effects of losartan, ho-1 inducers or ho-1 inhibitors on erectile signaling in diabetic rats

Effects of Losartan, HO-1 Inducers or HO-1 Inhibitors on ErectileSignaling in Diabetic Ratsjsm_1517 3254..3264

Mohamed Talaat Abdel Aziz, MD,* Mohamed Farid El Asmer, MD,† Taymour Mostafa, MD,‡

Hazem Atta, MD,* Soheir Mahfouz, MD,§ Hanan Fouad, MD,* Laila Rashed, MD,* Dina Sabry, MD,*Amira Hassouna, MD,* Ahmed Talaat Abdel Aziz, MD,* Amira Senbel, MD,¶ and Ahmed Demery, MD**

*Faculty of Medicine, Cairo University, Medical Biochemistry, Unit of Biochemistry & Molecular Biology, Cairo, Egypt;†Faculty of Medicine, Ain Shams University, Medical Biochemistry Department, Cairo, Egypt; ‡Faculty of Medicine, CairoUniversity, Andrology Department, Cairo, Egypt; §Faculty of Medicine, Cairo University, Pathology Department, Cairo,Egypt; ¶Faculty of Pharmacy, Alexandria University, Pharmacology Department, Alexandria, Egypt; **Faculty ofMedicine, Six October University, Medical Biochemistry Department, Cairo, Egypt

DOI: 10.1111/j.1743-6109.2009.01517.x

A B S T R A C T

Introduction. Activation of the renin-angiotensin system which is common in diabetes mellitus might affect hemeoxygenase (HO-1) gene expression.Aim. Assessment of the effects of administration of angiotensin II (Ang II) receptor antagonist (losartan) with HO-1inducer or inhibitor on erectile signaling in diabetic rats.Materials and Methods. Seventy male rats were divided equally into seven groups; healthy controls, streptozotocin-induced diabetic rats, rats on citrate buffer, diabetic rats on losartan, diabetic rats on HO-1 inducer (cobaltprotoporphyrin [CoPP]), diabetic rats on losartan and CoPP, and diabetic rats on losartan and HO-1 inhibitor(stannus mesoporphyrin [SnMP]).Main Outcome Measure. HO enzyme activity, HO-1 gene expression, cyclic guanosine monophosphate (cGMP)assay, intracavernosal pressure (ICP), and cavernous tissue sinusoids surface area.Results. HO-1 gene expression, HO enzymatic activity, and cGMP were significantly decreased in the cavernoustissue of diabetic rats. These parameters were significantly elevated with the use of CoPP that restored the normalcontrol levels of HO enzyme activity. Administration of losartan exhibited a significant enhancing effect on theseparameters compared with the diabetic group, but not restored to the control levels, whereas administration of CoPPcombined with losartan led to the restoration of their normal levels. ICP demonstrated significant decline in diabeticrats. The use of CoPP and/or losartan led to its significant improvement compared with diabetic rats. Administrationof either losartan and/or CoPP led to a significant increase in the cavernous sinusoids surface area of diabetic rats.Administration of losartan with SnMP significantly decreased the enhancing effect of losartan on the studiedparameters.Conclusion. The decline in erectile function in diabetes mellitus could be attributed to the downregulation of HO-1gene expression. HO-1 induction added to Ang II receptor antagonist could improve erectile function. Abdel AzizMT, El Asmer MF, Mostafa T, Atta H, Mahfouz S, Fouad H, Rashed L, Sabry D, Hassouna A, Abdel AzizAT, Senbel A, and Demery A. Effects of losartan, HO-1 inducers or HO-1 inhibitors on erectile signalingin diabetic rats. J Sex Med 2009;6:3254–3264.

Key Words. Losartan; HO-1 Inducer; HO-1 Inhibitor; Erectile Signaling; Diabetes

Introduction

H eme oxygenase (HO)-1 has been shown tobe expressed in vascular smooth muscle cells

(VSMCs), including those found in the cavernous

tissues [1]. HO-1 gene expression exerts antioxi-dant actions, which participate in defense mecha-nisms against agents that induce oxidative injury,such as heme and other inflammatory molecules[2]. These antioxidant actions originate in the

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capacity of HO-1 to degrade heme released fromoxidant-destabilized heme proteins [3], productionof bilirubin, which has potent antioxidant effects,and generation of the vasodilator gas carbon mon-oxide (CO) [4] that can bind to the heme moiety ofheme proteins causing either enzyme activation orinhibition. HO-derived CO has anti-apoptoticproperties and provides cellular cyto-protectionagainst oxidants and inflammatory molecules.HO-1 also recruits iron-binding proteins, whichlower intracellular iron levels [5].

