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International Journal of Impotence Research (2004) 16, 459–469 & 2004 Nature Publishing Group All rights reserved 0955-9930/04 $30.00 www.nature.com/ijir

Review –cyclic GMP pathway with some emphasis on cavernosal contractility

IF Ghalayini1*

1Faculty of Medicine, Jordan University of Science and Technology, Irbid, Jordan

Nitric oxide (NO) is formed from the conversion of L- by (NOS), which exists in three isoforms: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). nNOS is expressed in penile innervating the corpus cavernosum, and eNOS expression has been identified primarily in both cavernosal smooth muscle and . NO is released from nerve endings and endothelial cells and stimulates the activity of soluble (sGC), leading to an increase in cyclic -30,50-monophosphate (cGMP) and, finally, to depletion from the cytosolic space and cavernous smooth muscle relaxation. The effects of cGMP are mediated by cGMP dependent protein , cGMP-gated channels, and cGMP-regulated (PDE). Thus, cGMP effect depends on the expression of a -specific cGMP- receptor protein in a given cell type. Numerous systemic vasculature diseases that cause (ED) are highly associated with , which has been shown to contribute to decreased erectile function in men and a number of animal models of penile erection. Based on the increasing knowledge of intracellular signal propagation in cavernous smooth muscle tone regulation, selective PDE inhibitors have recently been introduced in the treatment of ED. 5 (PDE5) inactivates cGMP, which terminates NO-cGMP-mediated smooth muscle relaxation. Inhibition of PDE5 is expected to enhance penile erection by preventing cGMP degradation. Development of pharmacologic agents with this effect has closely paralleled the emerging science. International Journal of Impotence Research (2004) 16, 459–469. doi:10.1038/sj.ijir.3901256 Published online 1 July 2004

Keywords: nitric oxide; cGMP; soluble guanylate cyclase; phosphodiesterase; corpus cavernosum

Introduction NO and cGMP together comprise an especially wide-ranging system when one considers the many roles of cGMP in physiological Nitric oxide (NO) was first described by Stuehr and regulation, including smooth muscle relaxation, 1 Marletta (1985) as a product of activated murine visual transduction, intestinal ion transport, and macrophages. Also, the substance known as en- function.5 dothelium-derived relaxing factor (EDRF), described Erectile dysfunction (ED) is defined as the con- 2,3 by Furchgott and Zawadzki (1980), has been sistent inability to achieve or maintain an erection identified as NO. sufficient for satisfactory sexual performance, and is Soluble guanylate cyclase (sGC), responsible for considered to be a natural process of aging.6 Studies the enzymatic conversion of guanosine-5-tripho- have shown that ED is caused by inadequate sphate (GTP) to cyclic guanosine-30,50-monopho- relaxation of the corpus cavernosum with defect in sphate (cGMP), was first identified as a constituent NO production.7 4 of mammalian cells almost three decades ago. It is clear that NO is the predominant neurotrans- mitter responsible for cavernasal smooth muscle relaxation and hence penile erection. Its action is mediated through the generation of the second *Correspondence: IF Ghalayini, MD, FRCS, PO Box messenger cGMP. Neurally derived NO has been 940165, Amman 11194, Jordan. established as a mediator of smooth muscle relaxa- E-mail: [email protected] tion in the penis, and it is thought that constitutive Received 25 October 2003; revised 25 May 2004; accepted forms of nitric oxide synthase (NOS) work to 3 June 2004 mediate the erection.8 Released NO activates sGC, Nitric oxide–cyclic GMP pathway IF Ghalayini 460 which catalyzes the conversion of GTP to the and release of NO by these cells.14 Bivalacqua et al intracellular second messenger cGMP in smooth (2001)15 found in their study that in vivo adenoviral muscle cells. An increase in cGMP modulates transfer of CGRP can physiologically improve cellular events, such as relaxation of smooth muscle erectile function in the aged rat, while others cells.9 reported that intracavernosal injections of CGRP in This review will describe current knowledge of combination with adrenomedullin (ADM) or pros- cellular events involved in cavernosal relaxation taglandin E1 (PGE1) induce penile erection by and the range of putative factors involved in NO- activating different receptors.16,17 mediated relaxation. The combination of molecular and the arginine in the presence of reduced adenine dinucleotide Synthesis of NO (NADPH) and NO synthase (NOS) yields and NO, through a 5-electron oxidation of the 18 guanidine of L-arginine (Figure 1). Recent observations suggest that the main site of NO L-citrulline can be converted by arginine synthase biosynthesis in human corpus cavernosum is within (AS) to form L-arginine, the precursor for NO. Each the terminal branches of cavernosal nerves supply- of these , cofactors, or transport systems ing the erectile tissue. It is strongly suggested that could be an eventual target of pharmacologic NO released from nonadrenergic–noncholinergic intervention in the NO cascade. (NANC) neurons increases the production of cGMP, Oral administration of L-arginine in high doses which in turn relaxes the cavernous smooth mus- seems to cause significant subjective improvement cle.10,11 Endothelial-derived NO plays a major role in sexual function in men with organic ED only if in the sustained erectile response and is a major they have decreased production of plasma and urine source of NO in the penis.8 Some suggest that NO and , which are stable metabolites of is highly labile, therefore, it cannot be stored as a NO.19 preformed .12 Alternatively, an- There are at least three isoforms of NOS (neuronal, other neurotransmitter such as vasoactive intestinal endothelial, and macrophage)18,20 (Table 1). A polypeptide (VIP) may interact with either endothe- constitutive form of NOS is found in endothelium lial or smooth muscle cells in the corpus caverno- and neurons, and is calcium dependent.21 The sum to trigger the local formation of NO.13 Other constitutive NOS found in endothelial and smooth proerectile mediators, such as , calci- muscle cells has been named NOS-3, whereas the tonin gene-related peptide (CGRP) or substance P, constitutive NOS found in neural and epithelial act via endothelial cells by promoting the synthesis tissue has been named NOS-1. The latter has also

