Erectile dysfunction is characterized by the inability to achieve and/or maintain a suitable penile erection for a satisfactory sexual intercourse (NIH, 1993) and represents the sexual dysfunction most studied in men (Ückert et al., 2007). The first reports of this clinical disorder were found in ancient Egyptian writings dating back over 5,000 years, where reductions in both number and quality of erections of different Egyptians were described (Smith, 1974; Shah, 2002).

The international consultation committee for sexual medicine on definitions, epidemiology and risk factors for sexual dysfunction conducted an extensive analysis of the worldwide prevalence of erectile dysfunction. In this view, the prevalence of the disease was 1–10% in men under 40 years of age, 29% in those aged 40–49 years, 20–40% in men aged 60–69 years and 50–100% in men over 70 years of age (Nicolosi et al., 2003). In addition, the worldwide prevalence of erectile dysfunction is estimated at 322 million men by 2025 (Costa and Potempa, 2012).

Data regarding the erectile dysfunction incidence are less abundant. However, the number of new cases of the disease per year varies from 19 to 66 cases per 1,000 men, according to studies conducted in the United States, Netherlands, and Brazil (Johannes et al., 2000; Moreira et al., 2003; Schouten et al., 2005).

Erectile dysfunction is, predominantly, a vascular and benign disease, but it affects both physical and psychological health and has a significant impact on the quality of life of men and their partners, mainly due to the reduction of self-esteem and the commitment of the interpersonal relationship. The prevalence of the disease increases with age and can be seen as a serious public health problem (Medeiros-Júnior et al., 2014). The etiology is multifactorial, and, among the risk factors, the presence of cardiovascular diseases, sedentary lifestyle, smoking, diabetes, depression, anxiety and obesity are prominent (Alves et al., 2012).

Currently, erectile dysfunction is not limited to the reduction of sexual activity but acts as an indicator of systemic endothelial dysfunction. From the clinical point of view, this disease can precede cardiovascular events and can be used as an initial marker to identify men with high cardiovascular risk. In this case, patients with ED and no medical history of cardiovascular disease should be screened for cardiovascular disease (Gandaglia et al., 2014; Yafi et al., 2016; Hatzimouratidis et al., 2019).

Therefore, since alterations in the cellular signaling of cavernous smooth muscle cells can lead to this disease, this review focused on both contraction and relaxation processes that regulate penile erection, as well as highlights different targets as new approaches toward the development of new drugs for erectile dysfunction treatment.

Penile erection is a neurovascular phenomenon modulated by psychological and hormonal factors, resulting in relaxation of cavernous smooth muscles from the penis. This phenomenon involves a complex interaction between the central nervous system and local stimuli. It is basically mediated by spinal reflexes, by processing information in the hypothalamus and by integrating tactile, visual, olfactory, auditory, and imaginary stimuli (Thomas, 2002).

Peripheral erection control depends on neuronal and local factors that ultimately influence the processes of cavernous smooth muscle contraction or relaxation. In this context, a muscle tone is generated and, thus, the functional state of the penis is maintained (Andersson, 2011).

Cavernous muscle tone modulation occurs through molecular mechanisms that depend on the action of agonists, such as neurotransmitters and endothelial-derived factors, and on the integrality of intracellular signaling. Specifically, an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) is the primary cause for the production of contraction, so the regulation of the intracellular levels of this ion and the sensitivity of contractile machinery are the key points for the regulation of smooth muscle cell (Maggi et al., 2000).

Sympathetic stimulation is the primary mechanism responsible for maintaining the penis in the flaccid state. In this case, noradrenaline (NA) released from noradrenergic neurons stimulates its receptors in penile vessels and cavernous smooth muscle cells from penis to induce contraction (Mills et al, 2001).

