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Review |
From * The James Buchanan Brady Urological
Institute, Department of Urology, and the
Division of Cardiology, Department of
Medicine, Johns Hopkins Medical Institutions, Baltimore, Maryland.
| Correspondence to: Dr Trinity J Bivalacqua, The James Buchanan Brady Urological Institute, Johns Hopkins Medical Institutions, 600 N Wolfe Avenue, Marburg 143, Baltimore, MD 21287 (e-mail: tbivala1{at}jhmi.edu). |
| Received for publication October 11, 2008; accepted for publication April 2, 2009. |
Erectile dysfunction (ED) is a common men's health problem characterized by
the consistent inability to sustain an erection sufficient for 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, neurogenic dysfunction, or both.
The constitutive forms of nitric oxide synthase (NOS, endothelial [eNOS] and
neuronal [nNOS]) are important enzymes involved in the production of nitric
oxide (NO) and thus regulate penile vascular homeostasis. Given the effect of
endothelial- and neuronal-derived NO in penile vascular biology, a great deal
of research over the past decade has focused on the role of NO synthesis from
the endothelium and nitrergic nerve terminal in normal erectile physiology, as
well as in disease states. Loss of the functional integrity of the endothelium
and subsequent endothelial dysfunction plays an integral role in the
occurrence of ED. Therefore, molecular mechanisms involved in dysregulation of
these NOS isoforms in the development of ED are essential to discovering the
pathogenesis of ED in various disease states. This communication reviews the
role of eNOS and nNOS in erectile physiology and discusses the alterations in
eNOS and nNOS via posttranslation modification in various vascular diseases of
the penis.
Key words: eNOS, nNOS, corpus cavernosum, phosphorylation
Underlying disease processes such as diabetes mellitus, hypercholesterolemia, hypertension, and the natural aging process are recognized to lead to abnormal function and responsiveness of the penile vascular bed, particularly reductions in NO biosynthesis. The following review examines the role of eNOS and nNOS in erectile physiology and the effect of posttranslational modification, as well as protein-to-protein interactions of the constitutive NOS isoforms in the development of penile vascular dysfunction.
eNOS and nNOS Definition and Description
Three isoenzymes of NOS exist and share the same basic structural and
catalytic mechanisms. Each functions as a homodimer, with the interface
between subunits being formed by the oxygenase domain. In their active forms,
NOS enzymes are associated with 2 molecules of calmodulin, as well as
cofactors, which allow for 2 sequential mono-oxygenation reactions that lead
to the generation of NO (Cartledge et al,
2001). The differences between the isoenzymes mainly involve their
distribution and regulation.
eNOS and nNOS, the constitutive forms of the enzyme, are thought to play primary roles in NO formation during erection. Immunohistochemical and functional studies have localized nNOS to nerve terminals of penile autonomic nerves (pelvic plexus, cavernous and dorsal nerves) and eNOS to the endothelium of the cavernosal cisternae and arteries (Podlasek et al, 2001; Stanarius et al, 2001; Azadzoi et al, 2004). Although the interplay between the NOS isoenzymes continues to be a matter of study, existing evidence points toward a model in which nNOS initiates the erectile response, which is then maintained and increased by eNOS activity (the latter being activated by shear stress; Busse and Fleming, 1998; Hurt et al, 2002; Burnett 2004; Musicki et al, 2005b; Bivalacqua et al, 2007).
PnNOS, the nNOS variant expressed in the penis and prostate, exists as 2 spliceoforms, a full-length alpha spliceoform and a beta spliceoform which lacks the N-terminal PDZ domain, important for protein-protein interactions. Evidence suggests that the alpha splice variant is active in NO formation at nerve terminals, whereas the functional role of the beta variant in vivo is unclear and might not be substantial (Gonzalez-Cadavid et al, 2000).