Another HO isoenzyme, HO-2 is constitu-tively expressed in the endothelium and VSMCsof the cavernous tissue. It functions as a “hemesensor” by fine tuning the activity of transcrip-tional factors and genes that are heme-responsive,including HO-1. Heme bound by the HO-2heme regulatory motif (HRMs) would be pro-tected from degradation and that when the con-centration of heme exceeds the binding capacityof HRMs, then excess heme would become avail-able to serve as substrate for catalysis throughinteractions with the “catalytic pocket.” As such,HO-2 could control expression of genes that areresponsive to heme, and protects vascular endot-helium and VSMCs against apoptotic changesinduced by oxidative stress and cytokine-mediatedinflammation [6,7]. In an experimental study onrats, Abdel Aziz et al. [8] showed that HO-2 geneexpression was not changed by the use of eitherHO inducers or inhibitors.

HO-1 gene expression was found to bedecreased in both type I and type II diabetes withor without micro-angiopathy [2,9]. Reduction ofthe antioxidant capability of the vessel wall byHO-1 downregulation may lead to augmentationof oxidative stress induced by hyperglycemia. COgenerated by HO has paracrine effects anddecreases endothelin-1 and platelet-derivedgrowth factor-b in endothelial cells, suggestingthat it also plays an important role in signal trans-duction [10]. It is therefore also possible thatHO-1 gene regulation may have physiologicaleffects on endothelial as well as smooth musclecells [11]. Li et al. [12] and Nicolai et al. [13] statedthat the induction of HO-1 induces arterial anti-oxidant actions and subsequent vasoprotection in amouse and a rat model of diabetes, and HO-1 geneupregulation ameliorate insulin resistance viaupregulation of adiponectin. Type II diabetes mel-litus and its related oxidant damage hamper theinteraction between the vascular wall and normalendothelial progenitors by mechanisms that are, atleast partially, reversed by the induction of HO-1

gene expression, adiponectin, and PhosphorylatedAMP kinase (pAMPK) levels [14].

HO-1 molecule was proved to play a significantrole in mediating erectile function. Abdel Aziz et al.[15,16] demonstrated that HO-1 generates COthat has a positive effect on soluble guanylatecyclase (sGC) and cyclic guanosine monophos-phate (cGMP) levels in cultured lung and rat vas-cular endothelial cells as well as in the cavernoustissue. Marks et al. [17] suggested an interactionand a signaling interplay between the Nitric oxidesynthase (NOS)/nitric oxide (NO) and HO/COsystems, i.e., when one system is on, the other is offand vice versa. Ryter et al. [18,19] and Ahmad et al.[20] stated that HO protects NO through the scav-enging of reactive oxygen species (ROS), prevent-ing NO from reacting with ROS and formation ofperoxynitrite and its subsequent degradation, con-firming that CO tissue levels parallel NO levels.Abdel Aziz et al. [15] proved that sildenafil citrateactions may be partially mediated through theinduction of HO-1 gene expression and HO-1enzymatic activity. This study clarifies the associa-tion between HO-1 gene induction and sildenafilcitrate-mediated signaling.

Diabetes mellitus and arterial hypertension arefrequently coexisting. There is evidence that acti-vation of the renin-angiotensin system (RAS), oneof the most potent factors in blood pressure regu-lation is very common in diabetes mellitus [21].The corpus cavernosum produces and secretesphysiologically relevant amounts of angiotensin II(Ang II). Intracavernosal injection of Ang II wasshown to terminate spontaneous erection in anes-thetized dog, while administration of an Ang IIreceptor antagonist results in smooth muscle relax-ation and thus erection [22]. In diabetic rats, RAScomponents were upregulated, resulting in theincreased concentration of Ang II in the corpora. Apositive feedback loop for Ang II formation incavernosum was also identified, which could con-tribute to overactivity of cavernous RAS in diabeticrats [23,24]. Ishizaka and Kathy [25] stated thatAng II leads to HO-1 gene downregulation in ratVSMCs that might augment vasoconstriction invivo. Therefore, this downregulation was com-pletely blocked by the Ang II type I receptorantagonist (losartan). Downregulation of HO-1gene in diabetes could party explain the decline inerectile function associated with diabetes.