Figure 1 NO generation from L-arginine and NO donors and the formation of cGMP. L-citrulline can be converted by arginine synthase(AS) to form L-arginine. Nitric oxide synthase (NOS), in the presence of O2 and the cofactors converts arginine to NO, with the formation of citrulline. Cofactors include reduced nicotinamide adenine dinucleotide phosphate (NADPH), (BH4), (FMN), and flavin adenine dinucleotide (FAD). AS, NOS, , and L-arginine availability are all possible sites of pharmacologic intervention in this pathway.

International Journal of Impotence Research Nitric oxide–cyclic GMP pathway IF Ghalayini 461 Table 1 Properties of the three isoforms of nitric oxide synthase (NOS)

Property Isoform I Isoform II Isoform III

Name bNOS, cNOS, nNOS iNOS, mNOS eNOS Tissue Neuronal, epithelial, skeletal, Macrophages, smooth muscle Endothelial, smooth muscle cells cells cells Expression Constitutive Transcriptional induction Constitutive Calcium requirement Yes No Yes 12 17 7 Approximate mass of protein 150–160 kDa 125–135 kDa 133 kDa bNOS ¼ brain NOS, cNOS ¼ constitutive or Ca þ regulated NOS, nNOS ¼ neuronal NOS, iNOS ¼ inducible NOS, mNOS ¼ macrophage NOS, eNOS ¼ endothelial NOS.