Pharmacological characterization of adrenergic receptors has shown that the expression of a subtype is 10 times more abundant than ß subtype in cavernous tissue. In addition, α_1A, α_1B, α_1D, α_1L and α₂ (α_2A-C) adrenergic receptors are expressed in human corpus cavernosum (Maggi et al., 2000; Andersson, 2011). Interestingly, activation of all a-adrenergic receptor subtypes promotes smooth cavernous muscle contraction, since it was evidenced that presynaptic stimulation of α₂-receptors inhibits non-adrenergic non-cholinergic transmission (NANC) (Filippi et al., 2002). Other important mediators of cavernous contraction include endothelin-1 (ET-1), prostaglandin F_2α (PGF_2α), thromboxane A₂ (TXA₂) and angiotensin II (ANG II) (Tejada et al., 1991; Becker et al., 2001; Andersson, 2011).

In addition, the functional regulation of [Ca²⁺]_i to initiate a contractile response in the cavernous smooth muscle depends on two types of couplings: an electro- and a pharmacomechanical. The electromechanical coupling leads to the contractile response through membrane depolarization directly associated with increased extracellular K⁺ concentration ([K⁺]_e) (Figure 1) or the action of K⁺ channel blockers (Rembold, 1996).

Despite the importance of electromechanical coupling of cavernous smooth muscle cell contraction, the condition of penile flaccidity arises mainly from the pharmacomechanical coupling triggered by the binding of agonists to G protein coupled receptors (GPCRs) and the activation of the inositol cascade through the G_q/11. In this scenario, adrenaline, and NA binding to α₁, ET-1 to ET_A/B, PGF_2α to FP, TXA₂ to TP and ANG II to AT₁ receptors, which triggers the activation of phospholipase C_β1 (PLC_β1) pathway. In this signaling, PLC_β1 hydrolyzes the phosphatidylinositol 4,5-bisphosphate (PIP₂), producing inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG) (Berridge, 2008). IP₃ induces Ca²⁺ release from the sarcoplasmic reticulum (SR) by activating the IP₃ receptors (IP₃R). In addition, in SR are expressed ryanodine receptors (RyR), caffeine sensitive Ca²⁺ release channels, which are activated by Ca²⁺ previously released via IP₃R in a process called Ca²⁺-induced Ca²⁺ release (CICR) (Dellis et al., 2006; Mchale et al., 2006).

Following this signaling cascade, Ca²⁺ and DAG activate the Ca²⁺ dependent protein kinase (PKC), which, in turn, leads to [Ca²⁺]_i increase through phosphorylation and the direct activation of the voltage-dependent Ca²⁺ channels (CaV) present in plasma membrane (Fukata et al, 2001). The increase in [Ca²⁺]_i favors the interaction of this ion with calmodulin protein (CaM), leading to 4Ca²⁺-CaM complex formation. This complex activates myosin light chain kinase (MLCK), which phosphorylates the regulatory myosin light chain (rMLC), promoting the interaction of myosin and actin filaments, triggering the process of contraction of the cavernous smooth muscle (Figure 2) (Webb, 2003).

Although [Ca²⁺]_i increase is transient, cavernous smooth muscle cells are able to maintain the contracted state even after the reduction of the intracellular levels of this ion. In fact, studies have reported an alternative pathway that contributes to the maintenance of smooth muscle contraction, designated as a Ca²⁺ sensitization pathway (Figure 3). This pathway involves the modulation of myosin light chain phosphatase (MLCP), mainly by the small GTP binding protein G (RhoA) and its associated kinase (ROCK), a serine/threonine kinase (Hori and Karaki, 1998). In the inactivated state, RhoA is bound to guanosine diphosphate (GDP) and the guanine dissociation inhibitory protein (RhoGDI), forming a complex found in cytoplasm (Jin and Burnett, 2006).

Several contractile agonists, such as ET-1 and ANG II, which normally increase [Ca²⁺]_i via GPCRs lead to the direct activation of the RhoA guanine nucleotide exchange factor (RhoGEF) by the G_12/13 proteins, which activate RhoA by exchanging GDP for guanosine triphosphate (GTP) in this protein (Somlyo and Somlyo, 2003). Thus, RhoA-GTP is translocated to plasma membrane, where it remains anchored by geranilgeranilization, and activates the ROCK. The activity of RhoA is reduced by the action of Rho-GTPase activating protein (RhoGAP), which culminates with the GTP cleavage in GDP and the formation of the inactive RhoA-GDP/RhoGDI complex, returning to cytoplasm (Burridge and Wennerberg, 2004).

