ORIGINAL ARTICLE
Acetylsalicylic acid protects erectile function in diabetic rats G. Hafez1, U. Gonulalan2, M. Kosan2, E. Arioglu1, B. Ozturk2, M. Cetinkaya3 & S. Gur1 1 Faculty of Pharmacy, Department of Pharmacology, Ankara University, Ankara, Turkey; 2 Department of Urology, Baskent University, Konya Research and Training Hospital, Konya, Turkey; 3 Department of Urology, Ankara Numune Education and Research Hospital, Ankara, Turkey
Keywords Acetylsalicylic acid—diabetes mellitus—erectile dysfunction—in vitro—intracavernosal pressure Correspondence Umut Gonulalan, School of Medicine, Baskent University, Konya Research and Training Hospital, Saray Street, No: 1 Selcuklu, Konya, Turkey. Tel.: +90 332 2631494; Fax: +90 332 2570637; E-mail: [email protected]
Accepted: September 16, 2013 doi: 10.1111/and.12187
Summary We aimed to evaluate the effect of acetylsalicylic acid (ASA) treatment on diabetes-induced erectile dysfunction. Adult male Sprague–Dawley rats were divided into four groups as follows: (i) control (C), (ii) diabetic (D), (iii) ASA-treated control (C+ASA) and (iv) ASA-treated diabetic (D+ASA) groups. In groups 2 and 4, diabetes was induced by injection of 35 mg kg 1 streptozotocin. ASA (100 mg kg 1 day 1, orally) was administrated to rats in groups 3 and 4 for 8 weeks. Both intracavernosal pressure (ICP) and mean arterial blood pressure (MAP) were measured in in vivo studies. In organ bath, the relaxation responses to acetylcholine (ACh), electrical field stimulation (EFS) and sodium nitroprusside were tested in corpus cavernosum (CC) strips. The mRNA expression for neuronal nitric oxide synthase (nNOS) was calculated using reverse transcription polymerase chain reaction technique. In in vivo experiments, diabetic rats displayed reduced ICP/MAP values, which were normalised with ASA treatment. The relaxant response to high-dose ACh and EFS at low frequencies (1–8 Hz) in CC strips from the D+ASA group were significantly higher when compared to the D group. Treatment with ASA normalised the raised mRNA expressions of nNOS in diabetic penile tissues. ASA may be involved in mRNA of protein synthesis of NO released from nonadrenergic and noncholinergic cavernosal nerve in diabetes.
Introduction Penile erection is in control of a complex neural and vascular interaction that causes cavernosal smooth muscle relaxation. The nitric oxide (NO) is synthesised by neuronal (nNOS) and endothelial NO synthase (eNOS) and plays an important role in cavernosal smooth muscle relaxation with the NO/cyclic guanosine monophosphate (cGMP) cascade (Rajfer et al., 1992; Toda et al., 2005). Diabetes mellitus (DM) is the common risk factor for erectile dysfunction (ED), although the pathogenesis of ED in DM remains controversial because of the multifactorial nature of DM (Sasaki et al., 2003). The neuropathy and vascular dysfunction make it difficult to pinpoint a single aetiology for ED (Bennett et al., 2005). Acetylsalicylic acid (ASA) acts with significant efficacy in the treatment of coronary and cerebrovascular events (Awtry & Loscalzo, 2000). This effect is a result of © 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 997–1003
cyclooxygenase-1 inhibition in platelets and endotheliumdependent arterial relaxation (Awtry & Loscalzo, 2000; Monobe et al., 2001). In a recent report, ASA improved endothelium-dependent vasorelaxation through increased bioavailability of NO (Tauseef et al., 2008). On the other hand, indomethacin and diclofenac as nonsteroidal antiinflammatory drugs negatively affected the erectile process in rats due to reduction in the plasma level of NO (Senbel, 2011). nNOS is localised in penile autonomic nerves and arterioles. However, eNOS is detectable in endothelial cells of the corpus cavernosum (CC) (Andersson & Wagner, 1995). Both reduction in nNOS expression and endothelial morphological changes have been documented as causing ED in diabetes (Albersen et al., 2011). To date, there has been no study regarding treatment of ED with ASA in diabetic rats. The goal of the current study was to investigate the effect of ASA treatment on 997
Acetylsalicylic acid in erectile dysfunction
the CC in diabetes-induced ED in a rat model in vivo and in organ bath studies. Materials and methods Animals and induction of diabetes Sixteen-week-old Sprague–Dawley male rats, weighing 300–310 g, were provided by the Animal Care Facility of Selcuk University (Konya, Turkey). Rats were divided into four groups as follows: control (C) (n = 18), diabetic (D) (n = 12), ASA-treated control (C+ASA) (n = 12) and ASA-treated diabetic (D+ASA) (n = 13). Diabetes was induced by an intravenous (i.v.) injection of 35 mg kg 1 streptozotocin (STZ, dissolved in citrate buffer, pH 4.6) via the tail vein. After the fourth day of injection, blood glucose levels were measured by Accu-Check Go Roche glucometer. Rats with low levels of blood glucose (below 250 mg dl 1) were omitted from experiments. ASA was given orally at a dose of 100 mg kg 1 day 1 over 8 weeks (Kapetanovic et al., 2009). Experimental protocols were approved by the Ankara University Animal Experiments Ethical Committee (No: 2008-28-134). In vivo experiments The erectile function studies in groups in vivo were performed after 8 weeks of diabetes. Rats were anaesthetised with an intraperitoneal (i.p.) injection of 100 mg kg 1 ketamine. Animals were placed in the supine position, and bladder and prostate were exposed through a midline abdominal incision. The inferior hypogastric plexus and pelvic nerves were identified post-erolateral to the prostate on both sides, and stainless bipolar wire electrodes were placed around these structures for electrical stimulation. The penis was denuded of skin, and CCs were exposed by removing part of the overlying ischiocavernosus muscles. To monitor intracavernosal pressure (ICP), a 23-gauge cannula was filled with 250 IU ml 1 heparin solution connected to polyethylene-50 tubing and inserted into the left CC. Systemic arterial blood pressure and mean arterial pressure (MAP) were monitored via a 23-gauge cannula placed into the carotid artery (Christ et al., 2006). Both pressure lines were connected to a transducer, and pressures were recorded by Biopac Lab Pro MP35 System (BIOPAC Systems, Inc., Goleta, CA, USA) on the computer. Stimulation of the cavernosal nerve was performed with an electrode. Stimulation parameters measured by Grass S48 stimulator were frequency 15 Hz, voltage 7.5 volt, pulse width 30 ms, duration 1 min and amplitude 1.5 mA. All transducers were calibrated before each experiment. The ratio of ICP to MAP was calculated to reach exact erectile response. 998