Although the role of transcriptional regulation of constitutive NOS isoforms and long-term regulation of NO production in the penis has been documented in many studies, it is now apparent that changes in NO production do not necessarily require changes in NOS abundance. Penile erection requires rapid production of NO, making posttranslational regulation of constitutive NOS isoforms more relevant than their transcriptional regulation. In addition, NO production from constitutive activation of eNOS by phosphorylation and possibly nNOS phosphorylation contributes to vascular homeostasis in the penis.
eNOS Posttranslational Modification
Multiple posttranslational mechanisms regulate eNOS activity. In addition
to rapid regulation by calcium-calmodulin, posttranslational mechanisms
include substrate and cofactor availability, fatty acid modification (such as
myristoylation and palmitoylation), subcellular localization, dimerization of
the enzyme subunits, binding to other proteins (such as caveolin-1 and heat
shock protein [Hsp]90), and phosphorylation, as well as regulation by reactive
oxygen species (Fleming and Busse,
2003). These mechanisms regulate eNOS-mediated responses under
physiologic circumstances and provide various mechanisms whereby endothelial
NO availability may be altered in states of vasculogenic dysfunction. The
Figure summarizes the major
molecular mechanisms of eNOS posttranslational regulation that regulate eNOS
activity and NO bioavailability. Although listed as separate categories, these
regulatory mechanisms are not mutually exclusive; rather, they may be
interrelated to maintain the proper spatial and temporal organization of eNOS
signaling. For example, the intracellular localization of eNOS regulates
calcium-calmodulin–dependent activation of eNOS and the extent of eNOS
phosphorylation (Jagnandan et al,
2005). Similarly, eNOS targeting to the cell membrane appears to
be essential for the S-nitrosylation/denitrosylation cycling that
regulates eNOS activation (Erwin et al,
2006).
|
Posttranslational Modification of eNOS by Phosphorylation— Phosphorylation of eNOS at multiple sites is a critical mechanism regulating eNOS activity. The phosphorylation is regulated by diverse signaling pathways where specific protein kinases and protein phosphatases regulate phosphorylation at each site. A diverse and growing list of activators have been identified that regulate eNOS phosphorylation and NO synthesis and include mechanical (shear stress and cyclic strain), humoral (vascular endothelial growth factor [VEGF], bradykinin, estrogen, sphingosine 1-phosphate), and pharmacological (statins) stimuli (Table).
|
Six specific sites of phosphorylation have been identified in eNOS at Tyr-81, Ser-114, Thr-495, Ser-615, Ser-633, and Ser-1177 (human sequence, equivalent to Tyr-83, Ser-116, Thr-497, Ser-617, Ser-635, and Ser-1179 on bovine eNOS; Fleming and Busse, 2003; Sessa 2004; Fulton et al, 2005). Phosphorylation of these sites increases or decreases the enzyme's activity directly or by modulating other regulatory sites on eNOS. The best characterized eNOS phosphorylation site is Ser-1177, located in the reductase domain of the enzyme. The most important physiologic stimulator of eNOS phosphorylation at Ser-1177 is shear stress, a pressure exerted on endothelial cells by blood flow in the vessel at a constant flow rate. Shear stress, statins, as well as several hormones and growth factors (eg, VEGF, insulin-like growth factor 1 [IGF-1], and estrogen), phosphorylate eNOS and activate eNOS catalytic function by reducing calmodulin dissociation from eNOS on Ser-1177 and facilitating electron transfer from the reductase to the oxygenase domain (Dimmeler et al, 1999; Fulton et al, 1999; Michell et al, 1999; McCabe et al, 2000; Bauer et al, 2003). Depending on a given stimulus and vascular bed, Ser-1177 can be phosphorylated by numerous protein kinases, including Akt (protein kinase B), cyclic AMP-dependent protein kinase (PKA), AMP-activated protein kinase, cGMP-dependent protein kinases (PKG), calcium-/calmodulin-dependent protein kinase II, and protein kinase C (Sessa, 2004).
