Physiology of Erectile Function: An Update on Intracellular Molecular Processes

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1 eau-ebu update series 4 (2006) available at journal homepage: Physiology of Erectile Function: An Update on Intracellular Molecular Processes Annamaria Morelli a, *, Sandra Filippi b, Linda Vignozzi a, Rosa Mancina a, Mario Maggi a a Andrology Unit, Department of Clinical Physiopathology, University of Florence, Florence, Italy b Interdepartmental Laboratory of Functional and Cellular Pharmacology of Reproduction, Departments of Pharmacology and Clinical Physiopathology, University of Florence, Florence, Italy Article info Keywords: Erectile function Penile smooth muscle tone NO/cGMP signalling RhoA/ROK pathway PDE5 Androgens Erectile dysfunction Abstract Objectives: To provide a comprehensive update of current knowledge concerning the molecular mechanisms underlying the erectile physiology. Methods: Results from numerous investigations, including both basic and clinical studies, have been considered. In particular, we pointed out the advances concerning the peripheral control of erection that ultimately influence the functional state of the penis. Results: Numerous neurotransmitters and endothelial factors modulate the penile vasculature and smooth muscle tone of corpora cavernosa in the penis. The regulation of adequate intracellular calcium levels represents the key determinant of the smooth muscle tone. Following the sexual stimulation, the activation of smooth muscle relaxation in the penis is mainly mediated by nitric oxide (NO), which acts via cyclic guanosine monophosphate (cgmp)-mediated intracellular signalling. The pro-erectile NO/cGMP pathway is coupled to the anti-erectile RhoA/Rho-kinase calciumsensitizing pathway, which was recently highlighted as another important regulator of the erectile function. In the last decade, the enzyme phosphodiesterase 5 (PDE5), critically involved in the degradation of cgmp and thereby in the maintenance of penile detumescence, has gained attention as target enzyme of the most used drugs (PDE5 inhibitors) for the treatment of erectile dysfunction. Moreover, androgens play an important role in peripheral regulation of erection, acting positively either on the enzyme which synthesizes NO (NOS), and on that involved in the degradation of cgmp (PDE5). Conclusion: The recent advances added to new insights into our knowledge of erectile physiology and leaded to the improvement in the clinical management of men affected by erectile dysfunction. # 2006 European Association of Urology. Published by Elsevier B.V. All rights reserved. In this article, we review the physiological mechanisms that regulate penile erection. Particular emphasis is given to those intracellular molecular processes involved in regulation of penile smooth muscle tone, including signaling pathways leading to contraction and relaxation of erectile tissue. * Corresponding author. Andrology Unit, Department of Clinical Physiopathology, University of Florence, V.le G. Pieraccini, 6, Florence, Italy. Tel ; Fax: address: a.morelli@dfc.unifi.it (A. Morelli) /$ see front matter # 2006 European Association of Urology. Published by Elsevier B.V. All rights reserved. doi: /j.eeus

2 eau-ebu update series 4 (2006) Introduction Penile erection is a complex neurovascular event involving the interaction between different systems: the nervous system (central and peripheral) and the penile (arterial and trabecular) smooth muscle system. The tone of penile smooth muscle is a key determinant of the hemodynamic events that maintain penile flaccidity or allow erection. The cavernosal arteries supply blood to the corpora cavernosa of the penis (through the pudendal artery), while the emissary veins running through the tunica albuginea allow blood to flow out of the penis. In the flaccid penis, smooth muscle cells of corpora cavernosa are contracted and penile blood inflow is low. During erection, relaxation of trabecular smooth muscle results in an increased blood flow and pressure in the corpora cavernosa and an expansion of sinusoidal spaces. The expanded corpora cavernosa cause mechanical compression of the emissary veins, restricting the venous outflow from the cavernosal spaces and facilitating an entrapment of blood in the cavernosal sinusoids. This blood engorgement finally results in penile rigidity [1]. Several neurotransmitters and endothelial factors have been shown to control erectile function by modulating the penile vasculature and smooth muscle tone of corpora cavernosa [2]. The correct balance between relaxant and contractile factors is required to determine the functional state of the penis, finally resulting in a normal erectile function. In this article, we review the physiological mechanisms that regulate penile erection with particular focusing on those intracellular molecular processes involved in regulation of penile smooth muscle tone. 2. Neurophysiological control of