This study aimed to assess the effects of usingAng II type 1 receptor antagonists (losartan) andHO-1 inducer or HO inhibitor on erectile signal-ing in diabetic rats.

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Materials and Methods

Animal TreatmentSeventy white albino male rats (inbred strain) ofmatched age and weight (2 years old, 200–250 gm),from the experimental animal unit, Faculty ofMedicine, Cairo University were included in thestudy. They were maintained according to the stan-dard guidelines of Institutional Animal Care andUse Committee and after Institutional ReviewBoard approval. Animals were fed a semi-purifieddiet that contained (gm/kg): 200 casein, 555sucrose, 100 cellulose, 100 fat blends, 35 vitaminmix, and 35 mineral mix [26]. The rats were dividedinto seven groups. Group 1, included 10 healthycontrol rats and Group 2 received citrate buffer.

Fifty rats received a single intraperitoneal (IP)injection of 65 mg/kg streptozotocin (STZ) dis-solved in 0.1 mM sodium citrate buffer [27].After 1 week, plasma glucose level was estimatedto confirm induction of diabetes (241.9 �25.1 mg/dL in diabetic rats, 86 � 6.4 mg/dL incontrols). Four weeks after STZ injection, meanarterial blood pressure was measured for all rats.Then diabetic rats were categorized into the fol-lowing experimental groups (N = 10 rats/group):

• Group 3: STZ-induced diabetic rats (diabeticuntreated control group).

• Group 4: received losartan as Angiotensin con-verting enzyme (ACE) inhibitor (10 mg/kg IP, 3times/ week for 2 weeks) [28].

• Group 5: received cobalt protoporphyrin(CoPP) as HO inducer (0.5 mg/100 gm IP, 3times/week for 2 weeks) [29].

• Group 6: received both losartan and CoPP.• Group 7: received losartan and HO inhibitor;

stannus mesoporphyrin (SnMP, 50 mmol/kgsingle IP injection dissolved in 0.1 M NaOH)[26].

Six rats from each group were sacrificed by cer-vical dislocation. Cavernous tissues were dissectedand divided into four portions (50 mg each). Thefirst one was used for HO enzyme activity assayafter preservation at -80°C in homogenizationbuffer (sucrose [0.25 M], KCl [0.1 M], Tris-HCl[1 mM], and 0.4 mM phenyl methyl sulfonyl fluo-ride, pH 7.8). The second portion was put inguanidinium thiocyanate and b-mercapto ethanol-containing RNA extraction buffer to assess HO-1gene expression by reverse transcriptase poly-merase chain reaction (RT-PCR). The thirdportion was put in 0.1 normal HCl to inhibit phos-phodiesterase enzyme, stored at -80°C, for cGMP

assay. The fourth portion was fixed in 10% forma-lin and used for histopathological examination andimage analysis. Cavernous sinuses surface area wasmeasured by Lab-Works analysis software forseparate image analysis, Biodoc analyze (UVP, Inc.Upland, CA, USA) [30]. Four rats from eachgroup were subjected to assessment of intracaver-nosal pressure (ICP) after cavernous nerve (CN)stimulation.

RT-PCR of HO-1Total RNA was extracted from the frozen cavern-ous samples as described previously [31]. Two oli-gonucleotide primers were prepared to amplify a211 base pair stretch of rat HO-1 gene with thesequence: (sense) 5′ GGA AAG CAG TCA TGGTCA GTC A 3′ and (antisense) 5′ CCC TTC CTGTGT CTT CCT TTG T 3′. Five-microgramRNA was reverse transcribed using 12.5 mL oligo-nucleotide (dT)18 primer (final concentration0.2 mMol) and was denatured at 70°C for 2minutes. Denatured RNA was placed on ice and6.5 mL of reverse transcription mixture containing50 mM KCl, 50 mM Tris-HCl, pH 8.3, 0.5 mM ofDeoxyribonucleotide triphosphate (dNTP), 3 mMMgCl2, 1 U/mL RNase inhibitor, and 200 units ofmoloney murine leukemia virus reverse tran-scriptase. The reaction tube was placed at 42°C for1 hour, followed by heating up to 92°C to stop thereaction, then placed on ice. The PCR reaction wasperformed by adding the PCR mix to the reversetranscription tube to reach a final volume of100 mL. The PCR mix contained 10 mmol/L Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, and0.001% gelatin, 250 mM dNTP mix, 2.5 U Taqpolymerase, and 100 mM of each primer. This reac-tion mixture was subjected to 40 cycles of PCRamplification as follows: denaturation at 95°C for 1minute, annealing at 55°C for 1 minute, and exten-sion at 72°C for 1 minute. After the last cycle, a finalextension at 72°C for 10 minutes was done [32].