In contrast to Ser-1177, eNOS phosphorylation at Thr-495, which is within the calmodulin binding sequence of eNOS, reduces eNOS catalytic activity by interfering with binding of calcium/calmodulin to eNOS (Fleming et al, 2001; Harris et al, 2001; Michell et al, 2001). Reciprocal dephosphorylation of Thr-495 and phosphorylation of Ser-1177 appears to be essential for eNOS activity (Fleming et al, 2001; Harris et al, 2001; Michell et al, 2001). PKC signaling has commonly been accepted to promote eNOS phosphorylation at Thr-495 and dephosphorylation at Ser-1177, reducing the enzyme's activity (Michell et al, 2001). Agonist-induced dephosphorylation of Thr-495, associated with increased eNOS activity, is mediated by protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), and calcineurin (PP2B; Michel et al, 2001; Greif et al, 2002).
The physiological significance of phosphorylation of other sites on eNOS is less known. PKA-mediated phosphorylation of Ser-633, which is located in the autoinhibitory sequence of eNOS, increases eNOS activity (Michell et al, 2002; Bauer et al, 2003; Boo et al, 2003). Various agonists, such as shear stress, VEGF, bradykinin, and statins, increase phosphorylation of this site after initial activation of the enzyme by calcium flux, Ser-1177 phosphorylation, or both. Phosphorylation of eNOS at Ser-615 by PKA and Akt increases eNOS activity and appears to be important for regulating interactions between eNOS and other proteins and for phosphorylation of eNOS at other sites (Michel et al, 2002; Bauer et al, 2003). Phosphorylation of eNOS at Ser-114 inhibits enzyme activity (Li et al, 2007). This inhibition doesn't occur through a direct effect on catalytic activity of the enzyme, but rather indirectly through an increased association of eNOS with caveolin-1 (Li et al, 2007). eNOS phosphorylation at Tyr-81 by Src kinase contributes to an increase in eNOS activity by affecting the calcium-stimulated eNOS activation process, altering protein-protein interactions, or changing the subcellular localization of the enzyme (Fulton et al, 2005, 2008).
eNOS phosphorylation in penile physiology. The role of eNOS phosphorylation in the physiology and pathophysiology of penile erection is increasingly being recognized. It has recently been demonstrated that eNOS phosphorylation at Ser-1177 serves an indispensable role in penile erection (Hurt et al, 2002). Our current understanding is that nNOS and eNOS, respectively, mediate the initiation and maintenance of maximal penile erection. In response to sexual stimuli, depolarization-induced calcium entry and calcium/calmodulin activation of nNOS initiates erection. Vasorelaxation causes increased blood flow and physical expansion of penile vasculature and sinusoidal spaces. The resulting shear force on the endothelium of these structures activates the phosphatidylinositol 3-kinase (PI3-kinase)/Akt/eNOS (Ser-1177) phosphorylation, causing calcium-"independent" activation of eNOS and sustained endothelial NO release, accounting for the achievement and maintenance of full erection (Hurt et al, 2002). These in vivo findings were substantiated by in vitro results showing that fluid shear stress increases phosphorylation of eNOS (Ser-1177) and NO release in human corpus cavernosal endothelial cells (Wessels et al, 2006). In addition to blood flow–related shear stress, VEGF also activates eNOS in the penis by Akt-independent phosphorylation at Ser-1177 (Musicki et al, 2004). Furthermore, activation of human cavernous tissue and penile artery by sphingosine 1-phosphate, potentiates acetylcholine-induced eNOS phosphorylation on Ser-1177 and increases NO release (Di Villa et al, 2006). Interestingly, in the rat penis, electrical stimulation of the cavernous nerve increases phosphorylation of eNOS at Thr-495 (Musicki et al, 2005b), suggesting a role of eNOS in preventing its own overactivation, which would cause an excessive eNOS-dependent erection. Mitogen-activated protein (MAP) kinases 1 and 2 (extracellular signal-regulated protein kinase [ERK] 1/2) have been implicated in negative regulation of eNOS activity in the penis. Sommer et al (2002) demonstrated that the expression of ERK 1/2 is substantially greater in the corporal smooth muscle of men with ED associated with various primary diseases compared with potent men. The mechanism of eNOS inhibition by ERK 1/2 in the penis is not known.
