Erectile dysfunction (ED) is one of the most common disorders in males (Kaya et al., 2017). The prevalence of ED in the general population ranges from 30 to 65% in men aged 40–80 years (Campbell et al., 2019). The Massachusetts Male Aging study emphasized the close association between aging and ED by showing an almost 1.5 times increased prevalence of ED after the age of 70 years (Feldman et al., 1994).

The development of a penile erection can simply be summarized in two sequential steps: (1) the transportation of blood into the cavernosal sinusoids resulting in the enlargement and rigidity of the penis, and (2) the reduction of venous outflow via veno-occlusion to maintain the enlargement and rigidity of the penis (Ferrini et al., 2017). However, this two-step process depends on a complex balance and coordination of neurogenic, vascular, and humoral events (Jang et al., 2017), such as the parasympathetic, sympathetic, and nitrergic nerves, neurotransmitters, blood vessels, and cavernous muscles (Claudino et al., 2004). Nitric oxide (NO) plays a key role in this coordination, because the increase in the blood flow via dilation of arterial vessels as well as the increase in the size of corporal sinusoids via the relaxation of smooth muscles is regulated by the NO (Ferrini et al., 2017). Both the nitrergic fibers and vascular endothelial cells supply the corpus cavernosum with NO (Claudino et al., 2004). Decreased nerve and endothelium-mediated corpus cavernosum relaxation, reductions in smooth muscle cell content, and pathological remodeling of the pudendal artery were shown in age-related ED. Moreover, they were attributed largely to increased oxidative stress and endothelial dysfunction (Johnson et al., 2011). However, the underlying molecular mechanisms have not been fully identified (Johnson et al., 2011). Furthermore, conflicting results have been reported, especially in relation to NO-mediated mechanisms (Ferrini et al., 2001; Gonzalez and Rajfer, 2004; Johnson et al., 2011).

A causal connection between oxidative-stress, aging and age-related pathologies (Davalli et al., 2016), as well as the beneficial effects of calorie restriction (CR) and regular physical exercise (PE) on oxidative stress in age-related pathologies is known (Testa et al., 2014; Simioni et al., 2018). Again, the beneficial effects of CR and PE on erectile function have also been shown separately (Ferrini et al., 2001; Claudino et al., 2004; Gonzalez and Rajfer, 2004; Johnson et al., 2011; Testa et al., 2014; Davalli et al., 2016; Souza et al., 2017; Simioni et al., 2018). This study aims to investigate the NO-mediated mechanisms of CR and PE on the corpus cavernosum in aged rats.

It is well known that aging is closely associated with ED with significantly increased prevalence after the age of 70 years (Feldman et al., 1994). Here, we also showed that erectile function in aged rats was significantly impaired compared with their young littermates, as was evident by decreased cavernosal function.

