Down-regulation of KCa2.3 channels causes erectile dysfunction in mice

Modulation of endothelial calcium-activated K+ channels has been proposed as an approach to restore arterial endothelial cell function in disease. We hypothesized that small-conductance calcium-activated K+ channels (KCa2.3 or SK3) contributes to erectile function. The research was performed in transgenic mice with overexpression (KCa2.3T/T(−Dox)) or down-regulation (KCa2.3T/T(+Dox)) of the KCa2.3 channels and wild-type C57BL/6-mice (WT). QPCR revealed that KCa2.3 and KCa1.1 channels were the most abundant in mouse corpus cavernosum. KCa2.3 channels were found by immunoreactivity and electron microscopy in the apical-lateral membrane of endothelial cells in the corpus cavernosum. Norepinephrine contraction was enhanced in the corpus cavernosum of KCa2.3T/T(+Dox) versus KCa2.3T/T(−Dox) mice, while acetylcholine relaxation was only reduced at 0.3 µM and relaxations in response to the nitric oxide donor sodium nitroprusside were unaltered. An opener of KCa2 channels, NS309 induced concentration-dependent relaxations of corpus cavernosum. Mean arterial pressure was lower in KCa2.3T/T(−Dox) mice compared with WT and KCa2.3T/T(+Dox) mice. In anesthetized mice, cavernous nerve stimulation augmented in frequency/voltage dependent manner erectile function being lower in KCa2.3T/T(+Dox) mice at low frequencies. Our findings suggest that down-regulation of KCa2.3 channels contributes to erectile dysfunction, and that pharmacological activation of KCa2.3 channels may have the potential to restore erectile function.


Results
Expression studies. In corpus cavernosum from WT animals, mRNA expression of the K Ca channels was examined and showed that the K Ca 1.1 α subunit followed by K Ca 2.3 channels and the K Ca 1.1 β1 subunit were the most robustly expressed K Ca channel subtypes (Fig. 1A). Q-PCR showed a clear down-regulation of K Ca 2.3 mRNA in K Ca 2.3 T/T (+Dox) mice (n = 9) as compared to K Ca 2.3 T/T (−Dox) mice (n = 9) (Fig. 1B). Expression of K Ca 1.1 α subunits (coding for the pore forming subunit) were unchanged in corpus cavernosum from the three groups of mice (Fig. 1C), while K Ca 1.1 β1 subunit expression was upregulated in corpus cavernosum of K Ca 2.3 T/T (+Dox) versus WT mice (Fig. 1D). Immunoblotting was performed for quantification of K Ca 2.3 in the corpus cavernosum of K Ca 2.3 T/T (−Dox) (n = 5), K Ca 2.3 T/T (+Dox) (n = 5), and WT (n = 5) mice and also aorta samples (n = 3-5). The immunoreactive band for K Ca 2.3 channels in corpus cavernosum from WT mice is slightly heavier than the band for the general K Ca 2.3 T/T mice because of the loss of the n-terminal poly-glutamate stretch in these animals (Fig. 1E). We observed a linear relation of pan-actin immunoreaction to the amount of protein loaded (results not shown), and therefore immunoblotting results for K Ca 2.3 channels were normalized to pan-actin (Fig. 1F) and showed that K Ca 2.3 expression was lower in corpus cavernosum from the K Ca 2.3 T/T (+Dox) mice compared to expression in corpus cavernosum from K Ca 2.3 T/T (−Dox) mice (Fig. 1E,F and Supplemental Figure S1E). The same expression pattern was found in aorta samples from WT (n = 5), K Ca 2.3 T/T (−Dox) (n = 3), and K Ca 2.3 T/T (+Dox) (n = 3) mice (Supplemental Figure S1A and S1E).
Immunoblotting for the pore-forming alpha-subunit of K Ca 1.1 showed no differences comparing aorta and corpus cavernosum from all three groups of mice (Supplemental Figure S1B and S1C). Immunoreaction for the regulatory beta-subunit of K Ca 1.1 channels, K Ca 1.1 β1 , was observed at 28 kDa and 110 kDa. A positive and specific band was supported by previous test using the peptide applied to raise the antibody (Supplemental Figure S1D). The K Ca 1.1 β1 showed an expression apparently inverse to up-or down-regulation of K Ca 2.3 channel expression in corpus cavernosum (Supplemental Figure S1C).