Dysregulation of eNOS phosphorylation in penile pathophysiology. Various mechanisms can disturb eNOS regulation by phosphorylation and endothelial NO bioavailability in the penis, resulting in vasculogenic ED. Dysregulation of eNOS phosphorylation in the penis has been associated with aging, diabetes mellitus, and hypercholesterolemia.
In the aged rat penis Akt-dependent phosphorylation of Ser-1177 on eNOS is decreased, whereas phosphorylation of Thr-495 on eNOS is increased, in spite of increased eNOS protein expression (Musicki et al, 2005b). Phosphorylation of the Ser-1177 on eNOS in the aged rat penis is also decreased in response to shear stress elicited by electrical stimulation of the cavernous nerve, presumably because of increased phosphorylation of Thr-495 on eNOS, which prevents Akt-dependent phosphorylation of Ser-1177 (Musicki et al, 2005b). In contrast to shear stress-related signaling, the susceptibility of eNOS to VEGF-induced signaling in the aged penis is preserved. VEGF is known to have an important role in the modulation of penile vasculature and the improvement of erectile response (Henry et al, 2000; Byrne et al, 2001; Lee et al, 2002; Gholami et al, 2003; Rogers et al, 2003; Musicki et al, 2004). The protective effect of VEGF on erectile function in the aged rats apparently is due to eNOS activation through increasing phosphorylation of Ser-1177, but not by decreasing phosphorylation of Thr-495 residues (Musicki et al, 2005b). Conceivably, reduced VEGF-dependent eNOS activation in the aged penis results from limited endogenous VEGF production. Erectile function of aged rats can lastingly be enhanced by long-term inhibition of phosphodiesterase (PDE)5 with sildenafil by rescuing Akt-dependent phosphorylation of eNOS (Ser-1177) in the penis (Musicki et al, 2005a). In the continuous presence of sildenafil, increased levels of cGMP promote cavernous relaxation and increase penile blood flow, resulting in shear stress on endothelial cells. Shear stress promotes constitutive activation of eNOS in the penis of aged rats by increasing Akt-dependent phosphorylation of the enzyme on Ser-1177, potentiating spontaneous erectile ability and maintaining vascular homeostasis in the penis. In another model of ED, the cavernous nerve injury model, which is believed to simulate the neural injury that occurs during radical prostatectomy, chronic sildenafil treatment also induces Akt-dependent eNOS phosphorylation (Ser-1177) in the penis (Mulhall et al, 2008) and preserves erectile function, although the effect of nerve injury itself on eNOS phosphorylation has not been evaluated.
Diabetes mellitus causes altered endothelial-derived NO production in the penis of experimental animals as well as human corpora cavernosa (Bivalacqua et al, 2001, 2004). This has been attributed to impaired eNOS protein expression and activity and increased oxidative stress (Bivalacqua et al, 2005; Musicki and Burnett, 2007). One mechanism of decreased NO formation in diabetes mellitus involves the activation of the hexosamine biosynthetic pathway, likely through hyperglycemia-induced mitochondrial overproduction of superoxide, and formation of N-acetylglucosamine (O-GlcNAc). O-linked GlcNAc is then attached to Ser and Thr residues of proteins involved in diverse aspects of cellular physiology. This monosaccharide modification often competes in a ying-yang fashion with phosphorylation in the cell's regulatory pathways (Wells et al, 2001). In the diabetic rat penis, O-GlcNAc modification of eNOS prevents phosphorylation of the enzyme specifically at Ser-1177 (does not affect residues Thr-495, Ser-615, and Ser-633), both at baseline and in response to fluid shear stress stimuli and VEGF signaling (Musicki et al, 2005c). In vitro studies with human corpus cavernosal endothelial cells failed to reproduce these findings: high glucose down-regulated total eNOS expression and shear stress-induced NO release but did not alter eNOS (Ser-1177) phosphorylation (Wessels et al, 2006). The discrepancy might be related to endothelial cells isolated from the corpora cavernosa of men with ED, where further reduction of P-eNOS (Ser-1177) expression might not be obvious.