G. Hafez et al.
Isometric tension measurement in CC strips After anaesthesia, rats were sacrificed by exsanguination, and then, CC tissues were isolated for bath studies. The CC strips were placed in 20 ml organ baths under 1 g tension in Krebs solution and oxygenated with 95% O2 and 5% CO2. Tissues were allowed to equilibrate for 60 min at 37 °C, and the bath medium was replaced every 15 min. Acetylcholine (ACh, 10 8–10 3 M), electrical field stimulation (EFS, 1–32 Hz) and sodium nitroprusside (SNP, 10 8–10 3 M)-induced relaxation responses were evaluated after pre-contraction with phenylephrine (PE, 10 5 M). The contractile response to PE (10 8–10 3 M) was also recorded. Total RNA isolation, reverse transcription polymerase chain reaction (RT-PCR) and PCR experiments The cavernosal tissues were stored at 80 °C after they were taken in liquid nitrogen in sterile conditions. Cavernosal tissues were powdered with liquid nitrogen and homogenised with an ultrasonic homogeniser (Bandelin electronic, Berlin, Germany) prior to RNA extraction. Total RNA was extracted with the TRIzol reagent (SigmaAldrich Co., St. Louis, MO, USA) according to the manufacturer’s protocol. The optical density values and amounts of RNA were determined spectrophotometrically using Nanodrop (NanoDrop Products, Wilmington, DE, USA) at wavelengths of 260 nm (k 260) and 280 nm (k 280), and the OD k 260/k 280 ratio was used as a precursory estimation of RNA quality. In addition, formamide/formaldehyde agarose gels were used later to evaluate RNA integrity. RT-PCR experiments were performed using ImProm II Reverse Transcriptase System (Promega, Madison, WI, USA). 1 ll oligo dT (Promega) was added to equivalent amounts of total RNA from all groups. The mixtures were placed into thermocycler (Hybaid, UK) and held at 70 °C for 5 min. The samples were then transferred into ice for 5 min. Thereafter, 1 ll 10 mM deoxynucleotide triphosphate (dNTP), 6.1 ll nuclease-free water, 4 ll 5XMgCl-free buffer, 2.4 ll 25 mM MgCl2, 1 ll reverse transcriptase and 0.5 ll RNasin were added, and the final volume was 20 ll. The tubes were again placed into the thermocycler and heated for 60 min at 42 °C for RT, followed by 15 min at 70 °C for denaturation. First strand cDNA samples were then cooled to 4 °C and were stored at 80 °C until use. The single strand cDNAs were consequently amplified by PCR using gene specific primer (Zheng et al., 2005) and Taq polymerase. For this purpose, 5X MgCl2-free reaction buffer, MgCl2 (2.8–3.8 mM), dNTP (0.5 mM), Taq DNA polymerase (5u ul 1) (Promega), cDNA (1 ll), and 2 ll (from 10 pmol ml 1) of respective © 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 997–1003
G. Hafez et al.
Acetylsalicylic acid in erectile dysfunction
sense and antisense primers were added to PCR tubes. Nuclease-free water was then added to each tube for a final volume of 50 ll. The samples were then mixed, placed in the thermocycler and denatured for 3 min at 94 °C. Thereafter, segments were amplified using the sequence 1.5 min denaturation (94 °C) followed by 1 min annealing (58– 60 °C) and 2 min extension (72 °C), and this sequence was repeated for a total of 35 cycles. Beta-actin was amplified in each set of PCR reactions and served as internal references during quantitation to correct for operator and/or experimental variations. At the end of the reactions, 30 ll of each PCR product was then loaded onto a 1% agarose gel containing ethidium bromide and electrophoresed for 1 h at 100 V (Sci-Plas, UK). The resulting gels were then visualised using an UV transilluminator (Vilber Lourmat TFX 20 M UV) and photographed using UV gel camera (Kodak EDAS 290; Eastman Kodak Company, Rochester, NY, USA). Band intensities were calculated using Kodak 1D 3.6 program (Eastman Kodak Company).
(a)
(b)
Statistical analysis All data were expressed as means standard errors. The Statistical Package for the Social Sciences (16.0 for Windows, SPSS Inc., Chicago, IL, USA) software program was used for statistical analysis. One-way ANOVA with Tukey’s HSD test was used in comparison with data between the groups, and values of P < 0.05 were considered as significant. Results Body weights and blood glucose levels in rats The mean body weights in groups C, D, C+ASA and D+ASA were 326 16.6 g, 286 26.3 g, 286 3.83 g and 277 22.7 g respectively. The mean blood glucose levels in groups C, D, C+ASA and D+ASA were 91.7 0.81, 355 41.4, 88.1 1.33 and 350 32.1 mg dl 1 respectively. According to these results, the mean body weights in D, C+ASA and D+ASA groups at sacrifice were significantly lower than in C group (P < 0.05). The mean blood glucose levels at sacrifice were significantly higher in D and D+ASA groups in comparison with C and C+ASA groups (P < 0.05). In addition to these results, the tissue weights of each CC strip were measured, and there was no significant difference between the groups (P > 0.05). Relaxation response to ACh
between groups. However, the relaxant response of strips from the D+ASA group at 10 3M concentration of ACh was significantly higher than that of CC strips from the other groups (P < 0.01). Relaxation response to EFS The relaxant response to EFS (1–32 Hz) according to groups is displayed in Fig. 1b. As seen in Fig. 1b, the relaxant responses in CC from the D+ASA group at low frequency EFS (1–8 Hz) were significantly higher than the responses in the D group (P < 0.05). The relaxant response to EFS in the D+ASA group at higher frequencies was slightly higher than in the D group, but the differences were not statistically significant (P > 0.05). Furthermore, all relaxant responses of strips related to all frequencies of EFS were similar in the D+ASA, C and C+ASA groups (P > 0.05). Relaxation response to SNP
The relaxant response to ACh (10 –10 M) is seen in Fig. 1a. There was no difference in ACh responses 8
© 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 997–1003
Fig. 1 The endothelium and neurogenic relaxant responses to (a) acetylcholine and (b) electrical field stimulation in the corpus cavernosum strips according to groups. *P < 0.05 D+ acetylsalicylic acid (ASA) group vs D group. **P < 0.01 D+ASA group vs other groups.
3
The relaxation responses of strips related to SNP (10 8– 10 3 M) were statistically similar in all groups (Fig. 2a). 999
Acetylsalicylic acid in erectile dysfunction
(a)
G. Hafez et al.
significantly decreased in diabetic rats in comparison with the control group (P < 0.05). However, ASA treatment normalised the diminished ICP/MAP ratio in diabetic rats (P < 0.05). The mRNA expressions for nNOS using RT-PCR
(b)
The mRNA expressions of nNOS in C, D, C+ASA and D+ASA groups were 100%, 116%, 137% and 154%, respectively, and the difference between C and D groups was not statistically significant (P = 0.17). On the other hand, the mRNA expressions of nNOS in the D+ASA group were significantly increased in comparison with C and D groups (P < 0.01, Fig. 5). Discussion
Fig. 2 The endothelium-independent relaxant responses to (a) Sodium nitroprusside were similar between groups, and the contractile responses of strips related to (b) Phenylephrine were unaltered in all groups.