cGMP AssaysFrozen CC samples (50 mg) stored in 0.1 normalHCl were grounded with a stainless steel mortarthen homogenized and centrifuged at 600 ¥ g,at 4°C for 10 minutes. The supernatant was usedfor cGMP assay by ELISA kit (R&D Systems,Minneapolis, MN, USA) [33]. Cavernous tissueprotein content was determined by Bradfordprotein assay method [34].

Preparation of Penis MicrosomesAfter the removal of the corpus spongiosum anddorsal vein, corporus cavernosum (CC) tissues

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(50 mg) were pooled and homogenized in a buffercontaining 100 mM Tris-HCl and 1.15% KCl,pH 7.4. The homogenate was centrifuged at10,000 ¥ g for 30 minutes. The supernatant wascollected and centrifuged again at 100,000 ¥ g for90 minutes. The microsome-rich pellet was resus-pended in 0.25 M sucrose buffer and used for HOenzyme activity assay [26,35].

HO Enzyme Activity AssayCavernous tissue (50 mg) homogenized sampleswere incubated with heme (50 mmol/L), rat livercytosol (5 mg/mL), MgCl2 (2 mM/L), glucose-6-phosphate dehydrogenase (1 U), glucose-6-phosphate (2 mM/L), and reduced nicotinamideadenine dinucleotide phosphate (NADPH)(0.8 mM/L) in 0.5 mL of 0.1 M/L phosphatebuffer saline (pH 7.4) for 60 minutes at 37°C. Thereaction was stopped by putting the tubes on ice,and the reaction solution was extracted withchloroform. The rate of bilirubin formation wasmonitored at 464 nm and 520 nm by a spectropho-tometer [36].

ICP MeasurementMeasuring ICP changes elicited by electricalstimulation of CN was performed in rats, accord-ing to the method previously described [37,38].Briefly, the rats were anesthetized by IP injectionof thiopental (50 mg/kg). The skin overlying thepenis was incised, and the prepuce was degloved toexpose the CC where a 26-gauge needle filled withheparinized saline was carefully inserted into theCC on one side to measure the ICP. The needlewas inserted at mid-length of the penile shaftpointing toward its base. The needle was sealed toa polyethylene 50 tubing and connected to Gould-Statham pressure transducer (Oxnard, CA, USA)where ICP was displayed on a Grass polygraph(Model 7D, Grass Instruments Co, Boston, MA,USA). Through a lower suprapubic midline inci-sion, the lateral prostate was dissected and themajor pelvic ganglion was identified [39,40]. CNwas unilaterally freed from its facial attachmentsand was stimulated electrically using a bipolarplatinum electrode placed 3–4 mm distal to themajor pelvic ganglion [41] The two poles of theelectrode were separated by 2 mm and the elec-trode was connected to an electronic stimulator(Letica®, Panlab, Model 12106/150, Barcelona,Spain). The CN was stimulated for 1 minute withpulse parameters 5 volts, 1 ms duration, and 0.3–5 Hz frequency. Each period of stimulation wasseparated by a resting period of 5 minutes. A fre-

quency response curve was obtained, where themaximum rise in ICP during nerve stimulation wasmeasured and compared statistically to control anddiabetic untreated rats.

Mean Arterial Blood Pressure MeasurementIt was measured by rat tail cuff method using anelectronic electrosphygmomanometer after the ratwas pre-warmed to 37°C for 15 minutes. Anaverage of three readings was recorded for eachmeasurement while the animals were quietlyresting.

Statistical AnalysisStatistical Package for Social Studies (SPSS)program version 16.0.1 (SPSS Inc., Chicago, IL,USA) was used. Numerical data were expressedas mean � standard deviation. For comparisonsbetween treatment groups, the null hypothesis wastested by a single-factor anova for multiple groupsor unpaired t-test for two groups. Comparisonsand correlations were considered statistically sig-nificant if P < 0.05.

