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Endothelial Dysfunction in Erectile Dysfunction: Role of the Endothelium in Erectile Physiology and Disease

2003; Wiley; Volume: 24; Issue: S6 Linguagem: Inglês

10.1002/j.1939-4640.2003.tb02743.x

2047-2927

Trinity J. Bivalacqua, Mustafa F. Usta, Hunter C. Champion, Philip J. Kadowitz, Wayne J.G. Hellstrom,

Urinary Bladder and Prostate Research

Erectile dysfunction (ED) is defined as the consistent inability to obtain or maintain an erection for satisfactory sexual intercourse. Basic science research on erectile physiology has been devoted to investigating the pathogenesis of ED and has led to the conclusion that ED is predominately a disease of vascular origin. The incidence of ED dramatically increases in men with diabetes mellitus, hypercholesterolemia, and cardiovascular disease. Loss of the functional integrity of the endothelium and subsequent endothelial dysfunction plays an integral role in the occurrence of ED in this cohort of men. This communication reviews the role of the vascular endothelium in erectile physiology and the influence of endothelial dysfunction in the pathogenesis of ED. Future pharmacological and gene therapy interventions to restore endothelial function may represent exciting new therapeutic strategies for the treatment of ED. Penile erection is a neurovascular phenomenon that depends upon neural integrity, a functional vascular system, and healthy cavernosal tissues (Giuliano et al, 1995). Normal erectile function involves 3 synergistic and simultaneous processes: 1) neurologically mediated increase in penile arterial inflow, 2) relaxation of cavernosal smooth muscle, and 3) restriction of venous outflow from the penis. The corpus cavernosum of the penis is composed of a meshwork of interconnected smooth muscle cells lined by vascular endothelium. Of note, endothelial cells and underlying smooth muscle also line the small resistance helicine arteries that supply blood to the corpus cavernosum during penile tumescence. Pathological alteration in the anatomy of the penile vasculature or impairment of any combination of neurovascular processes can result in ED. Aging is most commonly associated with ED, but a number of underlying disease processes are recognized to lead to abnormal function and responsiveness of the penile vascular bed. ED was believed to be a psychological condition; however, in the past 2 decades, authorities have recognized that the majority of patients' erectile failure can be attributed to an organic etiology. ED may result from neurologic, arteriogenic, veno-occlusive, or cavernosal impairments and is therefore associated with vascular risk factors such as atherosclerosis, hypertension, hypercholesterolemia, diabetes mellitus, and cigarette smoking (Feldman et al, 1994; Laumann et al, 1999; McKinlay, 2000). Because ED is highly prevalent in men with cardiovascular disease, and because cardiovascular disease is well known to be associated with endothelial dysfunction, one can infer that endothelial dysfunction of the penile vascular tree may contribute to impairments in erectile function. Therefore, it has been hypothesized that endothelial dysfunction can result in ED (Maas et al, 2002; Solomon et al, 2003). Recent clinical and basic science investigations on aging, diabetes, hypercholesterolemia, and hypertension have shown that endothelial dysfunction is a major contributing factor to penile vascular pathology. The following review examines the role of the vascular endothelium in erectile physiology and demonstrates the importance of the endothelium in normal erectile physiology and how impairments in endothelial function can cause deleterious effects on erectile function. The vascular endothelium not only serves as a passive barrier for the arterial and venous blood, but also plays a pivotal role in modulating vascular tone and blood flow in response to humoral, neural, and mechanical stimuli. Furchgott and Zawadzki (1980) first reported the obligatory role of the endothelium in regulating local and basal control of vessel tone. It is now widely accepted that the vascular endothelium has a fundamental role in the regulation of vascular tone in the circulation by releasing a variety of factors that affect the contractile and relaxatory behavior of the