erection The nervous system is considered to be the primary regulatory site affording the control of penile erection. Multiple levels of the neuroaxis, from the brain and spinal cord to nerves terminating within the penis, release several neurotransmitters that induce penile response. The neurophysiological control of erections occurs both at central and peripheral level (Fig. 1) Central mechanisms Central mechanisms regulating erection are complex and only partially elucidated, at least in humans. They are essentially not revised here, because beyond the main purpose of this article. Briefly, the central nervous system coordinates sensory stimuli from a variety of sources (visual, auditory, imaginative, tactile, and olfactory). Strong evidences from animal models support an important involvement of the hypothalamic neurons within the medial preoptic area (MPOA) and paraventricular nuclei (PVN), including oxytocinergic neurons, whose activation by dopamine and by oxytocin itself, leads to penile erection in male rats [3,4]. The release of OT is secondary to production of nitric oxide (NO) by nitric oxide synthase (NOS) in the PVN. The interaction between nitric NO and paraventricular oxytocinergic neurons seems to be the crucial mechanism of the central regulation of erection [4]. Dopamine is the most important neurotransmitter which exerts at central level a facilitatory effect on penile erection. A second putative excitatory pathway, within both hypothalamic and spinal loci, is the melanocortin system. Preliminary data in animal models and even in humans indicate that melanocortin receptors play an important role in the control of sexual beavior and therefore may represent a new pharmacological target for the treatment of ED [5] Peripheral mechanisms The pro-erectile and anti-erectile messages deriving from the integration and processing of sexual stimuli in the brain, descend in the spinal cord and activate nervous peripheral systems (Fig. 1). Peripheral control of erection depends on both neuronal and local factors that ultimately influence the vascular events in the penis. The different structures of the penis receive autonomic (sympathetic and parasympathetic) and somatosensory innervations [6]. They are innervated mainly by pudendal nerves, which originate from the sacral tract of the spinal cord (S2 S4) and contain the primary afferent sensory and motor pathways, and by cavernosal nerves, which originate in the pelvic plexuses containing the primary efferent sympathetic (hypogastric nerves from the thoracic-lumbar tract, T11-L2, of the spinal cord) and parasympathetic (pelvic nerves from the sacral tract, S2 S4, of the spinal cord) pathways. The nerve populations have been categorized as adrenergic, cholinergic and nonadrenergic noncholinergic (NANC), which release transmitters acting on vascular endothelium and smooth muscle components of corpora cavernosa and thereby modulate the functional state of the penis. Erections are inhibited by basal sympathetic tone, mainly mediated by noradrenaline (NA), whereas pro-

3 98 eau-ebu update series 4 (2006) Fig. 1 Schematic representation of neurophysiological control of erection. NO, nitric oxide; T11-L2, thoraco-lumbar tract of the spinal cord; S2-S4, sacral tract of the spinal cord. erectile dopamine-activated oxytocinergic neurons stimulate the parasympathetic system and promote the release of neuronal relaxing factors, mainly acetylcholine and nitric oxide (NO). Sexual arousal is the result of both increased parasympathetic activity and decreased sympathetic activity. Fig. 2 summarizes contraction/relaxation mediating transmitters or modulators in the penis. predominance [1,8]. a1-adrenoceptors subtypes (a1 A, a1 B, a1 D and a1 L ) as well as a2-adrenoceptors subtypes (a2 A, a2 B, and a2 C ) have all been demonstrated in human corporal smooth muscle and it seems that a1 A -, a1 L - and a2 A -adrenoceptors are predominant [9]. In the penile vasculature, both a1- and a2-adrenoceptors can contribute to contraction, 2.3. Factors leading to smooth muscle contraction A tonic sympathetic neural input is the main mechanism leading to smooth muscle contraction in the penis. NA and endothelins (ETs) are the main neurotransmitters that contribute to contraction in penile tissue [7]. Released NA from simpathetic adrenergic nerves stimulates post-junctional receptors (adrenoceptors) in the penile vasculature and trabecular smooth muscle of the corpora cavernosa to induce contraction [1]. The pharmacological characterization of such receptors indicates that the a subtype is 10-times more abundant than b subtype and that both a 1 and a 2 are present in penile tissue, although they are differentially expressed, with the a 1 adrenoceptors having the functional Fig. 2 Summary of the contraction/relaxation mediating transmitters in the penis. NPY, neuropeptide Y; PGF 2a, prostanoid F 2a ; TXA 2, tromboxane A 2 ; VIP, vasointestinal peptide; PGE 1 and PGE 2, prostaglandin E 1 and E 2.