Results

Mean physiological as well as chemical parameterswere represented in Table 1. Cavernous tissuecGMP was significantly decreased in diabetes-induced rats. Administration of either losartan orCoPP led to a significant increase of cGMP levelscompared with the diabetic rats, but its levels werenot restored to the healthy control levels(Figure 1). There was a significant decrease in HOenzyme activity in the cavernous tissue of diabeticrats where administration of Losartan led to a sig-nificant enhancing effect compared with diabeticrats, but its level was not restored to the healthycontrol values. However, administration of CoPPeither separately or in combination with losartanled to a restoration of the healthy control levels ofHO enzyme activity (Figure 2). Administration oflosartan and SnMP abolished the effect of losartanon the studied chemical parameters.

ICP demonstrated significant decrease indiabetes-induced rats, whereas the use of eitherlosartan and/or CoPP led to its significant increasecompared with the diabetic group. However,administration of CoPP either separately or incombination with losartan led to a significantelevation of ICP in comparison to the controlgroup. Administration of losartan in combinationwith SnMP diminished the effect of losartan onICP (Figure 3).


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Figure 1 Cyclic guanosine mono-phosphate (cGMP) levels in the cav-ernous tissue (pmol/mg protein) in thestudied rat groups (N = 42 rats, sixrats/group). *Significant difference incomparison with the healthy controlgroup. **Significant difference in com-parison with the streptozotocin group.Results are expressed as mean �standard deviation. CoPP = copperprotoporphyrin; DM = diabetes melli-tus; SnMP = stannus mesoporphyrin.

Figure 2 Heme oxygenase activityin the cavernous tissue (pmolbilirubin/mg protein/minute) in thestudied rat groups (N = 42 rats, sixrats/group). *Significant difference incomparison with the healthy controlgroup. **Significant difference in com-parison with the streptozotocin group.Results are expressed as mean �standard deviation. CoPP = copperprotoporphyrin; DM = diabetes melli-tus; SnMP = stannus mesoporphyrin.

Table 1 Physiological and chemical parameters of the studied rat groups; body weight, blood glucose and mean arterialblood pressure

Body weight (gm)Blood glucoselevels (mg/dL)

Mean arterial bloodpressure (mm Hg)

Control 223 � 8.8 86.0 � 6.4 101.6 � 2.1Rats received citrate buffer 219 � 17.9 89 � 7 98.3 � 10.1STZ-induced DM 226 � 15.7 241.9 � 25.1* 146.7 � 1.3*STZ + Losartan 224.7 � 17.6 241.9 � 25.1* 114.9 � 2.8*,**STZ + CoPP 226 � 18.1 241.9 � 25.1* 126.1 � 1.5*,**STZ + Losartan + CoPP 228.5 � 15.8 241.9 � 25.1* 101.7 � 2.1**STZ + Losartan + SnMP 223.5 � 16.8 241.9 � 25.1* 123.3 � 9.9**

*Significant difference in comparison with the control group.**Significant difference in comparison with the streptozotocin (STZ) group.CoPP = copper protoporphyrin; DM = diabetes mellitus; SnMP = stannus mesoporphyrin.


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Results of RT-PCR showed that HO-1 genewas not expressed in diabetic rats group, althoughit was expressed in the rat groups that receivedeither losartan and/or CoPP and in rats thatreceived losrtan with SnMP (Figure 4). There was

no change in the cavernous tissue sinusoids surfacearea of diabetic rats. Administration of losartanand/or CoPP significantly improved the sinusoidssurface area of the cavernous tissue (Figure 5).

Discussion

HO-1 has been shown to be expressed in VSMCs,including those found in the cavernous tissues [1].However, little is known about HO regulation inVSMCs by vasoactive agents. In the present study,we examined the effect of administration of eitherAng II receptor antagonist, losartan with HO-1inducer (CoPP), or inhibitor (SnMP) on erectilesignaling in diabetic rats. HO-1 gene was notexpressed in the cavernous tissue of diabetic ratsand its enzymatic activity was significantly sup-

Figure 3 Intracavernosal pressure(ICP) after electric current stimulationwith increasing frequencies in thestudied rat groups (N = 28 rats, fourrats/group). Results are expressed asmean � standard deviation. CoPP =copper protoporphyrin; DM = diabetesmellitus; SnMP = stannus mesopor-phyrin.