Fig. 3 The mean intracavernosal pressure/mean arterial blood pressure ratio in rats. *P < 0.05 D group vs C group or D+ acetylsalicylic acid group.
Contractile response to PE The contractile response to PE (10 8–10 3 M) was unaltered in CC strips from all groups (Fig. 2b). In vivo erectile responses The mean ICP/MAP ratios and graphics of rats are exhibited in Figs 3 and 4. The mean ICP/MAP ratio was 1000
Acetylsalicylic acid (ASA) is widely used in the treatment of cardiovascular diseases. It decreases vascular smooth muscle cell proliferation and proinflammatory mediators and improves endothelium-dependent vasorelaxation mediated by NO (Kodama et al., 2000; Cyrus et al., 2002; Tauseef et al., 2008; Furuno et al., 2011). On the other hand, there are several diabetic animal models that reported reduced expression of nNOS in CC (Cellek et al., 1999; Xu et al., 2001; Garcia et al., 2010). We investigated the possible protective effects of ASA on ED induced with diabetes. Several studies have reported about the administration and appropriate doses of ASA in rats. A study by Kapetanovic et al. compared different routes of administration of ASA (gavage versus dietary) and demonstrated that the route of ASA administration did not change the pharmacokinetic or pharmacodynamic parameters (Kapetanovic et al., 2009). The relationship between ASA and endothelial function has also been clarified (Furuno et al., 2011). Furuno et al. evaluated the effective dose of ASA on platelet aggregation and endothelial function in healthy volunteers (Furuno et al., 2011). They reported that ASA at all doses suppressed platelet activity, and endothelial-mediated arterial dilatation at higher doses of ASA was worsened. In rat models, an optimal dose of 100 mg kg 1 day 1 was determined for evaluating the effects of ASA (Wu et al., 2002; Tauseef et al., 2007, 2008). In the present study, ASA (dose of 100 mg kg 1 day 1, orally) was given by gavage for 8 weeks in treated animals. ASA is a cardioprotective agent via the inhibition of platelet activity, decrease in vascular smooth muscle cell proliferation and reduction in proinflammatory mediators (Kodama et al., 2000; Cyrus et al., 2002; Furuno et al., 2011). Bornman et al. (1987) reported that ASA delays penile atherosclerosis. It has been suggested that ASA with © 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 997–1003
G. Hafez et al.
Acetylsalicylic acid in erectile dysfunction
(a)
(b)
(c)
(d)
Fig. 4 The intracavernosal pressure/mean arterial blood pressure graphics of rats in (a) Control, (b) Diabetic, (c) Acetylsalicylic acid (ASA) treated Control and (d) ASA treated Diabetic groups.
Fig. 5 The change in the percentage of neuronal nitric oxide synthase mRNA expression according to groups. *P < 0.05 C+ acetylsalicylic acid (ASA) group vs C group or D group. **P < 0.01 D+ASA group vs C group or D group.
antioxidant activity ameliorated endothelium-dependent vasorelaxation because of the raised bioavailability of NO (Tauseef et al., 2008). On the other hand, several studies in diabetic models indicated reduction in the expression of nNOS enzyme in the CC (Cellek et al., 1999; Xu et al., 2001; Garcia et al., 2010). Thus, decreased endogenous NO release or synthesis in the cavernous nerve endings © 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 997–1003
causes ED in diabetic rats. A reduction in nNOS expression and endothelial morphological changes leading to ED in 12-week diabetic rats was observed by Albersen et al. (2011). Furthermore, a significant reduction in ICP/ MAP ratio in diabetic rats with neurostimulation was seen at 8–11 weeks of diabetes (Christ et al., 2006). In fact, the diabetes-induced harmful effect on the efferent neurons was correlated with the severity of ED (Rehman et al., 1997). In the present results, the ICP/MAP ratio in the ASA-treated diabetic group significantly increased when compared with diabetic rats in in vivo studies. Most importantly, this normalised effect shows the protective effect of ASA in diabetes. Our study showed no difference between control and diabetic groups concerning nNOS levels as measured with RT-PCR. In addition, we detected a significant increased expression in the nNOS levels in ASA-treated diabetic rats. Thus, the functional and molecular activities with ASA treatment in diabetes could be different in in vivo and in vitro experiments at 8 weeks of diabetes induction. The increased nNOS expression might be an important factor in the improvement in ED in the ASA-treated diabetic rat penis. Studies have reported that ACh-induced relaxation response was seen in diabetic CC smooth muscle, and it was mediated by endothelium-dependent mechanisms 1001
Acetylsalicylic acid in erectile dysfunction
(Azadzoi & Saenz de Tejada, 1992; Gur et al., 2000). In our study, we detected a significantly increased relaxant response in the D+ASA group in comparison with other groups at high doses of ACh. The response to EFS of isolated CC strips is a frequency-dependent and a neurogenic relaxation and is mediated by nonadrenergic and noncholinergic neurotransmitters (Saenz de Tejada et al., 1988). In previous experimental studies, the responses to EFS in diabetic tissues were significantly less pronounced at all frequency levels (Saenz de Tejada et al., 1989; Azadzoi & Saenz de Tejada, 1992). However, in our findings, the relaxant responses to EFS at 1–8 Hz in CC from ASA-treated diabetic rats were significantly increased when compared with the diabetic group. Furthermore, relaxant responses of strips related to all frequencies of EFS in the ASA-treated diabetic group were similar to those of the control group in our study. We considered that ASA treatment could improve the impaired neurogenic relaxation and endothelium-dependent mechanisms in diabetic animals. Previous studies reported that penile corporal smooth muscle relaxed normally with SNP in diabetes (Azadzoi & Saenz de Tejada, 1992; Gur et al., 2000), while SNP is an endothelium-independent vasodilator agent. In the present study, there was no difference between the groups according to SNP-induced relaxation responses of strips. Perhaps the relaxation capacity of corpus cavernosal smooth muscle was not affected by diabetes. It is noted that PE is an a-adrenergic receptor agonist and produces a dose-dependent contractile response in penile CC (Tauseef et al., 2008). The PE-induced contractile responses in ASA-treated control and diabetic groups were insignificantly higher than other groups in our study. ASA might have an impact on a-adrenergic receptor response of corpus cavernosum. On the other hand, Tauseef et al. (2008) determined that relaxation and contraction responses of aortic rings to SNP and PE were not changed with ASA treatment in hypercholesterolaemic rats. There were some limitations in our study. We did not evaluate the eNOS with RT-PCR. The alterations on eNOS with ASA treatment might explain the significant increased ICP/MAP ratio in ASA-treated diabetic rats. Another limitation of our study was the absence of Western blot quantification of nNOS protein expressions. The impact of different dosages of ASA on diabetes-induced ED should be investigated in future studies. Conclusions This is the first study investigating the effect of ASA treatment on diabetes-induced ED in rats. While there are multiple mechanisms in the progression of diabetic ED, the present study demonstrated that ASA treatment 1002