Penile erectile tissue is composed of two corporal bodies called the corpora cavernosa. These cavernosal bodies are composed of sinusoidal spaces with trabecular meshwork, which are lined by endothelium (Agarwal et al., 2006). A penile erection is a two-step mechanical process summarized as: (1) transportation of blood into the expanded cavernosal sinusoids, which results in the enlargement and rigidity of the penis for the initiation of the erection, and (2) maintenance of that enlargement and rigidity by cavernosal veno-occlusion (CVO) in order to prevent the leakage of the blood from the veins before the sexual act is completed (Johnson et al., 2011). In the first step, while the blood flows into the corporal sinusoids via cavernosal arteries, corporal sinusoids expand to provide a space for the pooling of the blood. In the second step, the increase in the intra-corporal pressure via pooling of the blood into the expanded sinusoids shuts the venous channels and decreases the outflow to prevent the leakage of the blood from the vein. The increase in the inflow of the blood via cavernosal arteries, located within the corporal bodies, and expansion of the corporal sinusoids is dependent on the relaxation of smooth muscles located both in the arterial system and corporal sinusoids (Lue and Tanagho, 1987). Therefore, a penile erection is a well-coordinated neuro-vascular process, which includes closed functional interaction between the penis vascular and nervous system (Andersson, 2011). NO plays an important role in this coordination via both nitrergic fibers and vascular endothelial cells (Ferrini et al., 2017; Jang et al., 2017). NO synthesized by nNOS, located in the terminal axons of the nerve innervating the corporal smooth muscle, enters the corporal smooth muscle cell and activates guanylyl cyclase to form cGMP from GTP and induces smooth muscle relaxation and initiates the erection (Burnett et al., 1992). The length of time of erection is dependent on the length of time for maintaining the corporal muscle in its relaxed state. Since the erection requires a balance between inflow and outflow of the blood within the corporal sinusoids, NO produced by eNOS plays a role both in the initiation and continuation of a penile erection (Hannan et al., 2010). NO, synthesized from the endothelial cells by eNOS, diffuses into the smooth muscle cells and causes relaxation with a mechanism similar to nNOS. Also, iNOS may originate from the smooth muscle cell, and NO produced by iNOS acts within the mitochondria. However, iNOS-mediated effects in the corpus cavernosum are quite controversial (Wang et al., 2013; Zhao et al., 2016). Although iNOS is generally considered to be expressed in an inflammatory condition, increasing evidence has demonstrated that iNOS has an important role in the protection of the penile corpus cavernosum either by combating the oxidative stress associated with the ongoing apoptotic process (Gonzalez and Rajfer, 2004; Ferrini et al., 2015) or by inhibiting the breakdown of cGMP (Ferrini et al., 2007). In our study, both three isoforms of NOS (eNOS, nNOS, and nNOS) protein levels in the Y-C group of rats were almost the same. Still, iNOS protein levels were almost doubled in the O-S group. Based on this knowledge, we can speculate that iNOS plays different roles in the penile erection depending on the level of the oxidative status of the penis.

It is known that about 15% loss of functional corporal muscle mass leads the symptomatic ED (Agarwal et al., 2006). The aging-dependent decrease in the amount of the functioning corporal smooth muscle, mainly by apoptosis, was shown (Grünewald and Beal, 1999). Again, the progressive loss of anti-apoptotic genes (Bcl-2 and Bcl-x) in the corpus cavernosum of aged rats was also shown (Yamanaka et al., 2002). In line with these studies, we also showed a decrease in the anti-apoptotic protein (Bcl-XL) levels together with the increase in pro-apoptotic protein (Bax, p53) levels in the O-S group. Again, in our study, decreased contraction and relaxation responses were observed in the functional study of the corpus cavernosum. These findings were consistent with the findings of endothelial and smooth muscle cell degeneration in the histological slices in the O-S group.

Oxidative stress is believed to be the primary trigger for apoptosis (Agarwal et al., 2006). Oxidative stress occurs when the cells are exposed to excessive levels of reactive oxygen spices (ROS) resulting from the imbalance between pro-oxidant and protective mechanisms (Zalba et al., 2000). Under physiological conditions, the tissue antioxidant enzymes (SOD, GSH, and catalase) prevent the continuous formation of ROS. For example, the detoxification of ROS by cavernosal endothelial cells by using these enzymes is known (Dobrina and Patriarca, 1986). Although the upregulation of these enzymes is the main compensatory mechanism, sustained high levels of ROS diminish the activity of these enzymes (Ushiyama et al., 2004; Lagoda et al., 2007). Age-related increase in ROS together with a decrease in antioxidant capacity, which is characterized by reduced levels of SOD and glutathione, have been shown in different study settings (Bivalacqua et al., 2003; Ferrini et al., 2004). Consistent with these findings, in our study, the O-S group of animals was characterized with decreased antioxidant capacity (statistically significant decrease in SOD and GSH levels) and increased oxidant status (statistically increase in MDA and 8-OHdG levels). It should be emphasized that high MDA levels are indicative of lipid changes in the lipid matrix and cell membrane, whereas high 8-OHdG levels indicate DNA damage. Thus, DNA damage and lipid peroxidation might be responsible for the damage to the cavernosal structure in our study. The observation of an increased level of pro-apoptotic protein levels together with the decreased antiapoptotic protein levels in the O-S group supports this argument.