Immunohistochemistry showed expression of K Ca 2.3 channels in helicine arteries in the corpus cavernosum of WT mice ( Fig. 2A,B, panel B shows an enlargement of the inset in panel A). Labelling was also seen in capillaries (arrows, Fig. 2A) between the skeletal muscle fibers surrounding the albuginea layer (asterisks, Fig. 2A) and Data are means ± SEM of 5-9 animals in each group. P ≤ 0.05 (*) versus K Ca 2.3 T/T (+Dox) mice. Compared with one-way ANOVA followed by a Tukey multiple comparisons test or with a Student's t-test. ≈Around.
Double labelling for K Ca 2.3 (green staining, Fig. 3A and C) and smooth muscle actin (red staining, Fig. 3B and C) showed specific K Ca 2.3 expression in the endothelium of blood vessels and corpus cavernosum (Fig. 3C). Figure 4 shows immunoelectron microscopical images of endothelial cells lining a sinusoid of the WT corpus cavernosum (Fig. 4A, black asterisk). Gold-particle-labelled K Ca 2.3 proteins were found on the apical plasma membrane domains of the endothelial cells (Fig. 4B,C). Occasionally, the lateral membrane of the endothelial cells was also labelled with K Ca 2.3 (Fig. 4D).
Functional studies in isolated corpus cavernosum strips. The optimal passive tension for the corpus cavernosum strips examined was similar when comparing preparations from the three groups of mice (Supplemental Figure S2A-F). Therefore, all the experiments were performed with a passive tension of 1.8 mN. Regarding active tension produced by norepinephrine, we found that compared to WT mice, contractions to norepinephrine were enhanced in strips from K Ca 2.3 T/T (+Dox) and reduced in strips from K Ca 2.3 T/T (−Dox) (Fig. 5). Concentration-response curves for acetylcholine (ACh)-induced nitric oxide (NO)-mediated relaxations were unchanged in corpus cavernosum strips from K Ca 2.3 T/T (−Dox) , K Ca 2.3 T/T (+Dox) and WT mice, but the slopes of the curves were different for K Ca 2.3 T/T (−Dox) against the WT, and at 0.3 μM ACh relaxation was significantly enhanced in corpus cavernosum strips from K Ca 2.3 T/T (−Dox) mice compared to the WT mice (Fig. 6A).
In corpus cavernosum, NS309, an opener of K Ca 2 and K Ca 3.1 channels, induced concentration-dependent relaxations independent of mouse model (Fig. 6B), where apamin significantly inhibited NS309 relaxation at 0.01 µM (Fig. 6E). Incubation with a combination of inhibitors of NO synthase, nitro-L-arginine (L-NOARG, 100 µM) and of cyclooxygenase, indomethacin (10 µM) significantly inhibited NS309 relaxation (Suppl. Figure S3). Although there was no shift in the concentration-response curves for ACh, pre-treatment in WT corpus cavernosum with NS309 in a concentration (0.5 µM) where it is considered selective for K Ca 3.1 and K Ca 2 channels 13 , enhanced relaxations induced by 0.3 μM ACh (Fig. 6C).
Concentration-response curves for the NO donor sodium nitroprusside (SNP) were similar in corpus cavernosum from all three groups of mice (Fig. 6D). ODQ, a guanylate cyclase inhibitor, inhibited SNP relaxation to the same degree in corpus cavernosum from all three groups of mice (Supplementary Figure S4A-C).

Discussion
The main findings of the present study are that 1) Genetically encoded down-regulation of the K Ca 2.3 channel in mice results in erectile dysfunction measured as lowered ICP/MAP at 4 and 8 Hz. 2) Electron microscopy revealed that K Ca 2.3 channels are located primarily on the luminal plasma membrane and occasionally on the lateral plasma membrane of endothelial cells in the corpus cavernosum, and that modulating the expression of these channels (up-or down-regulation) changes norepinephrine contraction in corpus cavernosum strips. 3) A non-selective opener of K Ca 2 and K Ca 3.1 channels, NS309, induced concentration-dependent relaxations and enhanced the response to 0.3 µM acetylcholine in corpus cavernosum strips of WT mice. Therefore, suggesting that modulation of these channels may hold the potential for developing a novel approach for treatment of erectile dysfunction.
Previous studies have shown mRNA expression of K Ca 2.3 channels in rat and human corpus cavernosum 14,15 . In the present study, Q-PCR showed expression of preferentially the K Ca 2.3 channel subtype, which suggests that K Ca 2.3 is the major K Ca 2 subtype in murine corpus cavernosum.
