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From the * Department of Urology, Korean Cancer
Center Hospital, Seoul, Korea; and the
Department of Urology, Seoul National
University Hospital, Seoul, Korea.
| Correspondence to: Dr Jae-Seung Paick, Department of Urology, Seoul National University College of Medicine, 28 Yon gon dong, Chongno-Gu, Seoul, Korea, 110-744 (e-mail: jspaick{at}snu.ac.kr). |
| Received for publication June 12, 2004; accepted for publication September 9, 2004. |
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Abstract |
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Key words: Erectile dysfunction, Sprague-Dawley rat, losartan
It is well known that aging is associated with diminished aortic compliance and resting cardiac output. In addition, such structural changes as intimal-medial thickening and increased arterial stiffness are usually associated with vascular aging (Lakatta and Levy, 2003), and this may contribute to the increased incidence of target organ changes such as nephrosclerosis, left ventricular hypertrophy, and decreased aortic compliance. In the penis, a decreasing trend in peak systolic velocity with aging has also been observed (Chung et al, 1997).
Structural and functional alterations of the aging penis consist of endothelial dysfunction (Cartledge et al, 2001), up-regulation of corporal smooth muscle tone (Christ et al, 1991), diminished cavernosal smooth muscle and elastic fibers, and an increase of cavernosal fibrosis (Wespes, 2002). The derangement of nitric oxide (NO) metabolism and the increased expression of transforming growth factor ß1 (TGF-ß1) may be implicated in these changes (Dahiya et al, 1999; Rajasekaran et al, 2002).
Angiotensin II (Ang II) is the main active metabolite of the renin-angiotensin system. It has been shown that physiological amounts of Ang II are produced and its receptors have been identified in the corpus cavernosum (Kifor et al, 1997). Therefore, Ang II and its receptors are thought to play a role in the regulation of cavernosal tone. For example, an intracavernosal injection of Ang II has been reported to terminate erection in anesthetized dogs (Kifor et al, 1997), and Ang II is thought to play a role in penile detumescence (Becker et al, 2001). Furthermore, Ang II regulates the vascular tone by counteracting the effects of NO, the principal mediator of penile erection.
Based on these research findings, there is speculation as to whether disturbances in the systemic or local secretion or degradation of Ang II may contribute to the development of male erectile dysfunction. In other vascular beds, recent studies (Kansui et al, 2002; Weber, 2002) have shown that the role of Ang II was not limited to local regulation of vascular smooth muscle tone; rather, it is also believed to play an important role in the cardiovascular pathophysiology, such as endothelial damage, cell proliferation, vascular remodeling, and oxidation. Therefore, drugs that antagonize the effects of Ang II, such as angiotensin converting enzyme inhibitor (ACEI) or angiotensin type 1 receptor (AT1) antagonist, have been reported to potentially prevent age-related endothelial dysfunction even in normotensive patients (Devereux et al, 2003).
We have hypothesized that Ang II antagonism would be helpful to correct age-related erectile dysfunction even if the erectile dysfunction is not associated with hypertension, and erectile function may be assisted by the improvement of the local penile molecular environment as well as the systemic blood flow.
To demonstrate our hypothesis, we examined the effects of AT1 antagonist on erectile function and on relevant molecular environment in the penises of aged normotensive rats.
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Materials and Methods |
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Drug Treatments
Young and aged animals were assigned to the control or treatment groups (n
= 6 in each group). The control animals had free access to water and standard
rat chow. The treated animals received the AT1 antagonist losartan
(CozaarTM, MSD, Seoul, Korea) (30 mg/kg/d) in their drinking water for 1
month. The concentration of the losartan contained in the drinking water was
determined based on the rats' previously established drinking patterns.
In Vivo Evaluation of Erectile Function
The rats were anesthetized with an intraperitoneal injection of urethane
(1.6 g/kg), and they were placed on a homeothermic blanket maintained at
37°C. The rats' left carotid artery was cannulated for the continuous
monitoring of the mean arterial blood pressure. The right corpus cavernosum
was cannulated to permit continuous monitoring of intracavernosal pressure
(ICP). Pressure data were collected and analyzed electronically
(PowerlabTM, ADInstruments, Colorado Springs, Colo).
