7) (3 times 10minus6) for 20min Following the incubation periodcumulative concentration response curves for carbachol wereestablished in the presence of Ang-(1-7)

223 Assay of Cyclic GMP Phosphodiesterase (PDE) ActivityExperiments were carried out to determine the levels ofphosphodiesterase in the corpus cavernosum tissues from thedifferent groups in the study PDE activity was measured inpenile tissue homogenates using a luminescence-based kitfrom Promega (Madison WI USA) Briefly the PDE assaywas started by adding substrate (cGMP 10 120583M final concen-tration) to the reaction mixture containing PDE buffer and aknown amount of tissue homogenate PDE-Glo terminationbuffer containing 3-isobutyl-1-methylxanthine (IBMX) wasadded to stop the reaction Luminescence was measuredusing a plate-reading luminometer following addition ofPDE-Glo detection solution provided in the kit Specificactivity PDE was calculated as Relative Light Units (RLU)120583gof tissue protein

224 Western Blotting Studies Western blotting for phos-phorylated forms of p38MAP kinase (p-p38Map kinase) wasperformed as described previously [15] Strips of the corpuscavernosum were cut into small pieces and homogenizedin appropriate amounts of lysis buffer protease inhibitorand phosphatase inhibitor cocktails A and B Lysates were






3 Results


ControlSTZ

lowast lowast

lowast

lowast

lowast

lowast

lowast

0

20

40

60

80

100

re

laxa

tion



Control STZ


Log [carbachol] (M)

0

20

40

60

80

100

re

laxa

tion

lowastlowast

lowast

lowast lowast






25

50

75

100

125

150

Nitr

ite le

vels

(as

of c

ontro

l)

lowastlowast



lowast

lowast

0

25

50

75

100

125

150

cGM

P le

vels

(as

of c

ontro

l)

lowast





100

200

300

400

500

PDE

activ

ity (a

s o

f con

trol)

lowast

lowast




4 Discussion



Control STZ

(a)

(b)Ang-(1-7) STZ + Ang-(1-7)

Ratio

of P

DE

GA

PDH

mRN

A

0

100

200

300

(as

of c

ontro

l) lowast

PDE-5GAPDH



lowast

0

1

2

3

4

5

6

Nor

mal

ized

leve

l of p

-p38

p-p38

Actin

(a)

(b)








Competing Interests




Acknowledgments


References






























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of






3 Results


ControlSTZ

lowast lowast

lowast

lowast

lowast

lowast

lowast

0

20

40

60

80

100

re

laxa

tion



Control STZ


Log [carbachol] (M)

0

20

40

60

80

100

re

laxa

tion

lowastlowast

lowast

lowast lowast






25

50

75

100

125

150

Nitr

ite le

vels

(as

of c

ontro

l)

lowastlowast



lowast

lowast

0

25

50

75

100

125

150

cGM

P le

vels

(as

of c

ontro

l)

lowast





100

200

300

400

500

PDE

activ

ity (a

s o

f con

trol)

lowast

lowast




4 Discussion



Control STZ

(a)

(b)Ang-(1-7) STZ + Ang-(1-7)

Ratio

of P

DE

GA

PDH

mRN

A

0

100

200

300

(as

of c

ontro

l) lowast

PDE-5GAPDH



lowast

0

1

2

3

4

5

6

Nor

mal

ized

leve

l of p

-p38

p-p38

Actin

(a)

(b)








Competing Interests




Acknowledgments


References






























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



25

50

75

100

125

150

Nitr

ite le

vels

(as

of c

ontro

l)

lowastlowast



lowast

lowast

0

25

50

75

100

125

150

cGM

P le

vels

(as

of c

ontro

l)

lowast





100

200

300

400

500

PDE

activ

ity (a

s o

f con

trol)

lowast

lowast




4 Discussion



Control STZ

(a)

(b)Ang-(1-7) STZ + Ang-(1-7)

Ratio

of P

DE

GA

PDH

mRN

A

0

100

200

300

(as

of c

ontro

l) lowast

PDE-5GAPDH



lowast

0

1

2

3

4

5

6

Nor

mal

ized

leve

l of p

-p38

p-p38

Actin

(a)

(b)








Competing Interests




Acknowledgments


References






























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Control STZ

(a)

(b)Ang-(1-7) STZ + Ang-(1-7)

Ratio

of P

DE

GA

PDH

mRN

A

0

100

200

300

(as

of c

ontro

l) lowast

PDE-5GAPDH



lowast

0

1

2

3

4

5

6

Nor

mal

ized

leve

l of p

-p38

p-p38

Actin

(a)

(b)








Competing Interests




Acknowledgments


References






























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




Competing Interests




Acknowledgments


References






























Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

















Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

(1-7) In Diabetes-Induced Erectile Dysfunctio

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