The role of impaired eNOS phosphorylation in the penis in association with hypercholesterolemia-induced ED is controversial. The amount of P-eNOS (Ser-1177) in the porcine (Musicki et al, 2008) and mouse (Musicki et al, unpublished data) penis was found not to be affected by hypercholesterolemia. In contrast, several studies showed decreased eNOS phosphorylation at Ser-1177 in the penes of cholesterol-fed rats (Ryu et al, 2006a,b) and rabbits (Xie et al, 2005). This disparity might pertain to different stages of atherogenesis progression and use of different animal models in these studies. In cultured endothelial cells exposed to atherogenic conditions, oxidized low density lipoprotein has been reported to stimulate (Pritchard et al, 2002), attenuate (Chavakis et al, 2001), or have no effect (Fleming et al, 2005) on phosphorylation of eNOS on Ser-1177, whereas it decreases phosphorylation on Thr-495, leading to uncoupling of eNOS and generation of superoxide (Fleming et al, 2005). It is conceivable that atherosclerosis could be associated with an increased electron flux from the reductase to the oxygenase domain of eNOS, increasing the activation status of eNOS but resulting in eNOS uncoupling. In a hypercholesterolemic rat model, combination of angiogenic factors angiopoietin-1 and VEGF rescues erectile function and eNOS (Ser-1177) phosphorylation and endothelial function in the penis (Ryu et al, 2006a).
eNOS trafficking and protein-protein interaction—caveolae and caveolin-1. The proper subcellular localization of eNOS is critical for optimal coupling of extracellular stimulation with NO production. The majority of functional eNOS in quiescent endothelial cells is targeted to caveolae by cotranslational N-myristoylation and posttranslational palmitoylation (Alderton et al, 2001). Caveolin-1, the resident membrane protein of caveolae, can directly interact with eNOS and inhibit its activity by occupying its calmodulin binding site (Feron et al, 1996; Garcia-Cardena et al, 1997; Ju et al, 1997). Stimuli such as shear stress and estrogen induce calcium increase, and the calcium/calmodulin complex displaces eNOS from caveolin-1 and away from its tonic inhibition. eNOS translocation is associated with interaction with other proteins, such as calmodulin and Hsp90, and eNOS activation (Michel et al, 1997; Feron et al, 1998).
Whereas eNOS bound with caveolin-1 is inactive, compartmentalization of eNOS in caveolae is necessary to localize regulatory and signaling proteins, including caveolin-1, certain G-protein–coupled receptors, Hsp90, Akt, estrogen and VEGF receptors, calcium pumps, and so on (Everson and Smart, 2001). Caveolin-1 is believed to be instrumental in the compartmentalization of signaling molecules within caveolae, thus enabling rapid and selective regulation of calcium- and phosphorylation-dependent signal transduction events that modify the response of the enzyme to extracellular stimuli (Feron et al, 1998; Shaul 2002). Caveolin-1 also binds to cholesterol and is involved in trafficking cholesterol from the endoplasmic reticulum to the plasma membrane to regulate the cholesterol content of caveolae, which in turn determines the structure and function of caveolae (Everson and Smart, 2001).
In addition to caveolin-1, which serves as a major negative allosteric regulator of eNOS, the NOS-interacting protein (NOSIP; Dedio et al, 2001) and the NOS traffic inducer (NOSTRIN; Zimmermann et al, 2002) also negatively regulate eNOS activity by promoting translocation of eNOS from the plasma membrane to intracellular sites. The carboxyl terminus of Hsp70-interacting protein (CHIP) inactivates eNOS by uncoupling its interaction with Hsp90 and by displacement of eNOS from the Golgi apparatus, which is otherwise required for trafficking of eNOS to the plasmalemma and subsequent activation (Jiang et al, 2003).