underlying vascular smooth muscle. The actions of the endothelium are not limited to regulating vascular tone, rather, they also play a pivotal role in the regulation of inflammation, platelet aggregation, vascular smooth muscle proliferation, and thrombosis (Behrendt and Ganz, 2002). The endothelium responds to chemical and hormonal signals as well as to physical hemodynamic changes caused by alterations in blood flow and shear stress by releasing mediators that modulate the tone of the underlying smooth muscle layer. In certain disease states, disruption of the functional integrity of the vascular endothelium plays an integral role in the ability of the endothelium to respond to local hemodynamic changes and paracrine and autocrine factors, a condition referred to as endothelial dysfunction (Cai and Harrison, 2000; Behrendt and Ganz, 2002; Maxwell, 2002). The regulatory role of the endothelium becomes attenuated during endothelial dysfunction, whereby there is either a decrease in responsiveness to vasodilator mediators or an increase in sensitivity to vasoconstrictors. Endothelial dysfunction refers to several pathological conditions, including altered anticoagulation and anti-inflammatory activities, impaired modulation of vascular growth, and dysregulation of vascular remodeling (Mombouli and Vanhoutte, 1999). However, the term endothelial dysfunction is most commonly used to refer to decreases in endothelium-dependent smooth muscle relaxation caused by a loss of or increased destruction of nitric oxide (NO) bioactivity in the vasculature. The decrease in NO bioavailability in endothelial dysfunction may be caused by reductions in the enzyme endothelial NO synthase (eNOS, NOS3); a lack of substrate or cofactors for eNOS; alterations in intracellular signaling such that eNOS is not appropriately activated or uncoupled; or accelerated degradation of NO by reactive oxygen species (ROS), such as superoxide anion. Importantly, in endothelial dysfunction, responses to the endothelium-independent vasodilator sodium nitroprusside (SNP) are usually unaltered, indicating that dysfunction arises from abnormal NO production or release from the endothelial cells. However, it is conceivable that other factors, such as superoxide anion, may be preventing NO from eliciting the normal vasodilator response. This condition causes a disruption in the balance of the vasoactive mediators, whereby the role of vasoconstrictors becomes even more prominent and the role of vasodilators diminishes, thereby affecting normal vascular tone. Various cellular processes are regulated through the release of NO from the endothelium, platelets, vascular smooth muscle cells, neurons, and other cell types (Ignarro et al, 1999). NO has many important physiological roles, including neurotransmission, regulation of vascular tone, immunomodulation, cell-mediated cytotoxicity against pathogens and tumor cells, and penile erection (Burnett, 1995; Ignarro et al, 1999; Bogdan, 2001). In addition to NO's direct toxic effects, it has been shown to exhibit an inhibitory effect on smooth muscle cell proliferation and collagen synthesis. The formation of NO and L-citrulline from its substrate L-arginine occurs in most tissues of the body. The enzyme that catalyzes this reaction in cells and neurons is termed NO synthase (NOS). This enzyme uses reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinculeotide, flavin mononucleotide, and tetrahydrobiopterin (BH4) as cofactors and heme as a prosthetic group. The constitutive forms of the enzyme, neuronal NOS (nNOS; NOS1) and eNOS, are coupled to Ca2+ and calmodulin and are the principal NOS isoforms involved in the induction of penile erection (Ignarro et al, 1990; Burnett et al, 1992; Rajfer et al, 1992), whereas inducible NOS (iNOS; NOS2) is independent of Ca2+ and calmodulin and requires new protein synthesis (Alderton et al, 2001). eNOS is predominately membrane bound, whereas nNOS is limited to the cytosol of central and peripheral neurons, although its mRNA is also localized to the skeletal muscle (Pollock et al, 1991). The concentrations of NO are continuously fluctuating at very low levels throughout the vascular system and are controlled predominately by eNOS. The constitutive enzymes eNOS and nNOS are regulated predominately at the posttranslational