The K Ca 2.3 mouse model used in the present study is a conditional model, in which doxycycline treatment causes suppression of the overexpressed channel resulting in expression levels below WT levels 16,17 . Only few studies had compared against the WT 17, 18 and biometrical vascular changes are expected on overexpression of K Ca 2.3 channels 19 . Consequently K Ca 2.3 currents in endothelial cells were found to be significantly lower than in the WT cells 18 . This is further confirmed by the immunoblotting for K Ca 2.3 channels in corpus cavernosum from transgenic animals showing upregulation in the vehicle-treated and down-regulation in the doxycycline-treated animals. Certainly, further research in complete K Ca 2.3 channels deficient models are advised in newer studies.
Immunohistochemical studies have suggested that K Ca 2.3 and K Ca 3.1 channels are expressed in the endothelium of rat and human penile arteries 20 . In the present study on murine penile tissue, immunohistochemical staining's confirmed expression of K Ca 2.3 channels in endothelial cells of penile arteries and corpus cavernosum. To the contrary, we did not see staining of smooth muscle, suggesting that K Ca 2.3 channels are mainly expressed in the endothelium of erectile tissue. K Ca 2.3 channels have been suggested to be compartmentalized within the endothelial cells of the systemic circulation, and to co-precipitate with caveolin-1, endothelial NO synthase and transient receptor potential channels 21,22 , suggesting that these proteins interact physically with each other perhaps in caveolae. Other studies have suggested that the K Ca 2.3 channels are connexin-37 associated and are localized close to endothelial-endothelial cell gap junctions 23 . In the present study, the electron microscopical examinations revealed that in endothelium of corpus cavernosum, there is expression of K Ca 2.3 channels on the apical or luminal plasma membrane of the endothelial cells and occasionally on lateral membranes of inter-endothelial junctions. In contrast, we found no or only few K Ca 2.3 channels on basal membranes of the endothelial cells in corpus cavernosum. This localization of the K Ca 2.3 channels on the endothelial cells in erectile tissue suggests that they may be physically coupled to calcium influx pathways (e.g. calcium-permeable TRPV4 or Piezo-1 channels) on the luminal membrane. On the lateral membrane K Ca 2.3 channels may be closely coupled to endothelial-endothelial cell communication, but other approaches e.g. measurements of endothelial cell calcium in situ and co-staining of channels and myoendothelial gap junctions will be required to clarify this issue.
Suppression of K Ca 2.3 channels and K Ca 3.1 channels either by genetic knockdown or their inhibition by the peptide blockers, apamin and charybdotoxin, respectively, have previously been reported to enhance the responses to vasoconstrictors in rat mesenteric arteries 18,24,25 , lamb coronary arteries 26 , and neurogenic contractions in rat penile arteries 27 . In the present study, down-regulation of the K Ca 2.3 channel in mouse corpus cavernosum, markedly enhanced norepinephrine contraction, while the norepinephrine contraction was reduced in corpus cavernosum from mice with up-regulation of K Ca 2.3. These results suggest that activation and presence of the K Ca 2.3 channels counterbalances the vasocontraction elicited by the sympathetic neurotransmitter, norepinephrine in corpus cavernosum, that may perhaps provide an important negative feedback on tone and thus favour erectile function.
In systemic arteries, K Ca 2.3 and K Ca 3.1 are involved in endothelium-dependent relaxations 18,28 . The channels also contribute to relaxation in rat penile arteries 27,29 . Experiments, in which only K Ca 2 channels were blocked, showed attenuation of acetylcholine relaxations in horse penile arteries as well as in rat corpus cavernosum 30,31 , suggesting major roles of K Ca 2.3 channels in relaxation of corpus cavernosum. However, often combined inhibition of both K Ca 2 and K Ca 3.1 channels is needed to effectively reduce acetylcholine relaxation 28,32 , suggesting important roles for both endothelial K Ca channel subtypes in endothelium-dependent relaxation in many vessels. Our present results, provide the first evidence that this may also be true for corpus cavernosum and penile arteries because in the present study down-regulation of K Ca 2.3 channel expression did not alter relaxation to acetylcholine or to the opener of K Ca 2/3.1 channels, NS309 in corpus cavernosum from K Ca 2.3 T/T (+Dox) mice. Interestingly, we found potentiating effects of NS309 and of upregulation of K Ca 2.3 channels on ACh-induced relaxation suggesting that K Ca 2.3 channels could add to acetylcholine relaxation under normal conditions. This further suggests that pharmacological modulation of K Ca 2.3 channels holds the potential for developing a novel approach for treatment of erectile dysfunction.