been found in a number of cell types including skeletal and cardiac muscle.22 An inducible form of NOS, now designated iNOS, is calcium indepen- dent.23 It is induced within 4–24 h of the appro- priate stimulus and can produce NO in a 100-fold greater amount than can constitutive NOS. Neuronal NOS has multiple regulator sites, in- cluding binding sites for nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin monoucleotide Figure 2 Modular structure of neuronal nitric oxide synthase (FMN). All of these are cofactors for the synthesis showing approximate locations of prosthetic groups and co- of NO.24 These cofactors bind to a domain factors. to process electron transfer. This is then linked to a and tetrahydrobiopterin (BH4)-containing catalytic domain by calcium– complex25 (Figure 2). The complete con- centrally from the brain, or peripherally by means verts L-arginine to L-citrulline and NO in the of the dorsal nerve spinal reflex, is assumed to be presence of molecular oxygen. In addition to the exerted through the activation of PnNOS activity.30 various protein modules or domains of neuronal This mechanism occurs mainly by Ca2 þ binding to NOS, which are involved in electron transfer, calmodulin by means of a Ca2 þ flux through the N- substrate binding, oxygen activation, and calcium methyl-D-aspartate receptor (NMDAR). Both the binding, a four-amino-acid motif (–leucine– NMDAR and inhibitors of nNOS activity, such as glycine–, GLGF) has been identified protein inhibitor of NOS (PIN)31–33 and carboxy- in amino terminal region of NOS-1. Although the terminal PDZ of nNOS (CAPON),34 also bind function of this amino-acid motif in NOS-1 has not to nNOS. PIN, NMDAR subunits, and a truncated been established, studies on other contain- variant of the NMDAR subunit 1mRNA (NMDAR1- ing this motif indicates that it may serve to target T) have been located in the peripheral penile nerves proteins to specific sites in the cell.25 nNOS has a and pelvic ganglion.27 recognition site for calmodulin that is also present The nitrergic activation of penile erection is not in eNOS and macrophage NOS. The constitutive restricted to peripheral nerves of the corpora isoforms are generally regulated by Ca2 þ –calmodu- cavernosa but is also dependent on central nervous lin, whereas inducible forms are not.23 system control. Copulatory behavior and ejaculation nNOS in the penis is expressed primarily as a are also central (CNS) regulated.35 variant of the brain form of nNOS and has been Erectile stimuli originate from the medial preoptic termed PnNOS. It has an additional 102-bp alter- area and paraventricular nucleus of the hypothala- native exon located between exons 16 and 17. The mus through the L6–S1 lumbosacral level of the function of this additional coding region is un- spinal cord containing the sacral parasympathetic known. PnNOS is thought to be responsible for nucleus (SPN).36 Pudendal nerve motor neurons triggering the nitrergic mechanism responsible for innervating the perineal striated muscles that cavernosal relaxation.26,27 A similar variant, nNO- induce maximum rigidity at the time of ejaculation Su, is present in the neuromuscular plates of are located in Onuf’s nucleus of the ventral sacral skeletal muscles,28 including the perineal muscles spinal cord and the dorsomedial and the dorsolat- involved in erectile rigidity and ejaculation in rats.29 eral nuclei of the lower lumbosacral spinal cord.29 The control of NO synthesis in the cavernosal nerve, Electrical stimulation of the medial preoptic area or whether due to sexual stimulation emanating microinjection of oxytocin, glutamate, or apomor-