4 eau-ebu update series 4 (2006) but the a2 subtype may have a more important role than in corpus cavernosum. Moreover, while postjunctional a-adrenoceptors mediate penile smooth muscle contractility and detumescence, prejunctional a 2 -adrenoceptors are usually involved in a negative regulation of NA transmission [10]. In addition, stimulation of prejunctional a 2 -adrenoceptors in horse penile resistance arteries was shown also to inhibit NANC nitrergic transmission, suggesting that this might be one of the mechanisms by which NA maintains detumescence [11]. Experiments in rabbits indicate that blocking a2- adrenoceptors with yohimbine resulted in an androgen-dependent increase in NO signaling [12]. Administration of a-adrenoceptor agonists cause detumescence, further confirming the role of a- adrenoceptor in the regulation of smooth muscle tone, whereas the employment of a adrenoceptors antagonists has been shown to be efficacious for induction and/or maintanance of penile erection [13]. ET-1 is the most potent contractile agent responsible for long-lasting contractions in the corpora cavernosa and penile vessels and contributes to the maintenance of the cavernosal smooth muscle tone [1,14]. ET-2 and ET-3 are less potent than ET-1 to induce contractions in penile tissue [15]. Other important contractile factors in erectile physiology inducing penile detumescence are neuropeptide Y (NPY) released by the nerve endings, prostanoids (PGF 2a ) and tromboxane A 2 (TXA 2 ) released from vascular endothelium, and angiotensin II synthesized by penile tissue, although their roles have not yet been fully established [16] Factors leading to smooth muscle relaxation Smooth muscle relaxation in the penis is dependent on the parasympathetic system, in which the most important neurotransmitter is NO. The other neurotransmitter released from parasympathetic nerve endings is acetylcholine which exerts an indirect smooth muscle relaxing action: (1) it stimulates the release of NO from the vascular endothelium of corpora cavernosa; (2) it reduces stimulation of the receptors on sympathetic endings, leading to decreased release of NA [16]. Thus, there are two major sources of NO in the penis: parasympathetic nerve endings and endothelium of corporal blood vessels and sinuses stimulated by acetylcholine. Synthesis of NO is catalyzed by the action of the enzyme nitric oxide synthase (NOS) that convert L-arginine an O 2 to L-citrulline and NO both in endothelial cells and in nerve endings. Three distinct isoforms of NOS have been identified and functionally they are grouped as: constitutive calcium-dependent NOS, including neuronal (nnos) and endothelial (enos), and inducible calcium-independent NOS (inos). All three NOS isoforms have been identified in the corpora cavernosa, with nnos and enos being preferentially expressed in the autonomic nerves and endothelium of the penis, respectively, and inos in virtually all cell types [17,18]. Postganglionic parasympathetic nerves whose transmitter function depends on the release of NO are now termed nitrergic nerves [19]. Both NANC and cholinergic nerves contain nnos [20]. The expression of penile variants of nnos (PnNOS) has been identified in nnos knock-out mice, which are fertile and have intact neurogenic NO production [21]. A recent publication documented that the alternatively spliced forms of nnos are major mediators of penile erection and that the b splice variant (nnosb) has unique structural properties that may explain the robust production of NO and the preserved erectile function in nnos knock-out mice [22]. Besides the action of acetylcholine for the activation of enos, also the shear stress induced by the increased blood flow in the penile vessels could contribute to activate the enos to produce continuously NO by a process involving PI 3 kinase/ Akt pathway. Therefore, it has been hypothesized that NO derived from nnos is responsible for the initiation of erection, whereas NO from shearstress-activated enos contributes to the maintenance of penile rigidity during erection [16]. Vasoactive intestinal polypeptide (VIP) and calcitonin gene related peptide (CGRP) are other substances released by parasympathetic nerve endings that contribute to cavernous smooth muscle relaxation. Prostanoids, such as prostaglandin E 1 and E 2 (PGE 1, PGE 2 ), released by the endothelium also have an important smooth muscle relaxing action [2]. PGE1 not only exerts a direct relaxant effect on the cavernous smooth muscle, but it also exerts a proerectile activity through the inhibition of NA release from adrenergic nerves [23]. For self-injection therapy, PGE 1 (alprostadil) represents one of the most effective and safest vasoactive drug in the treatment of ED [24]. 3. Regulation of penile smooth muscle tone In combination, all the neural pathways regulate the effectors sites of action within the penis which responds to the appropriate neuronal stimulus by generating a degree of smooth muscle tone and thereby the functional state of the penis. Therefore,