Hsp90. Hsp90 acts as a major protein activator of eNOS. Hsp90 is one of the most abundant cytosolic proteins in eukaryotic cells. It plays important roles in the maturation, folding, activation, and trafficking of proteins involved in signal transduction, cell cycle control, and transcriptional regulation. Low amounts of Hsp90 appear to be associated with eNOS in the quiescent state, and, on stimulation of endothelial cells with stimuli such as VEGF, estrogen, histamine, and shear stress, the association between the 2 proteins is increased, concomitant with enhanced NO production (Garcia-Cardena et al, 1998). The mechanism of this activation involves calmodulin-dependent disruption of eNOS binding with caveolin-1, recruitment of eNOS and Akt to adjacent regions on Hsp90, reduced dephosphorylation of Akt, and increased ability of Akt to phosphorylate Hsp90-bound eNOS (Garcia-Cardena et al, 1998; Sato et al, 2000; Fontana et al, 2002; Takahashi and Mendelsohn, 2003; Kupatt et al, 2004). The direct protein interaction between eNOS and Hsp90 also enhances eNOS-coupled activity and prevents superoxide production by eNOS (Pritchard et al, 2001).
Other proteins associated with increased eNOS activity or NO release are the intracellular trafficking protein dynamin and the voltage-dependent anion/cation channel porin. Dynamin-2 regulates eNOS activity through binding to the flavin adenine dinucleotide (FAD) domain of eNOS, promoting electron transfer between the bound flavins of the reductase domain (Cao et al, 2003). The interaction of eNOS and porin is direct and specific and is enhanced by stimulators of eNOS (Sun and Liao, 2002), but the mechanism of its action is not known.
eNOS-protein interaction in penile physiology. Little information is available regarding eNOS interaction with negative or positive protein modulators in the penis and its physiologic significance in penile erection. Caveolin-1 expression has been documented in the rat and porcine penis (Bakircioglu et al, 2000, 2001; Park et al, 2004, Linder et al, 2006; Musicki et al, 2008). Our preliminary data show that eNOS exists in a complex with Hsp90 and caveolin-1 in the penis, which is functionally relevant for regulation of penile erection by shear stress. Electrical stimulation of the cavernous nerve decreases the amount of caveolin-1 associated with eNOS. Accordingly, increasing the interaction between eNOS and caveolin-1 by intracavernosal injection of AP-Cav peptide, a caveolin surrogate that binds to eNOS, decreases the erectile response, suggesting that the eNOS and caveolin-1 interaction diminishes eNOS activation in the penis and accordingly opposes eNOS-mediated penile erection (Musicki et al, unpublished data).
Dysregulation of eNOS-protein interaction in penile pathophysiology. Several studies evaluated protein expression of caveolin-1 in the penis associated with vascular ED, and discrepant results were obtained. While aging and diabetes mellitus decreased caveolin-1 expression in the rat penis (Bakircioglu et al, 2001; Park et al, 2004), hypercholesterolemia increased (Bakircioglu et al, 2000) or did not affect (Musicki et al, 2008) caveolin-1 expression in the rat and porcine penis, respectively. It is, however, conceivable that caveolin-1 interaction with eNOS, and not necessarily total protein expression of caveolin-1, determines the activity of eNOS. A recent study showed that the levels of caveolin-1 bound to eNOS were increased in the hypercholesterolemic pig penis (Musicki et al, 2008). Increased interaction between eNOS and caveolin-1 apparently prevented eNOS activation and contributed to endothelial dysfunction in the penis associated with hypercholesterolemia. Low-fat diet and chronic exercise preserved endothelial function in the atherosclerotic pig penis by mechanisms involving decreased eNOS interaction with caveolin-1 (Musicki et al, 2008).
Whether dysregulation of eNOS interaction with caveolin-1 in the penis contributes to other vascular disease states associated with ED has not been evaluated. No studies have addressed the role of eNOS interaction with Hsp90 in the penis in normal physiology or pathophysiology.