G. Hafez et al.
is effective in improving ED in diabetic rats in vivo and in vitro. In addition, the significant increase in nNOS as well as eNOS expression with improved endothelial dysfunction after ASA treatment might be involved in the other factors which need to be investigated to understand the exact mechanism and effectiveness of ASA treatment in diabetic ED. This study might provide an initial evidence for the novel therapeutic approach of ASA in diabetic ED. ASA might be used in prophylactic treatment of diabetic ED to preserve the erection capacity of patients. References Albersen M, Lin G, Fandel TM, Zhang H, Qiu X, Lin CS, Lue TF (2011) Functional, metabolic and morphologic characteristics of a novel rat model of type 2 diabetesassociated erectile dysfunction. Urology 78:476.e1–476.e8. Andersson KE, Wagner G (1995) Physiology of penile erection. Physiol Rev 75:191–236. Awtry EH, Loscalzo J (2000) Aspirin. Circulation 101: 1206–1218. Azadzoi KM, Saenz de Tejada I (1992) Diabetes mellitus impairs neurogenic and endothelium dependent relaxation of rabbit corpus cavernosum smooth muscle. J Urol 146:1587–1591. Bennett NE, Kim JH, Wolfe DP, Sasaki K, Yoshimura N, Goins WF, Huang S, Nelson JB, de Groat WC, Glorioso JC, Chancellor MB (2005) Improvement in erectile dysfunction after neurotrophic factor gene therapy in diabetic rats. J Urol 173:1820–1824. Bornman MS, Franz RC, Jacobs DJ, Du Plessis DJ (1987) Effect of single dose aspirin on the development of penile hypercoagulability during erection. Br J Urol 59:267–271. Cellek S, Rodrigo J, Lobos E, Fernandez P, Serrano J, Moncada S (1999) Selective nitrergic neurodegeneration in diabetes mellitus- a nitric oxide dependent phenomenon. Br J Pharmacol 128:1804–1812. Christ GJ, Hsieh Y, Zhao W, Schenk G, Venkateswarlu K, Wang HZ, Tar MT, Melman A (2006) Effects of streptozotocin-induced diabetes on bladder and erectile (dys)function in the same rat in vivo. BJU Int 97:1076–1082. Cyrus T, Sung S, Zhao L, Funk CD, Tang S, Pratico D (2002) Effect of low dose aspirin on vascular inflammation, plaque stability and atherogenesis in low-density lipoprotein receptor-deficient mice. Circulation 106:1282–1287. Furuno T, Yamasaki F, Yokoyama T, Sato K, Sato T, Doi Y, Sugiura T (2011) Effects of various doses of aspirin on platelet activity and endothelial function. Heart Vessels 26:267–273. Garcia MM, Fandel TM, Lin G, Shindel AW, Banie L, Lin CS, Kue TF (2010) Treatment of erectile dysfunction in the obese type 2 diabetic ZDF rat with adipose tissue-derived stem cells. J Sex Med 7:89–98.
© 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 997–1003
G. Hafez et al.
Gur S, Ozturk B, Karahan ST (2000) Impaired endotheliumdependent and neurogenic relaxation of corpus cavernosum from diabetic rats: improvement with L-arginine. Urol Res 28:14–19. Kapetanovic IM, Bauer KS, Tessier DM, Lindeblad MO, Zakharov AD, Lubet R, Lyubimov A (2009) Comparison of pharmacokinetic and pharmacodynamic profiles of aspirin following oral gavage and diet dosing in rats. Chem Biol Interact 179:233–239. Kodama M, Yamasaki Y, Sakamoto K, Yoshioka R, Matsuhisa M, Kajimoto Y, Kosugi K, Ueda N, Hori M (2000) Antiplatelet drugs attenuate progression of carotid intimamedia thickness in subjects with type 2 diabetes. Thromb Res 97:239–245. Monobe H, Yamanari H, Nakamura K, Ohe T (2001) Effects of low-dose aspirin on endothelial function in hypertensive patients. Clin Cardiol 24:705–709. Rajfer J, Aronson WJ, Bush PA, Dorey FJ, Ignarro LJ (1992) Nitric oxide as a mediator of relaxation of the corpus cavernosum in response to nonadrenergic, noncholinergic neurotransmission. N Engl J Med 326:90–94. Rehman J, Chenven E, Brink P, Peterson B, Walcott B, Wen YP, Melman A, Christ G (1997) Diminished neurogenic but not pharmacological erections in the 2–3 month experimentally diabetic F-344 rat. Am J Physiol 272:1960–1971. Saenz de Tejada I, Blanco R, Goldstein I, Azadzoi K, de las Morenas A, Krane RJ, Cohen RA (1988) Cholinergic neurotransmission in human corpus cavernosum. Responses of isolated tissue. Am J Physiol 254:459–467.
© 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 997–1003
Acetylsalicylic acid in erectile dysfunction
Saenz de Tejada I, Goldstein I, Azadzoi K, Krane RJ, Cohen RA (1989) Impaired neurogenic and endothelium-mediated relaxation of penile smooth muscle from diabetic men with impotence. N Engl J Med 320:1025–1030. Sasaki K, Yoshimura N, Chancellor MB (2003) Implications of diabetes mellitus in urology. Urol Clin North Am 30:1–12. Senbel AM (2011) Functional inhibition of erectile process in rats by indomethacin. World J Urol 29:523–527. Tauseef M, Sharma KK, Fahim M (2007) Aspirin restores normal baroreflex function in hypercholesterolemic rats by its anti-oxidative action. Eur J Pharmacol 556:136–143. Tauseef M, Shahid M, Sharma KK, Fahim M (2008) Antioxidative action of aspirin on endothelial function in hypercholesterolaemic rats. Basic Clin Pharmacol Toxicol 103:314–321. Toda N, Ayajiki K, Okamura T (2005) Nitric oxide and penile erectile function. Pharmacol Ther 106:233–266. Wu R, Lamontagne D, de Champlain J (2002) Anti-oxidative properties of acetylsalicylic acid on vascular tissues from normotensive and spontaneously hypertensive rats. Circulation 105:387–392. Xu ZS, Fu Q, Zhao ST, Liu HN (2001) Effect of diabetes and insulin treatment on nitric oxide synthase content in rat corpus cavernosum. Asian J Androl 3:139–142. Zheng H, Li YF, Cornish KG, Zucker IH, Patel KP (2005) Exercise training improves endogenous nitric oxide mechanisms within the paraventricular nucleus in rats with heart failure. Am J Physiol Heart Circ Physiol 288:2332–2341.