level, whereas iNOS is expressed in response to an appropriate stimulus (eg, cytokines, inflammation) or transcriptional factors (nuclear factor kappa B; NF-κB) (Forstermann and Kleinert, 1995). Recently, the subcellular localization of eNOS to distinct microdomains of the plasma membrane, its interaction with the protein caveolin-1, and the phosphorylation state of specific serine and threonine residues of the enzyme have been found to play an integral part in the posttranslational regulation of eNOS activity (Feron et al, 1996; Fleming and Busse, 1999; Michell et al, 1999; Boo et al, 2002; Goligorsky et al, 2002). NO produced by 1) eNOS from endothelial cells lining the cavernosal smooth muscle cells and resistance helicine arteries in response to shear stress, 2) agonist-induced activation by acetylcholine released from cholinergic nerves, and 3) nNOS activity in nonadrenergic, noncholinergic (NANC) neurons is involved in signaling events that regulate neurotransmission and penile vascular tone. The most important physiological target of NO in the penis is the heme moiety of soluble guanylate cyclase (Mizusawa et al, 2002). NO diffuses to adjacent smooth muscle cells stimulating guanylate cyclase. This interaction converts guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP), which induces a substantial increase in intracellular cGMP, causing smooth muscle relaxation. Its action is primarily through the cGMP-dependent kinase I (cGKI, PKG), which alters intracellular Ca2+ levels by reducing Ca2+ channel activity and opening Ca2+-dependent K+ channels, leading to hyperpolarization of the smooth muscle cell (Christ et al, 1999). PKG can also phosphorylate other proteins to affect Ca2+ channels or lead to an alteration of the phosphorylation state of the myosin light chain (MLC) (Mills et al, 2002b). These second messengers reduce intracellular Ca2+ via Ca2+ sequestration and extrusion and activation of MLC phosphatases. The NO/cGMP-dependent smooth muscle relaxation results in entry of blood and engorgement of the corpus cavernosum and penile erection. The physiological actions of cGMP are terminated by the hydrolysis of the 3′5′ bond by the type 5 phosphodiesterase. The importance of the protein kinase cGKI in the erectile process was established in cGKI-deficient mice (Hedlund et al, 2000a). These knockout mice are unable to reproduce and have impaired cavernosal smooth muscle relaxation in response to neuronal and endothelial derived NO and exogenous NO. Until recently, the defined role of eNOS and nNOS in the regulation of NO-dependent penile erection has been a subject of great debate. Mice lacking the genes for both eNOS and nNOS were still able to exhibit normal erectile function and mating behavior (Burnett et al, 1996, 2002). The first explanation proposed for the maintenance of the NO-dependent erectile response in nNOS-alpha -/- mice was a compensatory up-regulation of eNOS to fulfill insufficient nNOS expression. A more recent explanation for the intact NO-dependent erectile response in these mice is the existence of nNOS gene variants resulting from alternative mRNA splicing of the nNOS-beta and nNOS-gamma alternative translation in exon 1 (Burnett, 2000; Gonzalez-Cadavid et al, 2000). eNOS -/- mice demonstrate normal erectile function to electrical stimulation of the cavernosal nerve even when their penises have only 60% of the NOS activity compared with wild-type mice (Burnett et al, 2002). Of particular interest, studies from eNOS -/- mice have shown direct physiologic evidence for the contribution of eNOS in mediating cholinergic-stimulated penile erections, thus documenting the importance of cholinergic stimulation and agonist-induced activation of the endothelium in cavernosal smooth muscle relaxation and erection (Burnett et al, 2002). Thus, transgenic mice studies with targeted deletion of the eNOS and nNOS genes have further elucidated the role of these NOS isoforms on the regulation of penile erection and have reinforced our current understanding of the importance of NO regulatory control of penile erection. NO production from nNOS in the NANC nerves innervating the penis is essential for the initiation of cavernosal smooth muscle relaxation and subsequent erection. The relative importance of endothelial-derived NO from eNOS