International Journal of Impotence Research Nitric oxide–cyclic GMP pathway IF Ghalayini 462 phine into the medial preoptic area and paraven- corpora cavernosa of diabetic ED patients compared tricular nucleus induces noncontact erections37 and with their nondiabetic counterparts.49 increased intracavernosal pressure,38 by means of an The activity of nNOS is controlled by a number NO-mediated process within the CNS.39 Nitrergic of mechanisms. A balance of various inhibitory and and oxytocinergic neurons have been located in the stimulatory transcription factors determines gene SPN and in Onuf’s nucleus.40 Retrograde tracing transcription of the enzyme. Enzyme activity can be from the corpora cavernosa has identified neurons halted by by a cyclic in the spinal SPN, in parvocellular region of the monophosphate (cAMP)-dependent protein paraventricular nucleus, and medial preoptic area.35 (PKA) or cGMP-dependent protein kinase (PKG), It was found that PnNOS, the brain-type nNOS, providing a negative feedback loop.50 The enzyme is and PIN, were expressed in the hypothalamus.41 In activated by increased intracellular calcium, which contrast, NMDAR1-T was expressed only in the binds to calmodulin to form the essential cofactor. penis, whereas the brain-type NMDAR1 was present It is also likely that co-transmitters influence in the brain and sacral spinal cord and not in the nNOS activity, perhaps by altering calcium concen- penis. PnNOS was found in the media preoptic area, tration by activation of prejunctional receptors. VIP posterior magnocellular, and the parvocellular re- is a probable stimulatory co-transmitter, while gions of the paraventricular nucleus, supraoptic noradrenaline acting on a-2 adrenoceptors inhibits nucleus, septohypothalamic nucleus, medial sep- NO formation.13 tum, cortex, and in some of the nNOS staining neurons throughout the brain.41 It was absent in the organum vasculosum of the lamina terminalis. PIN Inactivation staining was present in neurons of the medial preoptic area, paraventricular nucleus, medial sep- tum, and cortex, but not in the supraoptic nucleus, NO is inactivated by heme and the free , septohypothalamic nucleus, or organum vasculo- . Thus, scavengers of superoxide anion sum of the lamina terminalis.41 such as (SOD) may protect Inhibitors of NOS are substrate analogues of NO, enhancing its potency and prolonging its L-arginine, such as N-monomethyl-L-arginine duration of action. Conversely, interaction of NO (L-NMMA), N-nitro-L-arginine methyl with superoxide may generate the potent tissue- 42,43 À (L-NAME), and N-amino-L-arginine. damaging moiety, (ONOO ), which Among the NOS inactivating L-arginine deriva- has a high affinity for sulfhydryl groups and thus 5 tives, vinyl-L-NIO (N -(1-imino-3-butenyl)-L- inactivates several key sulfhydryl-bearing-enzymes. ; L-VNIO) is a potent, mechanism-based This effect of peroxynitrite is regulated by the inhibitor that attacks the heme cofactor of NOS with cellular content of . Since glutathione is a marked selectivity for nNOS.44,45 The use of the the major intracellular soluble sulfhydryl-contain- selective nNOS inhibitor, 7-nitroindazole (7-NI), has ing compound, factors that regulate the biosynthesis shown that inhibition of this isoform reduces the and decomposition of glutathions may have impor- erectile response and that if higher doses are used tant consequences.18 (which inhibit eNOS as well) the whole erectile Glutathione also interacts with NO under physio- response to cavernous nerve stimulation is re- logic conditions to generate S-nitrosoglutathione, a duced.46 more stable form of NO. Nitrosoglutathione may that inhibit the dephosphorylation of eNOS serve as an endogenous long-lived adduct or carrier might alleviate ED. eNOS abnormalities may play a of NO. Vascular glutathione is decreased in diabetes role in diabetic ED. Hyperglycemia decreases NO mellitus and atherosclerosis, and this may account production by eNOS via O-linked glycosylation of for the increased incidence of cardiovascular com- eNOS at the Akt target S1177 in hyperglycemic cell plications and ED in these conditions.18,51 culture conditions and in animal models of dia- Khan et al (2001) found that NO—and electrical betes.47 ED in diabetes is associated with peripheral field-stimulated (EFS)—mediated cavernosal nerve damage but may involve diminished endothe- smooth muscle relaxation is impaired in a rabbit lial production of NO as well.48 Numerous systemic model of diabetes but SOD significantly reversed the vasculature diseases (hypertension, atherosclerosis, impaired relaxation. Therefore, in diabetes, the hypercholesterolemia, diabetes mellitus, etc) that generation of reactive oxygen species may play an cause ED are highly associated with endothelial important role in the development of ED.52 dysfunction, which has been shown to contribute to Production of the superoxide radicals in rabbit decreased erectile function in men and a number of cavernous tissues increases during the state of animal models of penile erection.8 Deposition of hypercholesterolemia, which may lead to functional advanced glycation end products (eg pentosidine, impairment of cavernous smooth muscle relaxation pyrraline), which can quench NO both in animal in response to endothelium-mediated stimuli.53 models and cell cultures, was associated directly ED associated with aging is related in part to an and indirectly with declines in eNOS activity in the increase in cavernosal superoxide anion formation.

International Journal of Impotence Research Nitric oxide–cyclic GMP pathway IF Ghalayini 463 Gene-transfer of extracellular (EC)-superoxide dis- mutase (EC-SOD) may reduce superoxide formation and restores age-associated erectile function, and may represent a novel therapeutic target for the treatment of ED.54 Manipulation of physiological NO concentration is unlikely to give physiological benefit in ED, since higher levels will predispose to toxic effects. NO availability may be increased by the use of the enzyme superoxide dismutase (SOD), which causes decreased levels of superoxide anion.