In contrast to the effect of apamin on NS309 relaxation, genetic modulation of the K Ca 2.3 channel failed to cause marked changes in NS309 relaxation. Apart from the K Ca 2.3 channel we cannot exclude contribution from other apamin-sensitive channels to NS309 relaxation in corpus cavernosum. However, the expression of the K Ca 2.2 and K Ca 2.1 channels is markedly lower than of the K Ca 2.3 channels (Fig. 1A). Therefore, another possibility is that there is an upregulation of the K Ca 1.1 channel current due to upregulation of the K Ca 1.1 β1 subunit. NS309 can lead to release of endothelium-derived NO and prostaglandins followed by activation of smooth muscle K Ca 1.1 channels 10,13 . The marked expression of the K Ca 1.1 channels and our observation that combined inhibition of NO synthase, L-NOARG and cyclooxygenase inhibited NS309 relaxation (Suppl. Fig. S3) support that upregulation of K Ca 1.1 activity may counteract the effect of downregulation of the K Ca 2.3 channel on relaxations induced by acetylcholine in corpus cavernosum.
In line with specific roles of K Ca 2.3 channels in endothelium-dependent vasodilation and norepinephrine-induced tone, K Ca 2.3 channels have an impact on blood pressure regulation. Indeed, mice with down-regulation of K Ca 2.3 channel expression have a higher blood pressure 17,18,33 . In contrast, up-regulation of K Ca 2.3 channels has been found to have no effect on systemic blood pressure 18 . This normal blood pressure seems to be caused by higher levels of circulating norepinephrine 34 , which likely counterbalance the tonic vasodilator input provided by K Ca 2.3 overexpression as observed in vitro. Concerning pharmacological experiments, administration of another selective opener of K Ca 2 and K Ca 3.1 channels, SKA31, reduces blood pressure over several hours in mice 35 . In conscious dogs, intravenous infusion of SKA-31 produced a strong but short-lived depressor response 36 . In the present study, we also provide evidence that upregulation of K Ca 2.3 channel expression leads to significantly reduced blood pressure in the systemic circulation, this was evident in the anesthetized mice. Concerning down-regulation of K Ca 2.3 our study revealed a trend towards systemically elevated pressure, which is in line with previous reports on elevated pressures in the K Ca 2.3 T/T (+Dox) mice.
In contrast to the changes observed in the systemic blood pressure, we here found that the basal intracavernosal pressure were similar in the mice with either up-or down-regulation of K Ca 2.3 channel expression. The basal intracavernosal pressures were low, due to the pronounced activity of the sympathetic nerves to the erectile tissue, when the penis is in the flaccid state 37,38 . Down-regulation of K Ca channels has also been found to
up-regulate sympathetic norepinephrine activity 34 . Although our in vitro studies suggest that overexpression of the K Ca 2.3 channels can inhibit norepinephrine contraction in corpus cavernosum strips, this effect is not reflected in vivo, probably due to a low basal pressure in corpus cavernosum in vivo.
Drugs currently used to treat erectile function e.g. sildenafil or vardenafil, are phosphodiesterase type 5 inhibitors, which causes relaxation in corpus cavernosum, and penile arteries through increased cyclic GMP also involving activation of K Ca 1.1 channels 39 . Although the precise mechanism of action needs to be clarified, other drugs, such as calcium dobesilate, can enhance EDH type relaxation in human erectile tissue and restore erectile function in a diabetic rat model by activation of K Ca 2.3 and K Ca 3.1 channels 40,41 . Knockout mice of K Ca 1.1 channels have reduced erectile function 42 , and openers of K Ca 1.1 channels can enhance rat erectile function 43,44 , but so far this is the first study reporting that down-regulation of K Ca 2.3 channels causes erectile dysfunction in mice.