Figure 4 Heme oxygenase gene expression in the cavern-ous tissue of the studied rat groups (N = 42 rats, six rats/group). Lane 1 = control; Lane 2 = diabetes mellitus (DM);Lane 3 = citrate buffer; Lane 4 = DM and losartan; Lane5 = DM and copper protoporphyrin (CoPP); Lane 6 = DM,losartan, and CoPP; Lane 7 = DM, losartan, and stannusmesoporphyrin.

Figure 5 Lumen surface area ofcavernous sinusoidal vessels(mm2) in the studied rat groups(N = 42 rats, six rats/group). *Sig-nificant difference in comparisonwith the healthy control group.**Significant difference in com-parison with the STZ group.Results are expressed as mean� standard deviation. CoPP =copper protoporphyrin; DM =diabetes mellitus; SnMP = stan-nus mesoporphyrin.


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pressed. Studies on experimental type I diabetesalso revealed lower levels of HO-1 expressionand HO activity and that type II diabetes withor without microangiopathy is associated withdecreased HO-1 gene expression [2,9,42–44].Ishizaka et al. [25] showed that Ang II, which iscommon in diabetes mellitus, decreases HO-1mRNA in a dose-dependent and calcium-dependent manner in VSMCs. This effect wasblocked by an Ang II type 1 receptor antagonist,indicating that this is truly an Ang II-mediatedeffect. Removal of extracellular calcium preventedAng II-induced HO-1 downregulation, althoughcalcium-mobilizing agents promoted downregula-tion. Moreover, Alba et al. [45] stated that Ang IIprevented HO-1 synthesis by normal neutrophilsin vitro, and suppressed HO-1 gene expression inneutrophils from hypertensive patients in com-parison with cells from healthy subjects. In addi-tion, Ang II treatment led to a reduced HO-1enzyme activity to levels similar to those found inneutrophils from hypertensive patients. On thecontrary, Choi et al. [46] stated that intracellularCa2+ mediates expression of all stress responsivegenes, including HO-1. More recently, Ishizakaet al. [47] reported that HO-1 is upregulated in therat heart in response to chronic administration ofangiotensin II for 7 days.

The fact that Ang II receptor antagonistdecreased HO-1 gene expression in our study wasrather unexpected. HO-1 has been shown to beinduced by oxidative stress in various cell types, andAng II causes a delayed generation of superoxide[25,48] and hydrogen peroxide [49] in VSMCs. Inpreliminary studies, it was found that high concen-trations of hydrogen peroxide increased HO-1mRNA in VSMCs. However, low concentrations ofhydrogen peroxide decreased HO-1 mRNA asearly as 1 hour after stimulation [48]. Therefore,the effect of ROS on HO-1 regulation in VSMCsmay depend on the type and amount of generatedROS.

Hyperglycemia mediates local formation ofROS which is considered to be a major contribut-ing factor to endothelial dysfunction, includingabnormalities in cell cycling [31,50] and delayedreplication [51]. These abnormalities can bereversed by antioxidant agents [52] or by increasedexpression of antioxidant enzymes [53]. Du et al.[54] demonstrated that hyperglycemia stimulatesthe induction of apoptosis in endothelial cells by amechanism that involves the generation of ROSand superoxide anion. A reduction in antioxidantreserves has been related to endothelial cell dys-

function in diabetes, even in patients with well-controlled glucose levels [55,56].

Abraham et al. [31] demonstrated thatenhanced expression of HO-1 attenuates glucose-mediated cell growth arrest and reduces apoptosisin human microvessel endothelial cells. The pro-tective effect of the HO-1 signaling mechanism,which attenuated glucose-mediated endothelialcell apoptosis, was maintained through suppres-sion of p21 and p27 cyclin kinase inhibitorsor, perhaps, through its powerful antioxidantproperties [31]. Flammer et al. [57] stated thatangiotensin-converting enzyme inhibition orangiotensin receptor blockade may improveendothelial dysfunction, an early manifestation ofthe atherosclerosis in patients with diabetes. Theprotective effect was mediated through bloodpressure lowering effects or due to reduction inoxidative stress. Yang et al. [23] reported thatadministration of losartan blocked the effect ofAng II, downregulated the expression of AT1 andAng II generated locally, and partially restorederectile function. Park et al. [58] showed that losa-rtan treatment effectively restored the erectilefunction of aged rat to a level similar to that ofyoung rats. Toblli et al. [59] showed that the inter-actions against the RAS, either by ACE inhibitorsor angiotensin AT1 receptor blockers, produceconsiderable benefits regarding structural abnor-malities in cavernous tissue in an animal model ofrenal insufficiency beyond Blood pressure (BP)control. Llisterri et al. [60] demonstrated thatlosartan improved erectile function as well as bothsatisfaction and frequency of sexual activity incases with metabolic syndrome. Toblli et al. [61]demonstrated that a long-term combined therapyusing losartan and the phosphodiesterase type 5(PDE5) inhibitor sildenafil was a useful tool forfunctional and structural modification in cavern-ous tissue from spontaneously hypertensive rats.Lately, a role for HO inducers (CoPP) in improv-ing cavernous cGMP had been proved [62,63].Lau et al. [64] stated that in non-responders ofPDE5 inhibitors of ED patients, it is preferred toadd PGE1, doxazosin, endothelin-1 antagonists,angiotensin II antagonists (valsartan/losartan),adrenomedullin or Rho kinase inhibitors.