5 100 eau-ebu update series 4 (2006) the regulation of penile smooth muscle tone consists of molecular mechanisms that essentially depend on adequate levels of agonists (neurotransmitters, hormones, and endothelium-derived factors), integrity of intracellular transduction signalling, and interactions between effectors (contractile/relaxant) proteins. In particular, adequate intracellular levels of calcium and the sensitivity of the contractile machinery to calcium are required for the regulation of the smooth muscle tone. Smooth muscle cells contract when intracellular calcium concentration increases and relax when it falls. Moreover, though the nerve endings do not innervate each smooth muscle fiber, all cells contract and relax at the same time in the corpora cavernosa. This coordination process is allowed by intercellular communication channels called gap junctions which make possible the transfer of chemicals between the cytoplasm of adjacent cells and thereby is responsible for the synchronized erectile response [25] Intracellular molecular process of smooth muscle cell contractility As shown in Fig. 3, the binding of NA, ETs and prostanoids with their receptors on the surface of smooth muscle cells increases the intracellular activity of the membrane-bound enzyme phospholipase C (PLC) which converts phosphatidylinositol Fig. 3 Intracellular signalling leading to smooth muscle cell contraction in corpora cavernosa and determining the flaccid state of the penis. NA, noradrenaline; ET-1, endothelin-1; PLC, phospholipase C; PIP, phosphatidylinositol biphosphate; IP3, inositol-3- phosphate; DAG, diacylglycerol; PKC, protein kinase C; CaM, calmodulin; MLC, myosin light chain; MLCK, MLC kinase; SR, sarcoplasmic reticulum. biphosphate (PIP) to inositol triphosphate (IP 3 ) and diacylglycerol (DAG). IP 3 liberates calcium ions from intracellular stores (such as sarcoplasmic reticulum), while DAG stimulates protein kinase C (PKC) which opens the calcium channels on the smooth muscle cell membrane leading to calcium influx from extracellular space into the cell. This results in a transient rise in the cytoplasmic free calcium concentration, which induces smooth muscle contraction [26]. In details, calcium ions at elevated levels binds to calmodulin which changes its conformation and interacts with myosin light-chain kinase (MLCK). The resultant activation of MLCK determines phosphorylation of myosin light chains (MLC) and triggers the cycling of myosin crossbridges along actin filaments and the development of force. In addition, phosphorylation of the MLC also activates myosin ATPase providing energy for the muscle contraction. Although the increase of intracellular calcium is transitory, the smooth muscle cell is able to maintain the contracted status also after that the cytoplasmic calcium returns to the basal level. In fact, the so-called calcium-sensitizing pathways take over and consist of activation of receptors coupled to G-proteins that can also cause contraction by increasing calcium sensitivity without changing the intracellular calcium level. The most representative of such mechanisms involves the RhoA/Rho-kinase pathway, which is depicted in Fig. 4. RhoA is a member of the small monomeric GTPase family, including five subfamilies: Rho-like (RhoA, B, and C); Rac-like (Rac1, 2, and 3 and RhoG); Cdc42-like; RhoBTB-like and Rnd-like (Rnd 1, 2, and 3) [27]. In addition to its role in mediating smooth muscle contraction, RhoA is involved in the regulation of several cellular processes, such as stress fiber formation and cell migration. When inactive RhoA is GDP-bound and localizes within the cytoplasm. When NA and ETs bind to their excitatory receptors, RhoA is converted from the inactive GDP complex into an active GTP-bound complex that translocates to the plasma membrane where it binds through geranylgeranylation, initiating signaling transduction. The activity of RhoA is regulated by three proteins: RhoGDI (GDP dissociation inhibitor) which binds to RhoA-GDP to form the inactive cytoplasmic complex; RhoGEF (guanine nucleotide exchange factor) which facilitates the dissociation of RhoA- GDP from RhoGDI and thereby the traslocation of the active form RhoA-GTP to the plasma membrane; RhoGAP (GTPase-activating protein) which accelerates the intrinsic GTPase activity of RhoA with the subsequent re-association of RhoA-GDP/RhoGDI and re-localization in the cytoplasm [28]. The best