1003
Acetylsalicylic acid protects erectile function in diabetic rats G. Hafez1, U. Gonulalan2, M. Kosan2, E. Arioglu1, B. Ozturk2, M. Cetinkaya3 & S. Gur1 1 Faculty of Pharmacy, Department of Pharmacology, Ankara University, Ankara, Turkey; 2 Department of Urology, Baskent University, Konya Research and Training Hospital, Konya, Turkey; 3 Department of Urology, Ankara Numune Education and Research Hospital, Ankara, Turkey
Keywords Acetylsalicylic acid—diabetes mellitus—erectile dysfunction—in vitro—intracavernosal pressure Correspondence Umut Gonulalan, School of Medicine, Baskent University, Konya Research and Training Hospital, Saray Street, No: 1 Selcuklu, Konya, Turkey. Tel.: +90 332 2631494; Fax: +90 332 2570637; E-mail: [email protected]
Accepted: September 16, 2013 doi: 10.1111/and.12187
Summary We aimed to evaluate the effect of acetylsalicylic acid (ASA) treatment on diabetes-induced erectile dysfunction. Adult male Sprague–Dawley rats were divided into four groups as follows: (i) control (C), (ii) diabetic (D), (iii) ASA-treated control (C+ASA) and (iv) ASA-treated diabetic (D+ASA) groups. In groups 2 and 4, diabetes was induced by injection of 35 mg kg 1 streptozotocin. ASA (100 mg kg 1 day 1, orally) was administrated to rats in groups 3 and 4 for 8 weeks. Both intracavernosal pressure (ICP) and mean arterial blood pressure (MAP) were measured in in vivo studies. In organ bath, the relaxation responses to acetylcholine (ACh), electrical field stimulation (EFS) and sodium nitroprusside were tested in corpus cavernosum (CC) strips. The mRNA expression for neuronal nitric oxide synthase (nNOS) was calculated using reverse transcription polymerase chain reaction technique. In in vivo experiments, diabetic rats displayed reduced ICP/MAP values, which were normalised with ASA treatment. The relaxant response to high-dose ACh and EFS at low frequencies (1–8 Hz) in CC strips from the D+ASA group were significantly higher when compared to the D group. Treatment with ASA normalised the raised mRNA expressions of nNOS in diabetic penile tissues. ASA may be involved in mRNA of protein synthesis of NO released from nonadrenergic and noncholinergic cavernosal nerve in diabetes.
Introduction Penile erection is in control of a complex neural and vascular interaction that causes cavernosal smooth muscle relaxation. The nitric oxide (NO) is synthesised by neuronal (nNOS) and endothelial NO synthase (eNOS) and plays an important role in cavernosal smooth muscle relaxation with the NO/cyclic guanosine monophosphate (cGMP) cascade (Rajfer et al., 1992; Toda et al., 2005). Diabetes mellitus (DM) is the common risk factor for erectile dysfunction (ED), although the pathogenesis of ED in DM remains controversial because of the multifactorial nature of DM (Sasaki et al., 2003). The neuropathy and vascular dysfunction make it difficult to pinpoint a single aetiology for ED (Bennett et al., 2005). Acetylsalicylic acid (ASA) acts with significant efficacy in the treatment of coronary and cerebrovascular events (Awtry & Loscalzo, 2000). This effect is a result of © 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 997–1003
cyclooxygenase-1 inhibition in platelets and endotheliumdependent arterial relaxation (Awtry & Loscalzo, 2000; Monobe et al., 2001). In a recent report, ASA improved endothelium-dependent vasorelaxation through increased bioavailability of NO (Tauseef et al., 2008). On the other hand, indomethacin and diclofenac as nonsteroidal antiinflammatory drugs negatively affected the erectile process in rats due to reduction in the plasma level of NO (Senbel, 2011). nNOS is localised in penile autonomic nerves and arterioles. However, eNOS is detectable in endothelial cells of the corpus cavernosum (CC) (Andersson & Wagner, 1995). Both reduction in nNOS expression and endothelial morphological changes have been documented as causing ED in diabetes (Albersen et al., 2011). To date, there has been no study regarding treatment of ED with ASA in diabetic rats. The goal of the current study was to investigate the effect of ASA treatment on 997
Acetylsalicylic acid in erectile dysfunction
the CC in diabetes-induced ED in a rat model in vivo and in organ bath studies. Materials and methods Animals and induction of diabetes Sixteen-week-old Sprague–Dawley male rats, weighing 300–310 g, were provided by the Animal Care Facility of Selcuk University (Konya, Turkey). Rats were divided into four groups as follows: control (C) (n = 18), diabetic (D) (n = 12), ASA-treated control (C+ASA) (n = 12) and ASA-treated diabetic (D+ASA) (n = 13). Diabetes was induced by an intravenous (i.v.) injection of 35 mg kg 1 streptozotocin (STZ, dissolved in citrate buffer, pH 4.6) via the tail vein. After the fourth day of injection, blood glucose levels were measured by Accu-Check Go Roche glucometer. Rats with low levels of blood glucose (below 250 mg dl 1) were omitted from experiments. ASA was given orally at a dose of 100 mg kg 1 day 1 over 8 weeks (Kapetanovic et al., 2009). Experimental protocols were approved by the Ankara University Animal Experiments Ethical Committee (No: 2008-28-134). In vivo experiments The erectile function studies in groups in vivo were performed after 8 weeks of diabetes. Rats were anaesthetised with an intraperitoneal (i.p.) injection of 100 mg kg 1 ketamine. Animals were placed in the supine position, and bladder and prostate were exposed through a midline abdominal incision. The inferior hypogastric plexus and pelvic nerves were identified post-erolateral to the prostate on both sides, and stainless bipolar wire electrodes were placed around these structures for electrical stimulation. The penis was denuded of skin, and CCs were exposed by removing part of the overlying ischiocavernosus muscles. To monitor intracavernosal pressure (ICP), a 23-gauge cannula was filled with 250 IU ml 1 heparin solution connected to polyethylene-50 tubing and inserted into the left CC. Systemic arterial blood pressure and mean arterial pressure (MAP) were monitored via a 23-gauge cannula placed into the carotid artery (Christ et al., 2006). Both pressure lines were connected to a transducer, and pressures were recorded by Biopac Lab Pro MP35 System (BIOPAC Systems, Inc., Goleta, CA, USA) on the computer. Stimulation of the cavernosal nerve was performed with an electrode. Stimulation parameters measured by Grass S48 stimulator were frequency 15 Hz, voltage 7.5 volt, pulse width 30 ms, duration 1 min and amplitude 1.5 mA. All transducers were calibrated before each experiment. The ratio of ICP to MAP was calculated to reach exact erectile response. 998