The expression of K Ca 2.3 channels in corpus cavernosum from K Ca 2.3 T/T (+Dox) mice is downregulated compared to the K Ca 2.3 T/T (−Dox) mice, but the downregulation compared to WT mice does not reach significance. In previous studies of K Ca 2.3 T/T mice with and without doxycycline-treatment, only few studies have compared the results with wild type mice 17,18,45 , and it can be discussed whether the wild type or the upregulated K Ca 2. Although an instantaneous discharge frequency can reach 35 Hz, the pulse frequency in autonomic nerves rarely exceed 10 Hz 46, 47 , and therefore the findings of lower erectile responses at these frequencies seem relevant. The in vivo measurement in the present study were performed in anaesthetized animals and that may also influence the erectile responses, and consequently further investigation in conscious animals using other approaches will be required to confirm that K Ca 2.3 downregulation and/or pharmacological modulation of K Ca 2.3 channels play a role for erectile function. K Ca 1.1 channels consist of pore forming alpha-subunits and regulatory beta-subunits sensitive to calcium and membrane potential, respectively 48,49 . Post-transcriptional modulation e.g. sex hormones play a role in regulation of K Ca 1.1 expression 50 . As far as we understand this is the first time it is reported that a K Ca 1.1 beta-subunit can be up-regulated by a down-regulated gen and protein expression of K Ca 2.3 channels. It would be interesting in future studies to examine whether drugs targeting K Ca 1.1 channels may restore erectile function in mice with down-regulation of K Ca 2.3 channels.
In contrast to down-regulation of K Ca 2.3 channels, up-regulation of the channels gave normal intracavernosal pressure responses to low frequency stimulation of the cavernous nerve, while the maximal responses at 16 Hz were reduced compared to the responses in wild-type mice. However, the present study has been performed in healthy animals. Further studies in animal models for cardiovascular disease would be interesting to examine whether erectile function can be restored in diabetes by selective openers of K Ca 2.3 channels, once they become available.
K Ca 2.3 channels are also expressed in the brain and in the conduction system of the heart. The effect on the brain can be limited by development of drugs with hydrophilic groups preventing them from crossing the blood brain barrier. In the heart, blockers of K Ca 2.3 channels have been shown to prevent atrial fibrillation 51, 52 , but currently it is unknown whether specific openers of K Ca 2.3 channels will per se have pro-arrhythmic effects. So far results from experimentation using a non-selective K Ca 2/3-opener with moderate selectivity for K Ca 3.1 over K Ca 2 channels, SKA-31 and SKA-121, in mice and dogs 53 did not show pro-arrhythmic action of these openers. Regarding K Ca 2/3 negative gating modulators 54 , a recent study showed that the combined K Ca 2/3 negative gating modulator, RA-2, has no gross blood pressure elevating effects. However it is worth mentioning that the compound produced mild bradycardia in mice that may reflect a baroreceptor response or prolongation of cardiac action potential duration and thus the cardiac cycle.
In summary, the present study shows that K Ca 2.3 channels are located in the apical plasma membrane of endothelial cells, and occasionally at inter-endothelial junctions of the corpus cavernosum. We found that down-regulation of these channels increases norepinephrine contraction in corpus cavernosum strips, and it seems associated with erectile dysfunction. Moreover, pharmacological activation of K Ca 2 channels enhances acetylcholine-induced relaxations in corpus cavernosum, suggesting that modulation of these channels holds the perspectives for developing new drugs and a novel strategy to treat erectile dysfunction. The modified genetically mice used in the present study have a tetracycline-base genetic insertion in a 5′ untranslated region that can be activated by addition of doxycycline (Dox) in the water intake of the animals. For a minimum of 7 days before experimentation Dox (0.5 mg/mL) and sucrose (2%) was administered in dark bottles to the mice. Homozygous K Ca 2.3 targeted mice (K Ca 2.3 T/T ) with addition (K Ca 2.3 T/T (+Dox) ) or not (K Ca 2.3 T/T ) of Dox, together with their wild-type (WT) littermates were used for experiments. Dox in the water intake was maintained until the research protocols were performed.
For in vivo measurements, the mice were anesthetized with intraperitoneal pentobarbital (50 mg/Kg). Pain was assessed regularly during surgery and pressure measurements by pressing a needle against the paw. In case of reaction additional anesthesia (17 mg/Kg pentobarbital) was administered. The mice were cervical-dislocated-euthanized followed by exsanguination after in vivo studies or for isolation of tissues and in vitro studies.

PCR and Q-PCR.
Corpus cavernosum tissue was stored in RNA later (Sigma-Aldrich) until extraction and purification of total RNA was performed using the RNeasy Mini Plus Kit (Qiagen). cDNA was synthetized using SuperScript III Reverse Transcriptase (Life Technologies).