The NO receptor: soluble guanylate cyclase

Soluble GC is a heme-containing protein found in the cytosolic fraction of virtualy all mammalian cells, with the highest concentrations found in the lung and brain.4 Several isoforms of sGC have been cloned and characterized. Originally, sGC was purified (to apparent homogeneity) from bovine and rat lung and shown to exist as a heterodimer, consisting of 82 kDa (rat) or 73 kDa (bovine) and 70 kDa subunits, termed a1 and b1 respectively. Further subunits termed a2 and b2 have also been identified from the human foetal brain (82 kDa) and rat kidney (76 kDa), respectively. Recently, GUCIA2, the gene coding for the a2-subunit, has been localized to position q21–q22 on the human chro- mosome 11.55 Subunits isolated from the adult human brain, termed a3 and b3 (81 kDa and 70 kDa, respectively), may represent additional isoforms; Figure 3 Structure of soluble guanylate cyclase heterodimer. however, their close homology to the bovine a1 and The N-terminal constitutes a heme-binding domain with His105 b1 subunits suggests they may simply be species providing the axial ligand to the fifth coordinate of the heme-iron. variants of existing subtypes.56 Reverse transcrip- The central portion mediates dimerization of the monomers, tase-polymerase chain reaction (RT-PCR) has shown a prerequisite for catalytic activity. The C-terminal region forms the catalytic domains for conversion of GTP to cGMP. the existence of mRNA coding for both a3 and b3 subunits in vascular smooth muscle and endothelial cells in culture and in freshly isolated human vascular tissue.57 between NO and (ferrous) heme. On the other hand, Soluble GC is a heterodimer with at least three oxidizing agents such as methylene blue inhibit functional domains for each subunit (Figure 3). enzyme activation ( may also facilitate enzyme These domains are a heme-binding domain, dimer- activation by forming S-nitrosothiols with NO ization domain, and catalytic domain. The N- released from drugs).60 terminal portion of each subunit constitutes a The heme moiety is bound to the enzyme protein heme-binding domain and represents the least via an axial ligand, shown by point conserved region of the protein; it is the heme to be provided by His 105 in the b1- moiety that confers the NO-sensitivity of the subunit.61 At the C-terminus of each subunit is a enzyme.58 Heme-reconstituted sGC can be activated catalytic domain that exhibits a high degree of nearly 100-fold by NO.58 Soluble GC may contain 1 homology, both between sGC monomers and the mole of heme bound per monomer, depending on C-terminal regions of particulate GC and AC (ade- the purification protocol. The latter determination nylate cyclase).62 Intervening between the heme- appears to be considerably more NO sensitive than binding and catalytic regions is a dimerization an equivalent protein containing 1 mole heme per domain that is thought to mediate the subunit dimmer.59 association to form heterodimers, which is obliga- Oxidation of the heme group to a ferric state tory for catalytic activity. results in loss of the enzyme activity; thus, reducing Binding of NO to the heme-iron of sGC results in agents such as thiols or ascorbate enhance enzyme the formation of a pentacoordinate nitrosyl–heme activation, and thereby facilitating the reaction complex, which breaks the bond to the axial