G. Hafez et al.
Isometric tension measurement in CC strips After anaesthesia, rats were sacrificed by exsanguination, and then, CC tissues were isolated for bath studies. The CC strips were placed in 20 ml organ baths under 1 g tension in Krebs solution and oxygenated with 95% O2 and 5% CO2. Tissues were allowed to equilibrate for 60 min at 37 °C, and the bath medium was replaced every 15 min. Acetylcholine (ACh, 10 8–10 3 M), electrical field stimulation (EFS, 1–32 Hz) and sodium nitroprusside (SNP, 10 8–10 3 M)-induced relaxation responses were evaluated after pre-contraction with phenylephrine (PE, 10 5 M). The contractile response to PE (10 8–10 3 M) was also recorded. Total RNA isolation, reverse transcription polymerase chain reaction (RT-PCR) and PCR experiments The cavernosal tissues were stored at 80 °C after they were taken in liquid nitrogen in sterile conditions. Cavernosal tissues were powdered with liquid nitrogen and homogenised with an ultrasonic homogeniser (Bandelin electronic, Berlin, Germany) prior to RNA extraction. Total RNA was extracted with the TRIzol reagent (SigmaAldrich Co., St. Louis, MO, USA) according to the manufacturer’s protocol. The optical density values and amounts of RNA were determined spectrophotometrically using Nanodrop (NanoDrop Products, Wilmington, DE, USA) at wavelengths of 260 nm (k 260) and 280 nm (k 280), and the OD k 260/k 280 ratio was used as a precursory estimation of RNA quality. In addition, formamide/formaldehyde agarose gels were used later to evaluate RNA integrity. RT-PCR experiments were performed using ImProm II Reverse Transcriptase System (Promega, Madison, WI, USA). 1 ll oligo dT (Promega) was added to equivalent amounts of total RNA from all groups. The mixtures were placed into thermocycler (Hybaid, UK) and held at 70 °C for 5 min. The samples were then transferred into ice for 5 min. Thereafter, 1 ll 10 mM deoxynucleotide triphosphate (dNTP), 6.1 ll nuclease-free water, 4 ll 5XMgCl-free buffer, 2.4 ll 25 mM MgCl2, 1 ll reverse transcriptase and 0.5 ll RNasin were added, and the final volume was 20 ll. The tubes were again placed into the thermocycler and heated for 60 min at 42 °C for RT, followed by 15 min at 70 °C for denaturation. First strand cDNA samples were then cooled to 4 °C and were stored at 80 °C until use. The single strand cDNAs were consequently amplified by PCR using gene specific primer (Zheng et al., 2005) and Taq polymerase. For this purpose, 5X MgCl2-free reaction buffer, MgCl2 (2.8–3.8 mM), dNTP (0.5 mM), Taq DNA polymerase (5u ul 1) (Promega), cDNA (1 ll), and 2 ll (from 10 pmol ml 1) of respective © 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 997–1003
G. Hafez et al.
Acetylsalicylic acid in erectile dysfunction
sense and antisense primers were added to PCR tubes. Nuclease-free water was then added to each tube for a final volume of 50 ll. The samples were then mixed, placed in the thermocycler and denatured for 3 min at 94 °C. Thereafter, segments were amplified using the sequence 1.5 min denaturation (94 °C) followed by 1 min annealing (58– 60 °C) and 2 min extension (72 °C), and this sequence was repeated for a total of 35 cycles. Beta-actin was amplified in each set of PCR reactions and served as internal references during quantitation to correct for operator and/or experimental variations. At the end of the reactions, 30 ll of each PCR product was then loaded onto a 1% agarose gel containing ethidium bromide and electrophoresed for 1 h at 100 V (Sci-Plas, UK). The resulting gels were then visualised using an UV transilluminator (Vilber Lourmat TFX 20 M UV) and photographed using UV gel camera (Kodak EDAS 290; Eastman Kodak Company, Rochester, NY, USA). Band intensities were calculated using Kodak 1D 3.6 program (Eastman Kodak Company).
(a)
(b)
Statistical analysis All data were expressed as means standard errors. The Statistical Package for the Social Sciences (16.0 for Windows, SPSS Inc., Chicago, IL, USA) software program was used for statistical analysis. One-way ANOVA with Tukey’s HSD test was used in comparison with data between the groups, and values of P < 0.05 were considered as significant. Results Body weights and blood glucose levels in rats The mean body weights in groups C, D, C+ASA and D+ASA were 326 16.6 g, 286 26.3 g, 286 3.83 g and 277 22.7 g respectively. The mean blood glucose levels in groups C, D, C+ASA and D+ASA were 91.7 0.81, 355 41.4, 88.1 1.33 and 350 32.1 mg dl 1 respectively. According to these results, the mean body weights in D, C+ASA and D+ASA groups at sacrifice were significantly lower than in C group (P < 0.05). The mean blood glucose levels at sacrifice were significantly higher in D and D+ASA groups in comparison with C and C+ASA groups (P < 0.05). In addition to these results, the tissue weights of each CC strip were measured, and there was no significant difference between the groups (P > 0.05). Relaxation response to ACh
between groups. However, the relaxant response of strips from the D+ASA group at 10 3M concentration of ACh was significantly higher than that of CC strips from the other groups (P < 0.01). Relaxation response to EFS The relaxant response to EFS (1–32 Hz) according to groups is displayed in Fig. 1b. As seen in Fig. 1b, the relaxant responses in CC from the D+ASA group at low frequency EFS (1–8 Hz) were significantly higher than the responses in the D group (P < 0.05). The relaxant response to EFS in the D+ASA group at higher frequencies was slightly higher than in the D group, but the differences were not statistically significant (P > 0.05). Furthermore, all relaxant responses of strips related to all frequencies of EFS were similar in the D+ASA, C and C+ASA groups (P > 0.05). Relaxation response to SNP
The relaxant response to ACh (10 –10 M) is seen in Fig. 1a. There was no difference in ACh responses 8
© 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 997–1003
Fig. 1 The endothelium and neurogenic relaxant responses to (a) acetylcholine and (b) electrical field stimulation in the corpus cavernosum strips according to groups. *P < 0.05 D+ acetylsalicylic acid (ASA) group vs D group. **P < 0.01 D+ASA group vs other groups.
3
The relaxation responses of strips related to SNP (10 8– 10 3 M) were statistically similar in all groups (Fig. 2a). 999
Acetylsalicylic acid in erectile dysfunction
(a)
G. Hafez et al.
significantly decreased in diabetic rats in comparison with the control group (P < 0.05). However, ASA treatment normalised the diminished ICP/MAP ratio in diabetic rats (P < 0.05). The mRNA expressions for nNOS using RT-PCR
(b)
The mRNA expressions of nNOS in C, D, C+ASA and D+ASA groups were 100%, 116%, 137% and 154%, respectively, and the difference between C and D groups was not statistically significant (P = 0.17). On the other hand, the mRNA expressions of nNOS in the D+ASA group were significantly increased in comparison with C and D groups (P < 0.01, Fig. 5). Discussion
Fig. 2 The endothelium-independent relaxant responses to (a) Sodium nitroprusside were similar between groups, and the contractile responses of strips related to (b) Phenylephrine were unaltered in all groups.