As regards to mean arterial blood pressureassessment in the present study, there was a sig-nificant elevation of its levels in diabetic rats ascompared with control animals. This findingagreed with those of Li et al. [65] who reportedelevation of mean arterial blood pressure in experi-mental type I diabetes in rats.


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Administration of Losartan led to the upregu-lation of HO-1 gene expression and a significantenhancing effects of its enzymatic activity, but itslevels were not restored to the normal controlvalues. Administration of CoPP and/or losartanled to a significant improvement of ICP, cGMP,HO-1 gene expression, and HO enzymatic activityof the cavernous tissue. Abdel Aziz et al. [62] statedthat HO-1 gene and its enzymatic activity couldcomplement and even dominate NO in mediatingerectile function. HO-1 generates CO that acti-vates sGC with subsequent elevation of cGMPtissue levels. Moreover, the authors stated that theeffect of three of the commonly used and well-known phosphodiesterase inhibitors is partiallymediated through upregulation of HO-1 geneexpression in the cavernous tissue. Administrationof HO inhibitor, SnMP, abolished the effects oflosartan on the studied chemical parameters. It wasshown that SnMP did not inhibit HO-1 geneexpression because SnMP is an inhibitor of HOenzyme activity but it is not a suppressor of itsgene expression [66]. Abdel Aziz et al. [26]reported similar findings. The authors proved thatSnMP inhibited the enhancing effect of COdonor, carbon monoxide releasing molecule(CORM-3) and HO inducer, hemin on erectilesignaling molecule, cGMP.

Administration of losartan and/or CoPP signifi-cantly improved the surface area of the cavernoussinusoids. These findings are in agreement withthe fact that certain factors are associated withendothelial dysfunction in diabetes which includesactivation of protein kinase C, over expression ofgrowth factors and/or cytokines, and oxidativestress. These factors could enhance dysfunction ofdiabetic endothelial cells, with subsequent impair-ment of sinusoids relaxation and narrowing ofsinusoids surface area [55,67]. Calles-Escandonet al. [67] stated that the treatment of endothelialdysfunction with ACE inhibitors, antioxidants,or insulin sensitizers could improve endothelialfunction and could impair the progression toatherosclerosis.

Assessment of the ICP after electric currentstimulation showed its significant decline in dia-betic rats, whereas the use of CoPP and/or losar-tan led to a restoration of ICP normal levels.These findings are in agreement with Ushiyamaet al. [38] who stated that erectile dysfunction inhypertensive rats results from impaired relaxationevoked by NO and CO, a product of HO. In theirexperimental study, Park et al. [58] reported thatdespite the systemic pressure-lowering effect of

the losartan, the peak ICP was not significantlyreduced; rather, the ICP/systemic arterial pressurewas increased. They concluded that alteration ofthe RAS might be implicated in the erectile dys-function of elderly males with therapeutic value.

It is concluded that the decline in erectile func-tion in diabetics could be attributed to downregu-lated HO-1 gene expression. Therefore, thecombined use of HO-1 inducer(s) and angiotensintype-1 receptor blocker drugs may improve erec-tile function.

Corresponding Author: Hanan Fouad, MD,Medical Biochemistry Department, Faculty of Medi-cine, Cairo University, POB 11562, Cairo, Egypt.Tel: +202-23632297; Fax: +202-23632297; E-mail:[email protected]

Conflict of Interest: None.

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effects of losartan, ho-1 inducers or ho-1 inhibitors on erectile signaling in diabetic rats

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