International Journal of Impotence Research Nitric oxide–cyclic GMP pathway IF Ghalayini 464 and activates the enzyme.4,63 NO is the cGMP-regulated cyclic phosphodies- only nitrogen monoxide capable of stimulating sGC terases (PDEs). This means that cGMP can alter cell activity.64 Cofactors, including Mn2 þ and Mg2 þ , are function through protein phosphorylation or required for the catalytic conversion of GTP to through mechanisms not directly related to protein cGMP by sGC. The Mg2 þ is thought to act as the phosphorylation. most physiologically important cofactor. In the Two general classes of cGMP kinases exist in presence of Mg2 þ , stimulation of the enzyme by vertebrate cells: a type I and a type II form. The type NO results in a marked increase in Vmax (4200-fold) I cGMP kinase is more abundant and widely and a decrease in Km for the substrate GTP from distributed and has been isolated from vascular B100 to B50 mM.65 and other tissues while the type II form has been In addition to iron, sGC possesses a second metal detected in vertebrate intestinal epithelial cells.75 ion, copper, which is also thought to function as a Cyclic GMP kinases are found in a number of cofactor for enzyme activity.66 Free copper different cells but are most abundant in three cell inhibit purified sGC activity by reducing Vmax, types in vertebrates: smooth muscle, platelet, and although the potency of NO-stimulation is unaf- cerebellum.76 Appreciable amounts are also found fected. in cardiac muscle, and lesser amounts are found in Activation of sGC can be achieved satisfactorily leukocytes,77,78 , hepatocytes, vascu- with NO donors, such as glyceryl trinitrate, nitro- lar endothelial cells, and renal tubular epithe- prusside, or S-nitrosothiols. Agents like methylene lium.76,79 blue and LY83583 (6-anilinoqinoline-5,8-quinone) It is currently thought that at least one mechanism can be utilized for inhibition of the enzyme.4 Both of cGMP-induced relaxation is the reduction of compounds have been shown to release superoxide intracellular-free Ca2 þ levels ([Ca2 þ ]i), since in aqueous solution and a significant component of [Ca2 þ ]i is the signal for activation of light- their activity may therefore be via inactivation of chain kinase (MLCK) and contraction in smooth NO. Further, such compounds can directly inhibit muscle. This enzyme phosphorylates myosin reg- NOS activity.67 N-methyl hydroxylamine may re- ulatory light chains (MRLC), which then form cross present a rather more selective sGC inhibitor.68 bridges with thin filaments and so generate ODQ {1H-[1,2,4] oxadiazolol [4,3-a] quinoxaline- force. Cross bridges are dephosphorylated by myo- 1-one} has been demonstrated to block NO-depen- sin light-chain phosphatase (MLCP) resulting in dent smooth muscle relaxation in the respiratory relaxation (Figure 4).50,80 Calcium channel blockers and urogenital tracts69 and vasculature and inhibit predictably cause because they reduce the NO-mediated reduction in platelet reactivity.70 intracellular Ca2 þ . Substances that increase cAMP The mechanism of inhibition is thought to occur via like b2 agonist, may cause relaxation in smooth modulation of the heme-iron.70 muscle by accelerating the inactivation of MLCK YC-1 [3-(50-hydroxymethyl-20-furyl)-1-benzylinda- and facilitating the expulsion of calcium from the zole] has provided a non-NO-based activator of sGC cell.81 Theoretically, vasodilators should not cause that is effective in a biological milieu.71,72 YC-1 can ED; however, patients with severe atherosclerosis evoke erectile responses when given intracaver- may require higher blood pressure to deliver nously and it enhances erections induced by sufficient flow to the penis, and the lowering of cavernous nerve stimulation and apomorphine blood pressure by these agents may result in partial when given systemically.73 Activation of sGC by erection. YC-1 may provide an alternative means for enhan- The calcium-sensitizing Rho-A/Rho-kinase path- cing the activity of neurally derived NO during way may play a synergistic role in cavernosal sexual stimulation in the corpus cavernosum, vasoconstriction to maintain penile flaccidity. Rho- representing a novel approach for the treatment of kinase is known to inhibit MLCP, and to directly ED.73,74 phosphorylate myosin light-chain (in solution), Due to the ubiquitous nature of the NO–sGC– altogether resulting in a net increase in activated cGMP pathway, signal transduction by sGC also has myosin and the promotion of cellular contraction. profound pathophysiological significance. For ex- Although Rho-kinase protein and mRNA have been ample, and migraine may be due to detected in cavernosal tissue, the role of Rho-kinase overactivity of the pathway, and impotence, hyper- in the regulation of cavernosal tone is unknown.82 tension, and asthma as a result of underactivity. Chitaley et al (2001)82 found that Rho-kinase antag- onism stimulates rat penile erection independently of NO. Mills et al (2002)83 in their study support the Intracellular cyclic GMP receptor proteins hypothesis that NO inhibits Rho-kinase-induced cavernosal vasoconstriction during erection. These initial findings introduce a novel potential therapeu- Cyclic GMP interacts with three types of intracel- tic approach for the treatment of ED.84 lular receptor proteins: cGMP-dependent protein The mechanism by which cGMP kinase acts is kinases (PKGs), cGMP-regulated ion channels, and still not understood. Findings from several labora-