in the endothelial cells of the corpus cavernosum and arteries supplying the penis has recently been elucidated. Immunohistochemical and molecular probes for eNOS have demonstrated its presence in the trabecular lining of the corpus cavernosum and in the small intracavernosal resistance helicine arteries of the penis (Bloch et al, 1998; Hedlund et al, 1999; Bivalacqua et al, 2001a; Gonzalez et al, 2001; Mizusawa et al, 2001; Stanarius et al, 2001). Both human and animal studies have demonstrated that the corpus cavernosum is capable of relaxing in the presence of endothelium-dependent agonists (acetylcholine, carbachol, bradykinin) and impairment of endothelium-dependent cavernosal smooth muscle relaxation in vitro occurs in vascular-associated diseases, such as diabetes mellitus, hypertension, and hypercholesterolemia (Saenz de Tejada et al, 1989; Gur et al, 2000; Behr-Roussel et al, 2002, 2003). However, what role does endothelial-derived NO play in the regulation of normal penile erection in vivo? In the past 5 years there has been significant evidence supporting the vital role of endothelial-derived NO from eNOS in the regulation of penile erection both in normal physiology and in pathological disease states. Our current understanding on the mechanism for initiation and maintenance of penile erection is that penile erection is elicited by neural signals form the spinal cord, which stimulates nNOS activity and increases the production of NO from NANC nerves, thereby causing an increase in blood flow to the cavernosal tissue (Moreland et al, 2001b). eNOS is then activated by a shear stress/mechanical mechanism by increased blood flow from the arteries supplying the corpora and expansion of the sinusoidal spaces of the corpora. The continued shear stress on the endothelial lining of the intracavernosal smooth muscle cells and arteries continues to produce endothelial-derived NO, which maintains the tumescence phase of penile erection (Figure 1). . Schematic diagram of the independent roles of nNOS and eNOS in the initiation and maintenance of the erectile response. (Adapted from Hurt et al, 2002.) Hurt and colleagues (2002), using selective pharmacological inhibitors and eNOS knockout mice, first showed that penile erection-dependent processes to cavernosal nerve stimulation and drug-induced relaxation of the corpus cavernosum are mediated by phosphatidylinositol 3-kinase (PI3-kinase) and activation of the serine/threonine protein kinase Akt. This pathway phosphorylates eNOS to increase endothelial-derived NO (Michell et al, 1999). The use of pharmacological inhibitors of PI3-kinase in the penis of rats and eNOS -/- mice demonstrated that these inhibitors were able to reduce erections to electrical nerve stimulation and intracavernous papaverine. This signaling pathway was furthermore shown to be responsible for sustained NO production via a PI3 kinase/Akt-dependent activation of eNOS with subsequent increases in endothelial-derived NO and maintenance of maximal erection (Figure 1). The application of targeted genes involved in the erectile process has further enabled researchers to study the mechanisms involved in the pathophysiology of ED-associated conditions, such as aging, diabetes, and hypercholesterolemia. Both the natural aging process and diabetes cause significant impairment in erectile function that is contributed to numerous factors both in the central and in the peripheral nervous system as well as at the end organ. Most notably, changes in neural integrity of the cavernosal nerve and pelvic plexus as well as in endothelial cell function are well recognized. ED associated with these conditions is multi-factorial, but most authorities recognize that there is an overall reduction in NO biosynthesis (Sullivan et al, 1999; Maas et al, 2002). Until recently, the independent role of eNOS in these disease processes was unknown. By using adenoviral gene transfer of eNOS to the corpus cavernosum, it was determined that this NOS isoform was capable of restoring diminished erectile function in aged rats and rats with diabetes independent of nNOS expression (Champion et al, 1999; Bivalacqua et al, 2000b; Bivalacqua and Hellstrom, 2001; Bivalacqua et al, 2003b, in press). These studies have demonstrated that in disease states, in which eNOS expression is