S-Nitrosylation
S-nitrosylation is the covalent adduction of NO-derived nitrosyl
groups to the cysteine thiols of proteins. Protein
S-nitrosylation/-denitrosylation is now recognized as a regulatory
component of signal transduction comparable to
phosphorylation/dephosphorylation. In resting endothelial cells, eNOS is
tonically inhibited by S-nitrosylation at Cys-94 and Cys-99 (Cys-96
and Cys-101 in bovine eNOS) of the zinc tetrathiolate cluster, which comprises
the eNOS dimer interface (Erwin et al,
2005). Although the mechanism for S-nitrosylation of eNOS
is not well understood, it is possible that conformational changes alter the
dimer interface, decreasing the efficiency of electron transfer between the 2
subunits of eNOS (Erwin et al,
2005; Dudzinski et al,
2006). Agonists such as VEGF and insulin rapidly and transiently
decrease eNOS S-nitrosylation concomitant with the increase in eNOS
phosphorylation at Ser-1177 and enzyme activation. Although eNOS
denitrosylation occurs simultaneously with Ser-1177 phosphorylation, these
modifications do not appear to be interdependent
(Erwin et al, 2005). eNOS is
subsequently renitrosylated, corresponding to the decline in eNOS Ser-1177
phosphorylation and the return of the enzyme's activity to its basal state.
The source of NO for nitrosylation is eNOS localized in the membranes,
implying that this modification could be one of the final steps in returning
the enzyme to its basal state after its activation and translocation. The role
of eNOS S-nitrosylation/-denitrosylation, its relationship with
phosphorylation of eNOS in the penis, and its relation to penile erection were
not investigated.
An Endogenous Inhibitor of eNOS: ADMA
Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor for NO
synthesis. It is a naturally occurring amino acid resulting from proteolysis
of methylated arginine in proteins
(Vallance and Leiper 2004).
The methylation of arginine is catalyzed by arginine methyltransferase type I.
ADMA is eliminated largely through its metabolism to citrulline by
dimethylarginine dimethylaminohydrolase (DDAH)-1 and -2, type 2 being
predominant in endothelial cells. Both synthesis and degradation of ADMA are
regulated in an active manner; thus, dysregulation of either of these pathways
could result in elevated levels of ADMA. For example, DDAH is inhibited by
reversible S-nitrosylation under conditions of increased NO
generation, resulting in accumulation of ADMA and inhibition of NOS signaling
(Leiper et al, 2002).
Increased ADMA levels have been implicated in the pathogenesis of a variety of
cardiovascular diseases (Leiper et al,
2007). However, the mechanisms by which ADMA exerts its effect
have not been completely elucidated. ADMA has been shown to compete with
L-arginine, the NOS substrate
(Vallance and Leiper, 2004),
and recently it has been shown to induce eNOS uncoupling
(Sud et al, 2008).
Elevated plasma levels of ADMA were found in many conditions associated with ED (Maas et al, 2002). However, limited studies evaluated the role of ADMA in penile erection. Induction of ischemia in the penis (Masuda et al, 2002), and administration of cigarette smoke extract (Imamura et al, 2007) to the rabbit penis up-regulated ADMA content and increased DDAH activity in correlation with impaired NOS activity and cavernosal relaxation. However, more studies are needed to determine the mechanism of regulation of ADMA levels, and the mechanisms of ADMA effects on penile eNOS activity.
Phosphorylation and Activation of nNOS Through Association With NMADR
Mechanisms of regulation for nNOS activity in erection remain poorly
understood and evidence for activation and posttranslational modification of
PnNOS is particularly lacking. nNOS activity has been studied in other
contexts, however, and coexpression and colocalization data suggest that
pathways of nNOS activation are likely conserved in the penis.
Control of nNOS enzyme activity at the postsynaptic nerve terminal is exerted by the binding of calcium to calmodulin. The calcium influx responsible for this and subsequent activation of nNOS is in turn regulated by the N-methyl D-aspartate receptor (NMDAR), which is coupled to nNOS by the binding protein postsynaptic density-95 (PSD95) through interaction with the NOS PDZ domain (Bredt and Snyder, 1989; Garthwaite et al, 1989; Kornau et al, 1997; Christopherson et al, 1999). The association of nNOS with PSD95 seems necessary for NOS activation by the NMDAR, and its disruption might be the mechanism of inhibition of the NOS inhibitory protein CAPON (Sattler et al, 1999; Aarts et al, 2002; Gonzalez-Cadavid and Rajfer, 2004).