6 eau-ebu update series 4 (2006) Fig. 4 Intracellular molecular mechanism of smooth muscle cell contraction via RhoA/ROK mediated calcium-sensitizing pathway. NA, noradrenaline; ET-1, endothelin-1; GPCR, G protein coupled receptor; PLC, phospholipase C; GTP, guanosine triphosphate; GDP, guanosine diphosphate; GEF, guanine nucleotide exchange factor; GAP, GTPase-activating protein; GDI, GDP dissociation inhibitor; MLC, myosin light chain; MLCK, MLC kinase; MLCP, MLC phosphatase; ATP, adenosine triphosphate; SR, sarcoplasmic reticulum. characterized downstream effector of RhoA is Rhokinase (ROK) which is directly involved in smooth muscle contraction. ROK is a serine-threonine kinase that phosphorylates and thereby inhibits the regulatory subunit of MLC phosphatase. Consequently MLCs remain phosphorylated and sensitized to intracellular calcium. Alternatively, ROK prevents dephosphorylation of myofilaments through an indirect mechanism consisting of the phosphorylation/activation of a specific inhibitor of MLC phosphatase, CPI-17, or it directly phosphorylates MLC, thus maintaining contractile tone [14]. RhoA and ROK have been shown to be expressed in penile smooth muscle and, accordingly, a selective inhibitor of ROK (Y27632) has been shown to cause relaxation of human corpora cavernosa in vitro and to induce penile erection in animal models [29]. Moreover, it has been found that the transfection of dominant negative of RhoA enhanced erectile function in rats [30]. It has been proposed that the phasic contraction of penile smooth muscle is regulated by an increase in cytoplasmic calcium, while the tonic contraction is governed by the calcium-sensitizing pathway [16]. It is noteworthy that contractile mechanisms that take place in penile tissue are responsible for the predominant physiological condition of the penis which resides in the contracted/flaccid status for the majority of time. The predominant contracted status of the penis is associated to a low oxygenation of the penile tissue that is physiologically interrupted by the erectile episodes related and not related (nocturnal erections) to sexual activity. Thus the normal erectile function is important to allow the oxygenation of penile tissue to preserve tissue composition and the erectile function itself. Hypoxia is potentially dangerous in the penis, in fact, when protracted (more than 24 hours) it may damage penile tissue and compromise its erectile capacity [31]. It has been demonstrated in human and animal models that hypoxic condition in penile tissue induced the activation of compensatory and proerectile mechanisms in order to re-oxygenate the tissue, such as the increasing of vasorelaxant receptor subtype of ET-1 (ETB) and the reduction of RhoA/ROK expression [32] Intracellular molecular process of smooth muscle cell relaxation Although several vasodilators have been implicated in the erectile response, including VIP an prostaglandins, as well as acetylcholine, NO is considered the pivotal physiological stimulus for penile smooth muscle relaxation. Individual neurotransmitters act via different pathways, but the intracellular mechanism in every case is essentially based on the changing in the cytoplasmic calcium concentrations (Fig. 5). In response to sexual stimuli NO released from parasympathetic nerves and endothelial cells crosses the smooth muscle cell membrane and

7 102 eau-ebu update series 4 (2006) Fig. 5 cgmp-mediated intracellular signalling leading to smooth muscle cell relaxation in corpora cavernosa and determining the erection state of the penis. NANC, nonadrenergic noncholinergic; NO, nitric oxide; sgc, soluble guanylate cyclase; GTP, guanosine triphosphate; GMP, guanosine monophosphate; cgmp, cyclic GMP; PKG, cgmp-dependent protein kinase; PDE5, phosphodiesterase type 5; MLC, myosin light chain; MLCK, MLC kinase; MLCP, MLC phosphatase; SR, sarcoplasmic reticulum. binds to the soluble guanylate cyclase (sgc) in the cytoplasm. Binding of NO to sgc activates this enzyme, which converts guanosine triphosphate (GTP) into cyclic guanosine monophosphate (cgmp), the active second messenger which triggers a biochemical cascade of events culminating in smooth muscle relaxation [1]. These events include the activation of a cgmp-dependent protein kinase (PKG or cgki) which in turn phosphorylates several key target proteins, including ion channels, ion pumps, and enzymes all involved in the control of intracellular calcium level. Phosphorylation of these target proteins reduces the intracellular availability of calcium through the following mechanisms: (1) inhibition of cell membrane Ca 2+ -channel activity, by a direct mechanism or indirectly through the activation of K + channels