A stabilizing period of 20-30 minutes was allowed before recording of ICP and systemic arterial pressure (SAP). Apomorphine 100 µ/kg was given subcutaneously, and then erectile function was assessed by simultaneous SAP and ICP recordings for 30 minutes. The time to the first peak pressure, the maximal ICP, the mean number of ICP increases, the area under curve (AUC), and the maximal ICP/mean SAP were analyzed for each rat. The statistical analysis was then performed for all the animals included in each experimental group.
At the completion of the given observation periods, the animals were euthanized by an overdose of phenobarbital. The rats' penises were immediately removed, cleaned, frozen in liquid nitrogen, and stored at -70°C until the time of further processing.
Immunoblotting
The preparation of the penile tissue for immunoblotting was performed as
previously described (Akingba and Burnett,
2001). Homogenized penile tissue was centrifuged at 14 000 x
g for 20 minutes at 4°C. The protein concentration was determined
with BCA Protein Assay reagents (Pierce Chemical Company, Rockford, Ill)
according to manufacturer protocol. An equal amount (20 mg) of each sample was
diluted and fractionated onto 15% sodium dodecyl sulfate-polyacrylamide gel
for electrophoresis. After electrophoresis, the proteins were then transferred
to a Hy-bond-ECL (Amersham Life Sciences, Ghent, Belgium) nitrocellulose
membrane; they were blocked for 1 hour with blotto-Tween (5% nonfat dry milk
and 0.1% Tween-20) and then incubated for 1 hour at room temperature in a
1:2000 dilution of mouse anti-TGF-ß1 or mouse anti-endothelial nitric
oxide synthase antibody (Transduction Laboratories, Lexington, Ky) in 5%
nonfat dry milk. The bound antibody was detected with labeled goat anti-rabbit
immunoglobulin G secondary antibody conjugated to horseradish peroxidase, and
the results of this reaction were visualized using enhanced
chemiluminescence.
The eNOS and TGF-ß1 proteins corresponded to a 145-kd band and a 42-kd band, respectively. The density of respective bands was quantified by densitometric scanning of the Western blots using computer software (Media Cybernetics, San Diego, Calif).
Determination of Lipid Peroxidation by Reactive Oxygen Species
We used an MDA-586 colorimetric assay kit according to the manufacturer's
instructions (OXIS International, Portland, Oreg), which quantitatively
measured the product malondialdehyde (MDA) of the lipid peroxidative reaction
to assess the lipid peroxidation in the penile tissue homogenates. Briefly,
the samples were first homogenized in ice-cold 20 mM Tris-HCl buffer (pH 7.4).
After centrifugation at 3000 x g at 4°C for 20 minutes, an
aliquot of 200 µl of the supernatant was then used to detect a stable
chromophore that was produced by the reaction of a chromogenic reagent with
MDA in the supernatant at 45°C for 60 minutes. The absorbance of this
chromophore was measured at 586 nm on a microtiter plate reader (Dynex
Technologies, Chantily, Va). The degree of lipid peroxidation was determined
by a MDA standard curve as expressed by malondialdehyde equivalent content
(nmol MDA/mL).
Statistical Analysis
Measured data were expressed as means plus or minus standard error of the
mean. One-way analysis of variance (ANOVA) or the Mann-Whitney U test
using SPSS Windows (version 11) was used for comparisons between groups with
regard to the drug or aging effects. In the case of ANOVA testing, this was
followed by the Bonferroni correction. P values of less than .05 were
considered significant.
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Results |
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Losartan treatment significantly lowered the mean SAP of both groups of treated rats (Table 2). The ICP/SAP of the aged group, which is related to adequacy of the penile vascular system, was significantly increased by losartan treatment. The rank order of ICP/SAP is as follows; losartan-treated young rats > control young rats; losartan-treated aged rats > control aged rats (P > .05, using 1-way ANOVA with Bonferroni correction).