Fig. 3 The mean intracavernosal pressure/mean arterial blood pressure ratio in rats. *P < 0.05 D group vs C group or D+ acetylsalicylic acid group.
Contractile response to PE The contractile response to PE (10 8–10 3 M) was unaltered in CC strips from all groups (Fig. 2b). In vivo erectile responses The mean ICP/MAP ratios and graphics of rats are exhibited in Figs 3 and 4. The mean ICP/MAP ratio was 1000
Acetylsalicylic acid (ASA) is widely used in the treatment of cardiovascular diseases. It decreases vascular smooth muscle cell proliferation and proinflammatory mediators and improves endothelium-dependent vasorelaxation mediated by NO (Kodama et al., 2000; Cyrus et al., 2002; Tauseef et al., 2008; Furuno et al., 2011). On the other hand, there are several diabetic animal models that reported reduced expression of nNOS in CC (Cellek et al., 1999; Xu et al., 2001; Garcia et al., 2010). We investigated the possible protective effects of ASA on ED induced with diabetes. Several studies have reported about the administration and appropriate doses of ASA in rats. A study by Kapetanovic et al. compared different routes of administration of ASA (gavage versus dietary) and demonstrated that the route of ASA administration did not change the pharmacokinetic or pharmacodynamic parameters (Kapetanovic et al., 2009). The relationship between ASA and endothelial function has also been clarified (Furuno et al., 2011). Furuno et al. evaluated the effective dose of ASA on platelet aggregation and endothelial function in healthy volunteers (Furuno et al., 2011). They reported that ASA at all doses suppressed platelet activity, and endothelial-mediated arterial dilatation at higher doses of ASA was worsened. In rat models, an optimal dose of 100 mg kg 1 day 1 was determined for evaluating the effects of ASA (Wu et al., 2002; Tauseef et al., 2007, 2008). In the present study, ASA (dose of 100 mg kg 1 day 1, orally) was given by gavage for 8 weeks in treated animals. ASA is a cardioprotective agent via the inhibition of platelet activity, decrease in vascular smooth muscle cell proliferation and reduction in proinflammatory mediators (Kodama et al., 2000; Cyrus et al., 2002; Furuno et al., 2011). Bornman et al. (1987) reported that ASA delays penile atherosclerosis. It has been suggested that ASA with © 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 997–1003
G. Hafez et al.
Acetylsalicylic acid in erectile dysfunction
(a)
(b)
(c)
(d)
Fig. 4 The intracavernosal pressure/mean arterial blood pressure graphics of rats in (a) Control, (b) Diabetic, (c) Acetylsalicylic acid (ASA) treated Control and (d) ASA treated Diabetic groups.
Fig. 5 The change in the percentage of neuronal nitric oxide synthase mRNA expression according to groups. *P < 0.05 C+ acetylsalicylic acid (ASA) group vs C group or D group. **P < 0.01 D+ASA group vs C group or D group.
antioxidant activity ameliorated endothelium-dependent vasorelaxation because of the raised bioavailability of NO (Tauseef et al., 2008). On the other hand, several studies in diabetic models indicated reduction in the expression of nNOS enzyme in the CC (Cellek et al., 1999; Xu et al., 2001; Garcia et al., 2010). Thus, decreased endogenous NO release or synthesis in the cavernous nerve endings © 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 997–1003
causes ED in diabetic rats. A reduction in nNOS expression and endothelial morphological changes leading to ED in 12-week diabetic rats was observed by Albersen et al. (2011). Furthermore, a significant reduction in ICP/ MAP ratio in diabetic rats with neurostimulation was seen at 8–11 weeks of diabetes (Christ et al., 2006). In fact, the diabetes-induced harmful effect on the efferent neurons was correlated with the severity of ED (Rehman et al., 1997). In the present results, the ICP/MAP ratio in the ASA-treated diabetic group significantly increased when compared with diabetic rats in in vivo studies. Most importantly, this normalised effect shows the protective effect of ASA in diabetes. Our study showed no difference between control and diabetic groups concerning nNOS levels as measured with RT-PCR. In addition, we detected a significant increased expression in the nNOS levels in ASA-treated diabetic rats. Thus, the functional and molecular activities with ASA treatment in diabetes could be different in in vivo and in vitro experiments at 8 weeks of diabetes induction. The increased nNOS expression might be an important factor in the improvement in ED in the ASA-treated diabetic rat penis. Studies have reported that ACh-induced relaxation response was seen in diabetic CC smooth muscle, and it was mediated by endothelium-dependent mechanisms 1001
Acetylsalicylic acid in erectile dysfunction
(Azadzoi & Saenz de Tejada, 1992; Gur et al., 2000). In our study, we detected a significantly increased relaxant response in the D+ASA group in comparison with other groups at high doses of ACh. The response to EFS of isolated CC strips is a frequency-dependent and a neurogenic relaxation and is mediated by nonadrenergic and noncholinergic neurotransmitters (Saenz de Tejada et al., 1988). In previous experimental studies, the responses to EFS in diabetic tissues were significantly less pronounced at all frequency levels (Saenz de Tejada et al., 1989; Azadzoi & Saenz de Tejada, 1992). However, in our findings, the relaxant responses to EFS at 1–8 Hz in CC from ASA-treated diabetic rats were significantly increased when compared with the diabetic group. Furthermore, relaxant responses of strips related to all frequencies of EFS in the ASA-treated diabetic group were similar to those of the control group in our study. We considered that ASA treatment could improve the impaired neurogenic relaxation and endothelium-dependent mechanisms in diabetic animals. Previous studies reported that penile corporal smooth muscle relaxed normally with SNP in diabetes (Azadzoi & Saenz de Tejada, 1992; Gur et al., 2000), while SNP is an endothelium-independent vasodilator agent. In the present study, there was no difference between the groups according to SNP-induced relaxation responses of strips. Perhaps the relaxation capacity of corpus cavernosal smooth muscle was not affected by diabetes. It is noted that PE is an a-adrenergic receptor agonist and produces a dose-dependent contractile response in penile CC (Tauseef et al., 2008). The PE-induced contractile responses in ASA-treated control and diabetic groups were insignificantly higher than other groups in our study. ASA might have an impact on a-adrenergic receptor response of corpus cavernosum. On the other hand, Tauseef et al. (2008) determined that relaxation and contraction responses of aortic rings to SNP and PE were not changed with ASA treatment in hypercholesterolaemic rats. There were some limitations in our study. We did not evaluate the eNOS with RT-PCR. The alterations on eNOS with ASA treatment might explain the significant increased ICP/MAP ratio in ASA-treated diabetic rats. Another limitation of our study was the absence of Western blot quantification of nNOS protein expressions. The impact of different dosages of ASA on diabetes-induced ED should be investigated in future studies. Conclusions This is the first study investigating the effect of ASA treatment on diabetes-induced ED in rats. While there are multiple mechanisms in the progression of diabetic ED, the present study demonstrated that ASA treatment 1002