It is well known that NO is a highly reactive free radical that reacts with ROS, especially with the superoxide (O₂^–), to form ONO₂^– (Beckman and Koppenol, 1996). According to the body of evidence, ONO^–₂ affects the corpus cavernosum in four different ways. Firstly, ONO^–₂ causes smooth muscle relaxation, which is less potent than the NO, resulting in ineffective relaxation in the corpus cavernosum (Khan et al., 2001). We showed decreased relaxation and increased ONO₂^– together with decreased NO/cGMP levels in the O-S group of animals. Secondly, ONO₂^– increases the incidence of apoptosis in the endothelium, which leads to denudation of endothelium and a further reduction of available NO (Yamanaka et al., 2002; Fan et al., 2012). In the present study, increased NO and ONO₂^– together with decreased cGMP levels were consistent with the increased iNOS and decreased eNOS protein levels in the O-S group. In this group, the decreased anti-apoptotic protein levels together with the increased pro-apoptotic protein levels also supported these findings. Thirdly, reduced availability of NO by ONO₂^– induced the adhesion of the platelets and leukocytes in the vascular endothelium, which caused vasoconstriction and aggravated ED (Jeremy et al., 2000). Lastly, particularly in the presence of inflammation, produced ONO₂^– from a high level of NO can lead to a cytotoxic effect on cavernosal muscle (Wink et al., 1998). In our study, the observation of inflammatory cells in the histologic slices in the O-S group supported the argument that inflammation results in oxidative stress, and that, in increased oxidative stress conditions, the source of the high level of NO is the iNOS induced by inflammation in age-related ED. As discussed earlier, during the low level of oxidative stress, NO produced by iNOS within the smooth muscle cells combats the oxidative stress associated with the ongoing apoptosis. On the contrary, during the high level of oxidative stress, NO produced by iNOS, induced by inflammation, reacts with the reactive oxygen species to form ONO₂^–. This causes endothelial dysfunction, vasoconstriction, a decrease in the availability of NO, and the inhibition of SOD; all these lead to ED.

A causal connection between oxidative stress, aging, and age-related pathologies (Davalli et al., 2016) as well as the beneficial effects CR and PE on oxidative stress in age-related pathologies is known (Testa et al., 2014; Simioni et al., 2018). Again, the beneficial effects of CR and PE on erectile function have also been shown separately (Claudino et al., 2004; Souza et al., 2017). In our study, although the contractile response to phenylephrine (an alpha agonist) was attenuated in O-S, this was prevented both in the O-CR and O-PE groups. Moreover, when the CR and PE groups were compared, the response to phenylephrine was significantly higher in the O-CR than in the O-PE group. This might indicate that CR and PE show their beneficial effects on functional responses of corpus cavernosum through different mechanisms. Increased eNOS levels together with increased response to phenylephrine in the O-CR group support that the effect of CR is mediated by eNOS activation. Other researchers reported similar results (Whidden et al., 2011). On the contrary, lower eNOS protein levels together with mild phenylephrine response in the O-PE group shows the involvement of the NO-cGMP signaling pathway in an endothelium-independent manner. We speculate that this could be a direct stimulation of smooth muscle cells by nNOS, which is released from the cavernous nerve endings. Increased nNOS protein levels in this group supports this hypothesis. Similarly, Claudino et al. (2004) reported an eNOS-independent relaxation response in the corpus cavernosum from trained rats. Again, in the present study, alteration in the relaxation responses of corpus cavernosum to carbachol (a muscarinic agonist) also supports our hypothesis.

In the present study, CR and PE prevented age-related changes in the corpus cavernosum. Reducing nitrosative damage in the neurovascular structure was the main mechanism. CR and PE restored the endothelial and smooth muscle cells in the corpus cavernosum by decreasing apoptosis. The main mechanism of enhancing functional response in the corpus cavernosum with CR was the maintenance of endothelial function via eNOS activation (supported by increased eNOS levels together with the increased response to phenylephrine an alpha-1 agonist in O-CR group). However, it involves an increase in the NO-cGMP signaling pathway in an endothelium-independent manner with PE. This could be a direct stimulation of smooth muscle cells by nNOS, which is released from the cavernous nerve endings (supported by increased nNOS levels and mild response to carbachol a muscarinic agonist). As a result, lifestyle changes, such as CR and PE, should be considered in combination with pharmacological treatment strategies in the treatment of age-related changes in ED.

All datasets generated for this study are included in the article/supplementary material.

The animal study was reviewed and approved by the Yeditepe University Experimental Animals Ethics Committee.

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.