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Lipid Peroxidation
Compared to control young rats, the mean level of malondialdehyde in the
corporal tissues of control aged rats was significantly increased (12.5
± 1.2 nmol/gm vs 19.3 ± 1.5 nmol/gm, P < .001). This
increased rate of lipid peroxidation in aged rats was alleviated by losartan
treatment (13.8 ± 0.6 nmol/gm), while there was no significant change
observed in the losartan-treated young rats (11.8 ± 1.1 nmol/gm).
Immunoblot
Our results showed the expression of eNOS and TGF-ß1 in all the
animals. Compared to control young rats, there was approximately a 71%
decrease in the estimated eNOS expression in control aged rats
(Figure 2). These age-related
differences in eNOS were restored after losartan treatment. Like the results
of apomorphine-induced erection, the densitogram revealed the improved eNOS
expression of the losartan-treated aged rats to the level of the control young
rats and the losartan-treated young rats. Similar eNOS expressions were
observed between the losartan-treated aged rats and the control young
rats.
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In contrast, no age or losartan treatment-related differences in TGF-ß1 protein expression were observed in the penile tissues of any of the rat groups (Figure 3).
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Discussion |
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Another mechanism of erectile restoration is the alleviation of endothelial dysfunction that impairs penile vasodilation and adequate cavernosal relaxation. Ang II is known to induce endothelial dysfunction via the increased production of reactive oxygen species (ROS) and vasoconstricting eicosanoids, and both of these can counteract the vasodilating and vasoprotective effects of NO (Weber, 2002). The increased generation of superoxide is responsible for NO degradation because it mediates the uncoupling of NO synthase and inactivates NO via peroxynitrite formation. Since age-related endothelial dysfunction is a systemic process, NO (the main effecter of erection) inactivation is thought to occur in the aging penis. In our study, the control aged rats showed an increased production of ROS and decreased eNOS protein expression, and this indicates endothelial dysfunction, while losartan treatment restored these differences to the levels observed for the control young and the losartan-treated young rats.
We have studied the cavernosal expression of two proteins that have been implicated in age-related erectile impairment. Similar to the results of Rajasekaran et al (2002), the eNOS expression of the control aged rat was significantly decreased. The fact that restoration of erection is associated with increased expression of eNOS is also consistent with the results of Champion et al (1999), who demonstrated that the gene transfer of eNOS augmented the erectile response of the aged rat. However, contrary to our expectations, the expression of TGF-ß1 is not significantly different among all of the tested rats. This result is contradictory to that of Dahiya et al (1999), who demonstrated an increased mRNA expression of TGF-ß1 in senescent (30-month-old) rats. Although the reason for this difference in study results is not completely understood, part of the reason may lie in the age difference between the experimental rat populations. That is, given the possible mechanism of TGF-ß1-mediated cavernosal fibrosis that would result from chronic cavernosal ischemia, our aged rats may have been too young to show the increased expression of TGF-ß1.
Our findings are similar to those of Hale et al (2002), suggesting that drugs that inhibit Ang II, ACEI, and AT1 antagonists are beneficial to age-related erectile dysfunction; however, AT1 antagonist (such as losartan) has a theoretical benefit over an ACEI (Weber, 2002).
ACEI cannot completely inhibit the action of Ang II to the same degree as the AT1 antagonists. It is well known that ACE inhibitors do not fully prevent the conversion of Ang I to Ang II during chronic therapy, and this is probably because enzymes other than ACE (for example, chymase) may assume a greater role in facilitating this conversion when ACE is blocked. The same phenomenon has also been demonstrated in penile tissue (Iwamoto et al, 2001). This may indicate that long-term ACE inhibition might be ineffective because chymase can also generate Ang II. In contrast to ACE inhibition, AT1 receptor antagonism blocks Ang II completely via the inhibition of the AT1 receptor, and this stops the end results of the receptor activation, such as vasoconstriction and vascular hypertrophy. Furthermore, this may potentially stimulate AT2, which mediates vasodilation and has an antiproliferative effect.