After activation by RhoA-GTP, ROCK directly phosphorylates rMLC and the regulatory subunit MYPT1 of MLCP, making it inactive, promoting the maintenance of the phosphorylated state of rMLC and, consequently, the contraction force. In addition, it can activate a Ca²⁺ dependent kinase protein, also known as zipper-interacting protein kinase (ZIPK). The ZIPK phosphorylates directly the rMLC, however its main target is the Thr⁶⁹⁶ residue of MYPT1 which, when phosphorylated, inhibits the action of MLCP (Murthy, 2006).

CPI-17 (PKC-dependent phosphatase inhibitor of 17 kDa) represents a substrate for both ROCK and PKC. When phosphorylated, CPI-17 binds to the PP1c catalytic subunit of MLCP to inhibit its enzymatic activity and prolong smooth muscle contraction (Kitazawa et al., 2000).

RhoA-GTP also stimulates phospholipase D (PLD), this enzyme is mainly associated with intracellular membranes, but is also found in the plasma membrane and is specific for phosphatidylcholine (PC), releasing phosphatidic acid (PA) that through the action of phosphohydrolase enzyme is dephosphorylated to DAG leading to sustained activation of PKC. Activation of PKC may also be dependent on G_q/11-PLCβ1, which forms DAG by PIP₂ hydrolysis. PKC can phosphorylate the Thr³⁸ residue of CPI-17, thereby increasing its inhibitory potency over PP1c by more than 1000-fold, inhibiting the action of MLCP (Murthy, 2006; Berridge, 2009).

Mitogen-activated protein kinases (MAPK) have been associated with increased smooth muscle contractility, proliferation, and chemotaxis. Among them, MAPK ERK1/2 and p38 are especially noteworthy, especially for their activity on vascular smooth muscle, however, molecular mechanisms that directly alter cavernous contractility are not fully understood (Berridge, 2009).

Relaxation of penile corpus cavernosum occurs in response to NANC and cholinergic neurotransmission, with nitric oxide (NO) as the most important neurotransmitter. The synthesis of NO is catalyzed by nitric oxide synthase (NOS) enzyme that in the presence of O₂ converts l-arginine to l-citrulline and NO in both endothelial cells and nerve endings. Three distinct isoforms of NOS are expressed in human cavernous smooth muscle cells, the constitutive and calcium dependent isoforms include neuronal (nNOS) and endothelial (eNOS). In addition, the inducible isoform (iNOS) was identified, especially when there is tissue injury (Burnett et al., 1993; Gonzalez-Cadavid et al, 1999).

In endothelial cells, eNOS activation may occur in response to shear stress induced by increased blood flow in the penile vessels, leading to phosphatidylinositol 3-kinase (PI3K) activation, which phosphorylates PIP₂ and forms the 3,4,5-phosphatidylinositol trisphosphate (PIP₃). PIP₃ recruits and activates phosphoinositide-dependent kinase-1 (PDK-1) that, in turn, phosphorylates and activates Akt, also known as protein kinase B (PKB). Akt is a serine/threonine kinase that phosphorylates directly the eNOS on its Ser¹¹⁷⁷ residues, resulting in increased activity of this enzyme, a phenomenon that contributes to continuous NO production (Hurt et al., 2002; Sommer et al., 2002).

Other substances are responsible for modulating cavernous smooth muscle relaxation, such as vasoactive intestinal peptide (VIP) and calcitonin gene related peptide (CGRP), released by cholinergic nerve endings. The endothelium also releases relaxing prostanoids, such as prostacyclin (PGI₂) and prostaglandins E types 1 and 2 (PGE₁ and PGE₂) (Molderings et al., 1992; Porst, 1996).