that act hyperpolarizing cell membrane and inhibiting calcium influx; (2) activation of the plasma membrane Ca 2+ /ATPase pump with the extrusion of calcium across the membrane (this is thought to be the main mechanism mediating smooth muscle relaxation via intracellular calcium reduction); (3) activation of the sarcoplasmic reticulum Ca 2+ /ATPase pump responsible for the uptake of calcium from cytoplasm; (4) inhibition of IP 3 - mediated pathway by phosphorylation of an IP 3 regulatory protein associated with the receptor, leading to a decreased IP 3 -induced calcium release from intracellular stores [33]. Overall these mechanisms lead to a reduction of the Ca 2+ -/calmodulin complex which results in a decreased MLC kinase activity, reduced levels of phosphorilated MLC, breakdown of actin/myosin crossbridges and relaxation of the cavernosal smooth muscle. The other vasorelaxant factors, VIP and prostanoids (PGE1, PGE2) act via a different pathway that concomitantly to that mediated by cgmp takes place in penile smooth muscle tissue. Following the interaction with their specific receptors, these vasorelaxant factors act by activating adenylate cyclase, the enzyme which converts adenosine triphosphate (ATP) into cyclic adenosine monophosphate (camp), another intracellular second messenger which lowers cytosolic calcium levels. camp through the activation of the related protein kinase (PKA) leads to the opening of the K + channels causing plasma membrane hyperpolarization resulting in the inhibition of the voltage-gated Ca 2+ -channels and reduction of calcium influx into the cell. Moreover, activation of PKA may leads to the induction of transcriptional factors. Therefore, PGE 1 and other vasoactive agents which increase

8 eau-ebu update series 4 (2006) intracellular camp, may regulate mrna expression of a1- and a2-adrenoceptors [34]. On the other hand, an interaction between PGE 1 and the NO/cGMP signalling has been suggested by the observation that PGE 1 up regulates enos and nnos improving erectile response in rats [35] and that a synergistic interaction of PGE1 and NO is involved in relaxation of human corpus cavernosum [36]. However, the camp-mediated smooth muscle relaxation has only a marginal role in the erectile physiology, with the NO-activated cgmp-mediated pathway representing the most important. Besides calcium mobilization and calcium sensitization mechanisms, hyperpolarization of smooth muscle cells through the opening of K + channels represents one of the salient mechanisms known to be important in the regulation of penile smooth muscle tone. Corporal smooth muscle cells express distinct K + channels subtype, including the best characterized ATP-sensitive (K ATP ) and large-conductance Ca 2+ -activated K + channels, also termed maxi-k, whose activation may occur by different mechanisms. The opening of K + channels may be stimulated by PKA, PKG, or by cgmp itself. In addition it has been proposed that NO can directly stimulate K + -channels opening as well as Na + /K + - ATPase in the arteries and trabecular muscle [37]. Pharmacological or genetic manipulation of K + channels as therapy for ED provides a useful tool to regulate penile smooth muscle tone [38,39]. Given that the intracellular levels of both camp and cgmp second messengers are determinant to regulate the availability of cytoplasmic free calcium and thereby to trigger the contraction or relaxation of penile smooth muscle cells, a key role in the regulation of erectile function is played by the class of enzymes phosphodiesterases (PDEs) which converts camp and cgmp into their inactive metabolites, adenosine monophosphate (AMP) and guanosine monophosphate (GMP), respectively. This enzymatic degradation represents the crucial event terminating the above described cascade Phosphodiesterases PDEs are enzymes critically involved in regulation of cellular camp and/or cgmp levels by many stimuli. Eleven families of PDEs with varying selectivities for camp or cgmp have been identified in mammalian tissues [40]. Each PDE family includes multiple isoforms either as products of different genes or of the same gene through alternative splicing. PDE type 5 (PDE5) is the predominant PDE in the penis [41], responsible for the regulation of penile smooth muscle tone. The human PDE5 gene has been identified by three independent groups [42 44], it has been mapped to chromosome 4q26, and consists of 21 exons. Alternative splicing of this gene results in three transcript variants encoding distinct isoforms and it has been found that two alternate promoters regulate transcription of three PDE5 isoforms [45]. This enzyme contains a catalytic domain that specifically hydrolyzes cgmp and a regulatory domain