Since the aging rats' erectile function is restored by Ang II blockade, selective AT1 antagonist may be used for the aging male patients who are without cardiovascular disease. Although the current indication of losartan is limited to patients with overt cardiovascular disease, a substudy of the Losartan Intervention for Endpoint Reduction in Hypertension randomized trials has revealed that losartan prevented the incidence of strokes and diabetes even in normotensive patients (Devereux et al, 2003). As subclinical vascular dysfunction appears to play an important role in age-related erectile dysfunction, selective AT1 antagonists may improve vascular health and prevent the progression of clinical erectile dysfunction.
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Conclusions |
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Footnotes |
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References |
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Becker AJ, Uckert S, Stief CG, Scheller F, Knapp WH, Hartmann U, Jonas U. Plasma levels of angiotensin II during different penile conditions in the cavernous and systemic blood of healthy men and patients with erectile dysfunction. Urology. 2001; 58:805-810.[Medline]
Cartledge JJ, Eardley I, Morrison JF. Nitric oxide-mediated corpus cavernosal smooth muscle relaxation is impaired in ageing and diabetes. BJU Int. 2001; 87:394-401.[Medline]
Champion HC, Bivalacqua TJ, Hyman AL, Ignarro LJ, Hellstrom WJ, Kadowitz PJ. Gene transfer of endothelial nitric oxide synthase to the penis augments erectile responses in the aged rat. Proc Natl Acad Sci U S A. 1999; 116:48-52.
Christ GJ, Stone B, Melman A. Age-dependent alterations in the efficacy of phenylephrine-induced contractions in vascular smooth muscle isolated from the corpus cavernosum of impotent men. Can J Physiol Pharmacol. 1991; 69:909-913.[Medline]
Chung WS, Park YY, Kwon SW. The impact of aging on penile hemo-dynamics in normal responders to pharmacological injection: a Doppler sonographic study. J Urol. 1997; 157:2129-2131.[Medline]
Dahiya R, Chui R, Perinchery G, Nakajima K, Oh BR, Lue TF. Differential gene expression of growth factors in young and old rat penile tissues is associated with erectile dysfunction. Int J Impot Res. 1999; 11:201-206.[Medline]
Devereux RB, Dahlof B, Kjeldsen SE, et al. Effects of losartan or
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Feldman HA, Goldstein I, Hatzichristou DG, Krane RJ, McKinlay JB. Impotence and its medical and psychosocial correlates: results of the Massachusetts Male Aging Study. J Urol. 1994; 151:54-61.[Medline]
Hale TM, Okabe H, Bushfield TL, Heaton JP, Adams MA. Recovery of erectile function after brief aggressive antihypertensive therapy. J Urol. 2002; 168:348-354.[Medline]
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Kifor I, Williams GH, Vickers MA, Sullivan MP, Jodbert P, Dluhy RG. Tissue angiotensin II as a modulator of erectile function. I. Angiotensin peptide content, secretion and effects in the corpus cavernosum. J Urol. 1997; 157:1920-1925.[Medline]
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Manabe K, Heaton JP, Morales A, Kumon H, Adams MA. Pre-penile arteries are dominant in the regulation of penile vascular resistance in the rat. Int J Impot Res. 2000; 12:183-189.[Medline]
Rajasekaran M, Kasyan A, Jain A, Kim SW, Monga M. Altered growth
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Seftel AD. Erectile dysfunction in the elderly: epidemiology, etiology and approaches to treatment. J Urol. 2003; 169:1999-2007.[Medline]
Tarcan T, Azadzoi KM, Siroky MB, Goldstein I, Krane RJ. Age-related erectile and voiding dysfunction: the role of arterial insufficiency. Br J Urol. 1998; 82:26-33.
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Wespes E. Smooth muscle pathology and erectile dysfunction. Int J Impot Res. 2002; 14(suppl 1):S17-S21.
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