Although these substances are involved in cavernous smooth muscle relaxation, NO is considered the most important physiological mediator for penile erection. When there is a sexual stimulus, it is released by NANC neurons or endothelial cells in response to ACh and, as a soluble gas; it diffuses and activates its soluble guanylyl cyclase receptor (cGC) in cavernous smooth muscle cell (Figure 4) (Andersson, 2011).

The cGC converts GTP into cyclic guanosine monophosphate (cGMP) that activates cGMP-dependent protein kinase (PKG). This kinase phosphorylates several substrates to promote cavernous muscle relaxation (Figure 4). These include: 1) activation of K⁺ channels that indirectly, by repolarization or hyperpolarization, can inhibit Ca_V; 2) direct inhibition of Ca_V, decreasing Ca²⁺ influx (Rembold, 1996); 3) increase in Ca²⁺-ATPase kinetics of both SR (SERCA) and plasma membrane (PMCA), thus increasing Ca²⁺ sequestration and extrusion, respectively; 4) decrease of [Ca²⁺]_i by Na⁺/Ca²⁺ exchanger (NCX) activation that acts in a ratio of 3/1 (Blaustein, 1989); 5) inhibition of MLCK, reducing its affinity to 4Ca²⁺-CaM complex, preventing the phosphorylation of rMLC and, consequently, the contractile process (Rembold, 1996); 6) inactivation of IP₃R that reduces Ca²⁺ release from RS (Woodrum and Brophy, 2001); 7) RhoA inactivation by phosphorylation of Ser¹⁸⁸ residue, displacing it into the cytosol; 8) MLCP activation by phosphorylation of Ser⁶⁹⁵ residue from MYPT1 subunit which prevents phosphorylation of Thr⁶⁹⁶ residue by ROCK; 9) MLCP activation by telokin phosphorylation (endogenous activator of MLCP), being an independent mechanism of RhoA (Murthy, 2006).

From the physiological point of view, NO derived from nNOS is responsible for the onset of erection, whereas NO produced by eNOS in response to shear stress contributes to the maintenance of penile rigidity during erection (Andersson, 2011).

The other relaxing mediators, such as prostaglandins E_1-2 and prostacyclin act on EP₂, EP₄ and IP receptors, respectively, all coupled to Gs protein. The a-GTP subunit activates adenylyl cyclase (AC), which converts adenosine triphosphate (ATP) into cyclic adenosine monophosphate (cAMP), responsible for activating cAMP-dependent protein kinase (PKA). This kinase phosphorylates the same targets of PKG to promote cavernous muscle relaxation, with the exception of IP₃R (Figure 4) (Andersson, 2011).

Due to cGMP and cAMP importance in [Ca²⁺]_i reduction, highlights the essential role for the regulation of erectile function performed by the phosphodiesterases enzymes (PDEs), which hydrolyze cGMP and cAMP to their non-cyclic forms (GMP and AMP, respectively) and enclose the signaling cascade that results in cavernous smooth muscle relaxation. Each PDE family includes multiple isoforms that can act on a specific cyclic nucleotide or on both types. In penis, the predominant phosphodiesterase isoform is 5 (PDE5), which acts specifically on cGMP (Mancina et al., 2005).

As the balance between the contraction and relaxation processes of the cavernous smooth muscle regulates the processes of penile flaccidity and erection, any deregulation in steps of these signaling pathways may compromise penile function and thus contribute to erectile dysfunction development.

Balance among flaccidity and penile erection depends on the timing between the contraction and relaxation processes of cavernous smooth muscle cells. Thus, [Ca²⁺]_i is the central point of regulation of smooth muscle tone. In this view, the increase in [Ca²⁺]_i leads to contraction of these cavernous cells and, consequently, maintains the penis in the flaccid state, while relaxation, which promotes penile erection, is mediated by the reduction of [Ca²⁺]_i. In this sense, the imbalance of these mechanisms is related to ED development.

Currently, the main therapeutic lines for ED present as targets steps of contraction and relaxation signaling in cavernous smooth muscle cells. Therefore, further research targeting different components of electro- and pharmacomechanical couplings of cavernous smooth muscle cell arise as new targets for the development of promising drugs for the treatment of this disease.