reduced or unaltered, overexpression of this NOS isoform is capable of restoring erectile function to cavernosal nerve stimulation via increased endothelial-derived NO biosynthesis and cavernosal cGMP levels. Importantly, eNOS gene therapy has no effect on nNOS protein or gene expression, in that restoration of erectile function is solely dependent upon eNOS expression and endothelial-derived NO bioactivity. These studies document the importance of eNOS in the maintenance of the erectile response and its significance in pathological conditions associated with ED and endothelial dysfunction. A number of review articles address the mechanisms and pharmacology of penile erection (Bivalacqua et al, 2000a; Christ, 2000; Andersson, 2001b, 2003). The following section contains an overview of the endothelium-derived vasodilator and vasoconstrictor agents involved in the physiology of penile erection. Cavernosal smooth muscle cells in the penis are predominately found in the contracted state with minimal blood flowing through the cavernous sinuses. The balance between known contractile systems (RhoA/Rho-kinase, α-adrenergic, endothelin, angiotensin, thromboxane A2) and vasodilatory second-messenger systems (adenylate cyclase-cyclic AMP and guanylate cyclase-cyclic GMP) determines the overall tone of corpora cavernosa smooth muscle of the penis (Andersson, 2001a; Mills, 2002). This balance is controlled by both central and peripheral mechanisms and involves a plethora of neurotransmitters acting through various signal transduction pathways. Endothelial cells actively regulate basal vascular tone and vascular reactivity in physiological and pathological conditions by responding to mechanical forces and neurohumoral mediators with the release of a variety of relaxing and contracting factors. In the corpus cavernosum, the vascular endothelium and cavernosal arteries are a source of vasorelaxing factors such as NO; prostacyclin (PGI2); the not-yet identified endothelium-derived hyperpolarizing factor (EDHF); and the vasoconstrictor factors angiotensin II (Ang II), endothelin-1 (ET-1), and Rho-kinase. These endothelium-derived factors have a regulatory influence on cavernosal vascular tone. Normally, these factors act in concert to elicit an overall beneficial effect on cavernosal smooth muscle function, which is crucial in response to changes in blood flow, shear stress, and agonists in order to maintain physiological homeostasis throughout the penile vascular bed. The critical balance of vasodilators and vasoconstrictors is normally maintained during health and quickly responds to changes in blood flow and other factors. Decreases in NO-, prostaglandin-, and EDHF-mediated responses have been shown to be involved in cardiovascular diseases related to endothelial dysfunction, and increases in responses to Ang II and ET-1 have also been implicated (Harrison, 1997; De Vriese et al, 2000). Evidence now exists to demonstrate that this also occurs in the penile vascular bed. The principle mediator of cavernosal smooth muscle relaxation is NO released by the NANC neurons innervating the penis and by cavernosal endothelial cells (Burnett, 1995). NO is released from endothelial cells under the influence of agonists, such as acetylcholine from cholinergic neurons in the penis, and mechanical forces, such as shear stress caused by pulsital blood flow in the sinusoidal spaces of the corpus cavernosum (Hedlund et al, 2000b). NO diffuses to the underlying smooth muscle cells where it activates the soluble form of guanylate cyclase elevating intracellular levels of cGMP and the activity of cGKI protein kinase. The NO/cGMP-signaling cascade reduces contractile activity and promotes cavernosal smooth muscle relaxation (see previous section). NO can activate prostaglandin (PGE) synthesis in vivo. During shear stress blood flow in the penis, another mechanism involved to enhance cavernosal smooth muscle relaxation is activation of PGE synthesis via a NO-dependent mechanism (Ballermann et al, 1998). The vasodilators PGE and PGI2 are primarily produced by endothelial cells in the vasculature. PGI2 is the most abundant arachidonic acid product generated in vascular tissues (McNamara et al, 1998). Both isoforms of cyclooxygenase, COX-1 and COX-2, can convert arachidonic acid to PGH2. PGH2 is subsequently