that binds cgmp itself. Moreover cgmp transcriptionally regulates PDE5 gene. PDE5 is widely distributed, especially in those tissues with large smooth muscle component (visceral and vascular), with the corpora cavernosa being the tissue quantitatively expressing the highest level of PDE5 mrna, as demonstrated both in human and rat tissues [46,47]. The quantitative PDE5 tissue distribution may explain the selectivity of action of PDE5 inhibitor (PDE5i) drugs in the penis, representing the first line therapy for the oral treatment in patients affected by erectile dysfunction (ED). In Fig. 6 is shown the PDE5 activity in human corpora cavernosa extracts, as measured by inhibition efficacy of the most used PDE5i (sildenafil, tadalafil, vardenafil). Because PDE5 is the predominant enzyme responsible for cgmp degradation in penile smooth muscle, the activation of this enzyme terminates the NO-induced cgmp-mediated vasorelaxation, restoring the basal smooth muscle tone and penile Fig. 6 Inhibition curves of cgmp-hydrolysing activity induced by PDE5 inhibitors (vardenafil, sildenafil, tadalafil) in corpora cavernosa extracts from human penile biopsies. Aliquots of tissue homogenates (0.1 mg protein/ml) were incubated with 0.5 mm cgmp and 0.1 mm [ 3 H]cGMP in absence or in presence of vardenfil (10 S13 10 S6 M), sildenafil (10 S11 10 S6 M) and tadalafil (10 S11 10 S6 M). Ordinate: cgmp hydrolysing activity expressed as conversion percentage; Conversion (%) = [products count/ (substrate + products counts)] 100. Abscissa: inhibitor concentration. Inhibition curves obtained in three different experiments were fitted simultaneously with the program ALLFIT using the four-parameter logistic equation.

9 104 eau-ebu update series 4 (2006) flaccidity. In response to sexual stimulation, PDE5i act to potentiate the NO-mediated vasorelaxation in the erectile tissue by maintaining increased intracellular cgmp levels in vascular and corporal smooth muscle cells, thus improving penile erection in ED patients. It seems clear that the correct tight regulation of every enzyme involved in the NOmediated pathway, especially NOS and PDE5 responsible for cgmp formation and degradation, respectively, is required to assure a normal erectile function. Recent publications showed that dysregulation of PDE5 expression/activity is involved in etiology of priapism, a condition in which abnormally prolonged penile erection occurs, unassociated with sexual interest [48,49]. As a result of PDE5 downregulation, the penile tissue may become hypersensitive to cgmp signalling causing unrestrained erectile tissue relaxation after an episode of sexual stimulation Androgens and erectile function The role of androgens in the molecular mechanisms underlying the erectile function has become clearer during the past decade (Fig. 7). Besides the well known role that androgens play at the central nervous system level as important modulators of male sexual behaviour, including libido [50], recent advances about the relationship between androgens and erectile function at peripheral level have also Fig. 7 Mechanisms of action of testosterone on erectile function. By maintaining the correct balance between NOS and PDE5 expression/activity, T regulates intracellular cgmp levels in penile tissue and thereby smooth muscle relaxation for erectile function. On the other hand, T exerts a trophic and differentiating action for cavernous smooth muscle cells to maintain integrity of erectile structures. NOS, nitric oxide synthase; PDE5, phosphodiesterase type 5. been gained [51]. Although studies showed that not all castrated men become impotent [52] and that a high percentage (20 45%) of prostatic cancer patients preserved erectile function after medical or surgical castration [53,54], it has been demonstrated that androgen manipulation affects erectile function. A number of different studies on experimental animal models have shown that testosterone (T) regulate both formation and degradation of cgmp in the penis by acting on NOS and PDE5 expression. It has been demonstrated in the rat that androgen deprivation associated to several conditions, including ageing, and pathologies such as diabetes mellitus type I and II, adrenalectomy, hypophysectomy and castration, induced a reduction of penile NOS expression with decreased erectile function, that was reversed by androgen replacement therapy [50]. Moreover, a trophic effect of T on penile architecture has been demonstrated in different animal species [55]. Androgen deprivation in the animal model by surgical and medical castration resulted in loss of trabecular smooth muscle and increase in deposition of extracellular matrix, producing diffuse fibrosis and ED [56 58]. The androgen-dependent