The Q-PCR was performed in a MX3005 Q-PCR system (Agilent Technologies). The samples were run for a 40 cycles protocol. Ct-values for the gene of interest were normalised against Ct values for the housekeeping gene (GAPDH), after quantification with the program MxPro v.4.10. (Stratagene, Agilent Technologies). Values are expressed as a ratio of GAPDH. For genotyping of the mice, conventional PCR was performed in a Peqstar thermal cycler (Peqlab). The protocol followed a 'hot-start' procedure and thermal cycling conditions. Immunohistochemistry. Penile tissue was fixed in 2.5-3% paraformaldehyde overnight and paraffin embedded using standard protocols 56 . Sections were cut, fixed, target retrieve activated and labeled as described in the supplemental protocol for single labeling: primary rabbit anti-K Ca 2.3 antibody (1:400, Santa Cruz Biotechnology) and secondary goat anti-rabbit peroxidase-conjugated antibody (1:200) were used. Detection was done with 3,3′-diaminobenzidine (DAB) and images were taken using a light microscope (Leica DMRE). For double labeling, sections were incubated with rabbit anti-K Ca 2.3 antibody (1:400) and mouse anti-smooth muscle actin antibody (1:800, Dako). Visualization was performed with donkey anti-rabbit Alexa Fluor 488-conjugated and donkey anti-mouse Alexa Fluor 555-conjugated secondary antibodies (1:1000, Molecular Probes, Life Technologies). Imaging was obtained with a Leica TCS SL laser scanning confocal microscope and Leica confocal software (Leica). Electron microscopy. Penile tissue was maintained in 4% paraformaldehyde in a 0.1 M sodium cacodylate buffer overnight, followed by incubation in 2.3 M Sucrose for 2 hours and snap frozen. Ultrathin cryosections were obtained (Reichert Ultracut S, Leica) and incubated with rabbit anti-K Ca 2.3 antibody 1:1200) followed by incubation with goat-anti rabbit antibody conjugated to 10 nm gold particles (1:50). The sections were stained for 5 min in a 1.8% methylcellulose/0.4% uranyl acetate solution and observed with an electron microscope (Morgagni 268 from FEI Phillips Electron Optics).
Isometric tension recording in isolated corpus cavernosum. After dissection of corpus cavernosum as previously reported 43,57 , the strips were mounted between two wire clamps with one clamp connected to an isometric transducer (Danish Myo Technology), and immersed in 10 ml of physiological salt solution (PSS), bubbled with a gas mix (95% O 2 and 5% CO 2 ) while kept at 37 °C during the whole experiment 27 .
For strips from WT, K Ca 2.3 T/T (−Dox) or K Ca 2.3 T/T (+Dox) mice length-contraction curves were constructed with norepinephrine (3 μM) and then acetylcholine ((ACh)−1 μM) to obtain an optimal contraction and relaxation length.
To investigate whether opening of K Ca 2.1-3 and K Ca 3.1 channels enhances acetylcholine relaxation, the preparations were incubated with NS309 (5 × 10 −7 M) prior to construction of concentration-response curves for acetylcholine.
In vivo pressure measurements of intracavernous pressure. Mean arterial blood pressure (MAP) and intracavernous blood pressure (ICP) was measured using catheters placed, respectively, in the carotid artery and corpus cavernosum as previously described 11 . Maximal stimulation (6 V, 1ms, 16 Hz, 60 s) was applied to check maximal erectile function at the beginning of each experiment, before incremental frequencies (2,4,8 and 16 Hz) were applied at 1.5, 3, and 6 V. At the end of the experiment the maximal response was repeated to ensure that the cavernous nerve was intact and erectile function maintained.
Statistical analysis. Statistical comparisons were performed using Graphpad Prism-5.1 (GraphPad Software). Values are presented as means ± S.E.M. QPCR and immunoblotting results were compared with Student's t-test or in case of three groups with one-way ANOVA followed by Tukey test for multiple comparisons. Norepinephrine-induced-contractions were expressed as mili Newton (mN) of contraction over milligram (mg) of corpus cavernosum dry weight (mN/mg). The responses to ACh, NS309, or SNP were expressed as percentage of relaxation of norepinephrine-(3 μM)-contracted strips. Concentration-response curves were compared using two-way ANOVA followed by a Tukey test or a t-test, when a single concentration between two groups was compared. When the response of a single concentration was examined, with more than two groups, one-way ANOVA followed by Tukey test for multiple comparisons was used. Erectile function was analyzed as the ratio of peak ICP (PICP)(mmHg)/MAP (mmHg) × 100. For each frequency, two-way ANOVA with a Tukey test for multiple comparisons were used. Significance was accepted at P ≤ 0.05.