converted to PGI2 by the action of PGI2 synthase (PGIS) and PGE2 by PGE2 synthase. PGE synthesized by endothelial cells in the corpus cavernosum in response to mechanical shear stress blood flow in the penis binds to specific PGE (EP) receptors on smooth muscle cells (Traish et al, 1997; Meghdadi et al, 1999). Activation of EP receptors by PGE increases intracellular levels of cAMP via activation of adenylate cyclase, causing a reduction in intracellular levels of Ca2+ and cavernosal smooth muscle relaxation (Moreland et al, 2001a). Both PGE2 and its derivative PGE1 are potent vasorelaxing agents in human corpus cavernosum smooth muscle. There are 4 distinct EP receptors (EP1–4), and all 4 are expressed in the corpus cavernosum (Angulo et al, 2002). EP2 and EP4 are G-protein-coupled receptors and are responsible for an increase in cAMP synthesis in response to exogenous PGE1 administration to the human penis and in cultured cavernosal smooth muscle cells. PGE1 is a potent vasodilator of the penile vascular bed and is a highly efficacious local agent for the treatment of ED (Leungwattanakij et al, 2001). The currently unidentified EDHF likely plays an important role in erectile physiology. EDHF is released by endothelium-dependent agonists or shear stress and hyperpolarizes the underlying smooth-muscle-inducing relaxation by decreasing intracellular levels of Ca2+ within the smooth muscle cells (Busse et al, 2002). This mechanism involves decreasing the opening probability of the voltage-dependent Ca2+ channels and reducing the turnover of intracellular phosphotidylinositides. EDHF-mediated responses increase as the vessel size decreases (Mombouli and Vanhoutte, 1997). It was once believed that the final common pathway leading to smooth muscle hyperpolarization by EDHF required the opening of potassium channels on the smooth muscle cell membrane; however, this hypothesis has been modified in light of more recent experimental observations. Current evidence suggests that EDHF is formed after an increase in endothelial Ca2+, either induced by an agonist (acetylcholine, bradykinin) or shear stress that triggers the synthesis of a cytochrome P450 metabolite, which is essential for the subsequent EDHF-mediated response (Fisslthaler et al, 1999; Fleming, 2001). Epoxyeicostatrienoic acid (EETs) is an arachidonic-acid-derived product of cytochrome P450 epoxygenases that appears to play an important role in the regulation of vascular homeostasis (Fleming, 2001; Zhang et al, 2001). EETs are reported to be potent vasodilators in a number of peripheral vascular beds. In human coronary arteries, inhibitors of cytochrome P450 2C block EDHF-mediated responses, and EETs relax coronary arteries by hyperpolarizing smooth muscle cells through a K-channel-dependent mechanism (Miura and Gutterman, 1998; Miura et al, 1999). However, the role of EDHF-mediated cavernosal smooth muscle relaxation has not been determined until recently. Angulo et al (2003) revealed that an EDHF-mediated relaxation of human penile resistance arteries exists, which is resistant to NOS and COX inhibition, suggesting that EDHF may play an important role in the endothelium-dependent relaxation of the penile vascular bed. Additionally, our laboratory has found that the pharmacological inhibitor of cytochrome P450 2C, sulfaphenazole, attenuates cavernosal nerve mediated erectile responses in the rat. This suggests that a cytochrome P450 metabolite may mediate an EDHF-dependent smooth muscle effect in the penis that may contribute to the erectile response (Figure 2). Because this area of penile vascular biology is not fully elucidated, further research must be undertaken to evaluate the potential importance of this endothelium-derived relaxing factor in the regulatory control of penile erection. . Bar graph depicting the voltage-dependent erectile response (ICP/MAP) after cavernosal nerve stimulation at the 5 V setting for 1 minute in control rats before and after administration of the cytochrome P450 2C inhibitor, sulfaphenazole, in a dose of 100 mg/kg IV. The in vivo erectile response was measured at baseline and 30 minutes after administration of sulfaphenazole. n indicates number of experiments; *, (P < .05) response significantly different from baseline response. Ang II is a potent vasoconstrictor. In