International Journal of Impotence Research Nitric oxide–cyclic GMP pathway IF Ghalayini 465 enhancement of blood flow that can last for hours.90 Findings of Hurt et al8 support a model in which rapid, brief activation of neuronal NOS initiates the erectile process, whereas PI3-kinase/Akt-dependent phosphorylation and activation of eNOS leads to sustained NO production and maximal erection. Both electrical stimulation of the cavernous nerve and direct intracavernosal injection of the vaso- relaxant cause rapid increases in phosphorylated (activated) Akt and eNOS. Phos- phorylation is diminished by wortmannin and LY294002, inhibitors of PI3-kinase, the upstream activator of Akt. The two drugs also reduce erection. Penile erection elicited by papaverine is reduced profoundly in mice with targeted deletion of eNOS.8 Drugs that inhibit the dephosphorylation of eNOS might alleviate ED. In diabetes, ED is associated with peripheral nerve damage but may involve diminished endothelial production of NO as well.47,48 Figure 4 Control of smooth muscle contraction and site of action Other mechanisms for the lowering of [Ca2 þ ]i by of calcium channel-blocking drugs. Contraction is triggered by cGMP kinase include activation of Ca2 þ -ATPase by influx of calcium through transmembrane calcium channels. The the stimulation of phosphatidylinositol-4-phosphate calcium combines with calmodulin to form a complex that 77 converts myosin light-chain kinase to its active form (MLCK*). (PIP) formation by cGMP kinase and phosphoryla- The latter phosphorylates the myosin light chains, thereby tion of 240-kDa protein that mediates the activation initiating the interaction of myosin with actin. Activation of of Ca2 þ -ATPase by cGMP kinase.78 PKG may RhoA leads to the activation of Rho-kinase (ROK), which in turn catalyze the phosphorylation of phosphatidylino- phosphorylates the regulatory myosin-binding subunit of myosin phosphatase (MLCP), which results in the inhibition of the sitol kinase, leading to the formation of PIP and the 2 þ enzyme. b2 agonists (and other substances that increase cAMP) activation of Ca -ATPase by the lipid. The role of may cause relaxation in smooth muscle by accelerating the the 240-kDa protein is unknown. It is possible that inactivation of MLCK and facilitating the expulsion of calcium this protein is a component of the cytoskeleton that from the cell. MLC (myosin light chain). is involved in the recruitment of additional Ca2 þ - ATPase molecules from internal stores to the plasma membrane. Other mechanisms have been proposed tories have indicated that one effect of cGMP kinase for the effects of cGMP, and these include inhibition is stimulation of a Ca2 þ -pumping ATPase, an action of G protein function and inhibition of phospholi- that would be predicted to lower [Ca2 þ ]i in smooth pase C (PLC) activation.50 These effects could muscle cells activated with contractile agonists or contribute to [Ca2 þ ]i reduction in agonist-stimu- by depolarization.85 The generation of PKGs by lated smooth muscle, but they would have little cGMP leads to a number of events that decrease effect on depolarized cells or tissues because PLC [Ca2 þ ]i. It has been shown to phosphorylate and activation is not involved in the mobilization of 2 þ 81 therefore inhibit the 1,4,5-triphophate (IP3) [Ca ]i in this instance. There is evidence that receptor on the sarcoplasmic reticulum, thus pre- increases in cGMP also lower the Ca2 þ -sensitivity of venting calcium release from the store. In addition, cross bridge phosphorylation. This would contri- PKG increases activity of plasma and sarcolemmal bute to a decline in force through actions on the (mediated via the regulatory protein, phospholam- myosin light-chain kinase/phosphatase system.77 In ban) cation-ATPase pumps encouraging sequestra- addition, increased cGMP-dependent protein ki- tion of calcium into stores and out of the cell.86,87 nases decrease activity of plasma membrane calcium nNOS and eNOS are activated by calcium entry channels by phosphorylation (L-type channels) and into the cell, binding to calmodulin associated with by membrane hyperpolarization secondary to potas- the enzymes.88 Whereas physiologic penile erection sium (K þ ) channel activation (voltage-gated chan- lasts several minutes, the calcium-dependent acti- nels). There may also be a feedback loop within vation of nNOS or eNOS is quite transient. Recently, smooth muscle cells whereby a rise in [Ca2 þ ]i several groups showed that the phosphatidylinositol stimulates endogenous NO production, which will 3-kinase (PI3-kinase) pathway that activates the then lower [Ca2 þ ]i through the generation of / protein kinase Akt (also known as cGMP.50,91 PKB) causes direct phosphorylation of eNOS, redu- Vasodilatation and relaxation of cavernosal cing the enzyme’s calcium requirement and causing smooth muscle cells (SMC) engorges the corpora increased production of NO.89 This pathway is cavernosa with blood at arterial pressure. The responsible for both shear stress and growth-factor subcellular mechanism by which tumescence