In vitro experiments suggest that the transient activation of nNOS by the NMDAR involves phosphorylation of nNOS at activating and inhibitory Ser-1412 and Ser-847, respectively (Mount and Power, 2006). In a model put forth by Rameau et al (2007), activation of NMDAR leads to Akt-dependent phosphorylation of NOS at Ser-1412 (a site in the C-terminus of the molecule analogous to the Akt phosphorylation site of eNOS) and enzyme activation with increased nitric oxide and cGMP production (Rameau et al, 2007). Phosphorylation at Ser-1412 also serves to expose Ser-847 in the alpha helix autoinhibitory domain of nNOS, which is phosphorylated by calcium-calmodulin–dependent kinase II, resulting in enzyme deactivation (Hayashi et al, 1999; Komeima et al, 2000). As mentioned above, experiments by Magee et al (2003) have shown expression of NMDAR in penile nerves and its colocalization with PnNOS.
Inhibition of nNOS via PIN and Protein-Protein Interactions
Using the N-terminal domain of NOS in a yeast 2-hybrid system, Jaffery and
Snyder (1996) identified a 10-kd protein that interacted with and inhibited
nNOS, which they termed protein inhibitor of nitric oxide synthase (PIN;
Jaffrey and Snyder, 1996). In
vitro experiments suggest that the PIN might act to inhibit nNOS by preventing
its dimerization. In addition, because the PIN's structure is homologous to
the light chain of myosin and dynein, it has been proposed that the PIN could
be involved with nNOS's association with the neuronal cytoskeleton during
axonal transport (King et al,
1996; Gillardon et al,
1998; Jeong et al,
1998; Chang et al,
2000).
PIN interacts with the PDZ domain in the N terminus of nNOS and has been shown to be coexpressed and colocalized with PnNOS in the cavernosal and dorsal nerves of the penis as well as in the hypothalamic regions of the rat, particularly the paraventricular nucleus that controls penile erection (Fan et al 1998, Magee et al 2003). Interestingly, despite neuronal colocalization of PnNOS and PIN, no binding of recombinant PIN to nNOS or inhibition of NOS activity was observed in vitro using penile extracts (Magee et al, 2003). While these results may bring into question a role for nNOS inhibition by direct PIN binding, functional experiments help establish the significance of PIN in nNOS regulation (Magee et al, 2003). Specifically, overexpression of PIN leads to a reduced erectile response to electrical field stimulation, whereas reduction of PIN levels by treatment with anti-sense or short hairpin (sh)RNA PIN constructs allow for correction of age-related ED (Magee et al, 2007).
S-Nitrosylation of nNOS
As with eNOS, nNOS has been shown to undergo S-nitrosylation,
leading to its inhibition, and this has been proposed as a method of
autoregulation of nNOS by NO (Abu-Soud et
al, 1995; Hess et al,
2005). The role of this process in erectile function remains
undefined.
Summary
NO production from the constitutive forms of NOS is essential to normal
penile vascular physiology. Alterations in eNOS and nNOS via posttranslational
modification or protein-to-protein interaction occur in a number of disease
states that are associated with ED. Pharmacological or gene therapy strategies
can target these molecular events that are responsible for alterations in NO
production in the penis in order to improve cNOS function and thus restore
normal penile vascular homeostasis.
Footnotes
This paper is based on a presentation at a Special Symposium on April 12, 2008, "Therapeutic Strategies for Male Sexual and Hormonal Health," associated with the American Society of Andrology Annual Meeting, for which the presenting author received an honorarium.
Drs Musicki, Ross, Champion, and Bivalacqua have nothing to disclose. Dr Burnett has consulting and/or financial relationships with Pfizer, Eli Lilly and Co, and American Medical Systems, Inc.
Supported by NIH/NIDDK grants DK075782 and DK074826 to B.M. and the AUA Foundation Astellas USA Foundation MD/PhD Fellowship to T.J.B.
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