addition to the classical renin-angiotensin system (RAS) operating systemically, there is a functional RAS that generates Ang II locally in the vascular tissue of the penis (Kifor et al, 1997). Angiotensin receptors characterized in the penile vasculature suggest that endothelial cells in the corpus cavernosum may form a local Ang II-producing paracrine system that may modulate vascular tone by keeping the cavernosal smooth muscle cells in a constricted state (Park et al, 1997). In organ bath studies of isolated strips of corpus cavernosum, Ang II caused a dose-dependent contraction of the cavernosal tissue in vitro (Becker et al, 2001c). Elevated levels of Ang II have been noted in men with organic ED, suggesting this peptide may play a role in the pathogenesis of ED (Becker et al, 2001a). The endothelins (ET-1, ET-2, and ET-3) are a family of related peptides. ET-1 is a 21 amino acid peptide generated in the vascular endothelium that is recognized as a potent and sustained vasoconstrictor in the penile vasculature (Christ et al, 1995; Mills et al, 2001b). The effects of ET-1 are mediated through ETA receptors, which are located on the underlying smooth muscle, and through ETB receptors, which are located on the smooth muscle and vascular endothelium. ETA receptors mediate contraction and promote growth of smooth muscle. ETB receptors on smooth muscle also mediate contractions, whereas stimulation of ETB receptors on the endothelium promote NO and prostacyclin-mediated vasorelaxation. Of note, ET-1 acts as a vasodilator at low doses (ETB receptor activation) and as a vasoconstrictor (ETA receptor activation) at high doses when administered to cavernosal strips in vitro. Basal production of ET-1 by endothelial cells of the corpus cavernosum is hypothesized to contribute to sustained cavernosal smooth muscle contraction and maintenance of penile flaccidity. Both animal and human studies have not validated this hypothesis; therefore, despite the existence of ET receptors in the penis, most whole animal experimental and human clinical data do not support a central role for ET-1 in regulating the normal erectile response (Becker et al, 2000, 2001b; Dai et al, 2000; Kim et al, 2002). Diabetic corpus cavernosum obtained from animal models and humans have shown that ETA receptors are upregulated and ETB receptors are downregulated, suggesting that ET-1 may be involved in the pathogenesis of diabetic ED (Sullivan et al, 1997, 1998; Chang et al, 2003). However, future studies are warranted to establish the functional significance of these cavernosal molecular changes in the pathophysiology of diabetic-associated endothelial dysfunction. Arginine is a precursor for the synthesis of NO, urea, polyamines, creatine phosphate, and various proteins (Figure 3). This amino acid is transported from the circulation into mammalian cells by cationic amino acid transporter (CAT) isoforms (Durante, 2001). Cationic amino acids such as L-arginine are transported into cells via the y+ transport system. This systems activity is mediated by the CAT family, which is composed of 4 isoforms: CAT-1, CAT-2A, CAT-2B, and CAT-3. The existence of these CAT transporters has not been documented in the penis, but their existence is inevitable. The major site of arginine metabolism is the liver, where L-arginine generated in the urea cycle is converted to urea and ornithine by the enzyme arginase. Many additional tissues and cell types also contain the enzyme arginase, in particular endothelial cells (Li et al, 2002). . Arginine metabolism. L-arginine enters the endothelial cell through cationic amino acid transporter (CAT) and can react with the enzymes arginase or eNOS. When L-arginine reacts with eNOS, NO and L-citrulline is formed. NO can then bind to the soluble form of guanylate cyclase (sGC) to form cGMP. When L-arginine reacts with arginase, L-ornithine and urea are formed. Ornithine can react with ornithine decarboxylase (ODC) to form polyamines that contribute to cell proliferation or react with ornithine aminotransferase (OAT) to form pyrroline-5-carboxylate (P5C). Urea can form reactive oxygen specices (ROS) in some cell types. In endothelial cells, arginine is used as a substrate by both eNOS and arginase. Because both NOS and arginase use arginine as a common substrate, arginase ma