G. Hafez et al.
is effective in improving ED in diabetic rats in vivo and in vitro. In addition, the significant increase in nNOS as well as eNOS expression with improved endothelial dysfunction after ASA treatment might be involved in the other factors which need to be investigated to understand the exact mechanism and effectiveness of ASA treatment in diabetic ED. This study might provide an initial evidence for the novel therapeutic approach of ASA in diabetic ED. ASA might be used in prophylactic treatment of diabetic ED to preserve the erection capacity of patients. References Albersen M, Lin G, Fandel TM, Zhang H, Qiu X, Lin CS, Lue TF (2011) Functional, metabolic and morphologic characteristics of a novel rat model of type 2 diabetesassociated erectile dysfunction. Urology 78:476.e1–476.e8. Andersson KE, Wagner G (1995) Physiology of penile erection. Physiol Rev 75:191–236. Awtry EH, Loscalzo J (2000) Aspirin. Circulation 101: 1206–1218. Azadzoi KM, Saenz de Tejada I (1992) Diabetes mellitus impairs neurogenic and endothelium dependent relaxation of rabbit corpus cavernosum smooth muscle. J Urol 146:1587–1591. Bennett NE, Kim JH, Wolfe DP, Sasaki K, Yoshimura N, Goins WF, Huang S, Nelson JB, de Groat WC, Glorioso JC, Chancellor MB (2005) Improvement in erectile dysfunction after neurotrophic factor gene therapy in diabetic rats. J Urol 173:1820–1824. Bornman MS, Franz RC, Jacobs DJ, Du Plessis DJ (1987) Effect of single dose aspirin on the development of penile hypercoagulability during erection. Br J Urol 59:267–271. Cellek S, Rodrigo J, Lobos E, Fernandez P, Serrano J, Moncada S (1999) Selective nitrergic neurodegeneration in diabetes mellitus- a nitric oxide dependent phenomenon. Br J Pharmacol 128:1804–1812. Christ GJ, Hsieh Y, Zhao W, Schenk G, Venkateswarlu K, Wang HZ, Tar MT, Melman A (2006) Effects of streptozotocin-induced diabetes on bladder and erectile (dys)function in the same rat in vivo. BJU Int 97:1076–1082. Cyrus T, Sung S, Zhao L, Funk CD, Tang S, Pratico D (2002) Effect of low dose aspirin on vascular inflammation, plaque stability and atherogenesis in low-density lipoprotein receptor-deficient mice. Circulation 106:1282–1287. Furuno T, Yamasaki F, Yokoyama T, Sato K, Sato T, Doi Y, Sugiura T (2011) Effects of various doses of aspirin on platelet activity and endothelial function. Heart Vessels 26:267–273. Garcia MM, Fandel TM, Lin G, Shindel AW, Banie L, Lin CS, Kue TF (2010) Treatment of erectile dysfunction in the obese type 2 diabetic ZDF rat with adipose tissue-derived stem cells. J Sex Med 7:89–98.
© 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 997–1003
G. Hafez et al.
Gur S, Ozturk B, Karahan ST (2000) Impaired endotheliumdependent and neurogenic relaxation of corpus cavernosum from diabetic rats: improvement with L-arginine. Urol Res 28:14–19. Kapetanovic IM, Bauer KS, Tessier DM, Lindeblad MO, Zakharov AD, Lubet R, Lyubimov A (2009) Comparison of pharmacokinetic and pharmacodynamic profiles of aspirin following oral gavage and diet dosing in rats. Chem Biol Interact 179:233–239. Kodama M, Yamasaki Y, Sakamoto K, Yoshioka R, Matsuhisa M, Kajimoto Y, Kosugi K, Ueda N, Hori M (2000) Antiplatelet drugs attenuate progression of carotid intimamedia thickness in subjects with type 2 diabetes. Thromb Res 97:239–245. Monobe H, Yamanari H, Nakamura K, Ohe T (2001) Effects of low-dose aspirin on endothelial function in hypertensive patients. Clin Cardiol 24:705–709. Rajfer J, Aronson WJ, Bush PA, Dorey FJ, Ignarro LJ (1992) Nitric oxide as a mediator of relaxation of the corpus cavernosum in response to nonadrenergic, noncholinergic neurotransmission. N Engl J Med 326:90–94. Rehman J, Chenven E, Brink P, Peterson B, Walcott B, Wen YP, Melman A, Christ G (1997) Diminished neurogenic but not pharmacological erections in the 2–3 month experimentally diabetic F-344 rat. Am J Physiol 272:1960–1971. Saenz de Tejada I, Blanco R, Goldstein I, Azadzoi K, de las Morenas A, Krane RJ, Cohen RA (1988) Cholinergic neurotransmission in human corpus cavernosum. Responses of isolated tissue. Am J Physiol 254:459–467.
© 2013 Blackwell Verlag GmbH Andrologia 2014, 46, 997–1003
Acetylsalicylic acid in erectile dysfunction
Saenz de Tejada I, Goldstein I, Azadzoi K, Krane RJ, Cohen RA (1989) Impaired neurogenic and endothelium-mediated relaxation of penile smooth muscle from diabetic men with impotence. N Engl J Med 320:1025–1030. Sasaki K, Yoshimura N, Chancellor MB (2003) Implications of diabetes mellitus in urology. Urol Clin North Am 30:1–12. Senbel AM (2011) Functional inhibition of erectile process in rats by indomethacin. World J Urol 29:523–527. Tauseef M, Sharma KK, Fahim M (2007) Aspirin restores normal baroreflex function in hypercholesterolemic rats by its anti-oxidative action. Eur J Pharmacol 556:136–143. Tauseef M, Shahid M, Sharma KK, Fahim M (2008) Antioxidative action of aspirin on endothelial function in hypercholesterolaemic rats. Basic Clin Pharmacol Toxicol 103:314–321. Toda N, Ayajiki K, Okamura T (2005) Nitric oxide and penile erectile function. Pharmacol Ther 106:233–266. Wu R, Lamontagne D, de Champlain J (2002) Anti-oxidative properties of acetylsalicylic acid on vascular tissues from normotensive and spontaneously hypertensive rats. Circulation 105:387–392. Xu ZS, Fu Q, Zhao ST, Liu HN (2001) Effect of diabetes and insulin treatment on nitric oxide synthase content in rat corpus cavernosum. Asian J Androl 3:139–142. Zheng H, Li YF, Cornish KG, Zucker IH, Patel KP (2005) Exercise training improves endogenous nitric oxide mechanisms within the paraventricular nucleus in rats with heart failure. Am J Physiol Heart Circ Physiol 288:2332–2341.
1003