loss of erectile response was restored by androgen administration [56]. In addition, it has also been shown that T is involved in the maturation of penile tissue composition by promoting the commitment of pluripotent stem cells into the myogenic lineage and inhibiting their differentiation in adipogenic lineage [59 61].Traish et al. [62] have demonstrated a similar mechanism in the rabbit with an accumulation of adipocytes in the subtunical region of the corpus cavernosum, thus impairing the veno-occlusive mechanism, if T levels are low. This study also confirmed that androgen deprivation leads to loss of trabecular smooth muscle and increase of connective tissue fibers. Hence, androgens play an important role not only in the formation of the main relaxing factor, cgmp by regulating NOS activity, but also in the integrity of erectile apparatus. Recently, it has been demonstrated that, in several species including human, rabbit and rat, androgens are also important for the regulation of cgmp degradation by modulating PDE5 expression and activity [46,47]. This dual and antithetic role of androgens in promoting both the increase and the decrease of cgmp intracellular concentration in penile smooth muscle underlies the mechanism by which the erections are finalized to sexual act and thereby synchronized to sexual desire. Moreover, the androgen-dependent PDE5 expression

10 eau-ebu update series 4 (2006) might explain the occurrence of erections in presence of low T levels and in absence of sexual interest, as it occurs in children and in eunuchs, as well as in hypogonadal subjects when adequately stimulated (hard erotic movies). In fact, because of androgen deficiency, a reduced PDE5 activity favours an enhancement of cgmp signalling allowing penile smooth muscle relaxation and erection. Clinical studies recently evidenced a marginal effect of T on penile erections, suggesting that the decreased sexual activity in hypogonadal subjects is due to a decreased libido rather than to a direct effect of reduced T on erectile process [63,64]. This important role played by T in regulating both expression and activity of the main cgmp-catalysing enzyme in the penis (PDE5), reflects another important evidence concerning the responsiveness of patients with ED to PDE5i. In fact, in animal models, low levels of T severely hampered the PDE5i effectiveness, that was completely restored by T replacement [46,47]. The explanation is that the drug efficacy depends on the availability of the target enzyme. Accordingly, clinical studies demonstratedthatinedpatientswithlowtlevels who do not respond to PDE5i, T replacement was able to rescue therapy [65 67]. Hypogonadism is often associated with diabetes and both conditions represent major risk factors for ED [68]. Moreover, response to PDE5i is resulted less satisfactory in diabetic patients than in other subjects with ED [69]. A recent publication demonstrated in two different animal models of chemical diabetes that T deficiency underlies biochemical alteration, including nnos and PDE5 down-regulation, leading to ED and is responsible of unresponsiveness to sildenafil [70]. Indeed, T replacement was able to reinstate diabetes-associated ED and reduced responsiveness to sildenafil, suggesting the importance to test T plasma levels in the clinical management of diabetic patients. 4. Concluding remarks Penile erection is a complex neurovascular phenomenon that depends upon a tight regulation of several factors involved at multiple levels, from the neural control of sexual stimulation to the peripheral response occurring at penile tissue level. The coordinated functions of neurotransmitters and factors locally released in penile tissue are fundamental to regulate the degree of penile smooth muscle tone and allow normal erectile processes. The elucidation of the NO/cGMP/PDE5 pathway has provided a significant advancement not only in the comprehension of intracellular mechanisms basically involved in erectile functions, but also in the identification of those alterations responsible for ED, promoting the development of the most efficacious drugs (PDE5i) currently used for ED treatment. NO released by nerves and endothelium in the penis plays a crucial role in the initiation and maintenance of increased intracavernous pressure and penile erection, but the normal erectile function is warranted by a tight balance between relaxant and contractile factors. Conditions associated with reduced function of nerves and endothelium that alter a such balance (i.e., aging, hypertension, smoking and diabetes) cause changing in penile tissue resulting in impaired smooth muscle relaxation and erection. 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