International Journal of Impotence Research Nitric oxide–cyclic GMP pathway IF Ghalayini 466 occurs involves NO-induced activation of sGC, that inhibit PDE5 can enhance and prolong the increased cGMP levels, and activation of PKG. This smooth muscle relaxant effects of the NO–cGMP phosphorylates numerous ion channels and pumps, cascade in the corpus cavernosum, thereby poten- each promoting a reduction in cytosolic calcium. In tiating penile erection.93 The prototype of this new particular, PKG activates high-conductance Ca2 þ - therapeutic class of PDE5 inhibitors is , þ sensitive K (BKCa) channels, which hyperpolarize which was approved for treatment of ED in 1998. the arterial and cavernosal SMC membranes, caus- and are new agents in this ing relaxation. This mechanism appears to be class.93 compromised with age and vascular disease, leading Sildenafil is more selective for PDE599 than for to ED. Thus, increasing cavernosal NOS expression, other PDEs: 480-fold more than for PDE1; 41000- cGMP levels and/or BKCa channel expression is an fold more than for PDE2 to PDE4; and about 10-fold effective therapy for experimental ED. Future thera- more than for PDE6, an enzyme found in the pies may involve augmenting K þ channel expres- retina.99 The lower selectivity of sildenafil for sion by gene transfer or increasing channel function PDE5 over photoreceptor PDE6 may account for through the use of Type 5 phosphodiesterase (PDE5) the color visual disturbances observed with increas- inhibitors or phosphatase inhibitors.92 ing frequency with larger doses or higher plasma Regulation of phosphodiesterase (PDE) activity is levels of sildenafil.99 A potent, selective, reversible an important component of control of cGMP PDE5 inhibitor, tadalafil is under regulatory review concentration and hence activity of the NO–cGMP in Europe and North America as an oral therapy for pathway. Mammalian PDEs comprise 11 identified mild-to-severe ED.100 Tadalafil is highly selective for 101 families (PDE1–PDE11) and their isoforms, which PDE5 (IC50 ¼ 0.94 nmol/l) over other . are distinguished by their substrate specificities and In vitro studies with tadalafil have demonstrated a tissue concentrations.93 410 000-fold greater selectivity for PDE5 versus To date, five of these 11 isoenzymes (PDE1, 2, 3, 4, PDE1 to PDE4 and PDE7 to PDE10, as well as and 5) have been proven to be of pharmacological approximately 700-fold greater selectivity for PDE5 relevance. Currently, the presence of mRNAs spe- than for PDE6.93 Vardenafil is also selective for PDE5 cific for 14 different human phosphodiesterase in vitro and more selective for PDE5 than for PDE1 to isoforms in human cavernous tissue was shown by PDE4. It is 415-fold more selective for PDE5 than means of RT-PCR and Nothern blot analysis.94 The for PDE6.102 Vardenafil was described to be more expression of the following were detected in potent and selective than sildenafil on its inhibitory human cavernous tissue: PDE1A, PDE1B, PDE1C, activity on PDE5.102 PDE5 inhibitors augment the PDE2A, and PDE10A, which hydrolyze both cAMP response of exogenously applied nitrates, resulting and cGMP; the cAMP-specific PDEs PDE3A, PDE4A- in profound . For this reason, it is D, PDE7A and PDE8A, and the cGMP-specific PDEs contraindicated in patients using or PDE5A and PDE9A. The molecular identification of other -based , which are NO PDE isoenzymes was paralleled by efforts to detect donors.103,104 and characterize the hydrolizing activities of PDE It appears that no single mechanism explains all proteins expressed in human penile erectile tissue. the effects of cGMP on relaxation in the variety of In the early 1990s, Stief and co-workers reported the systems examined. The advantage for intracellular separation of hydrolytic activities of PDE isoen- signaling is that elevation in cGMP and activation of zymes 3, 4, and 5 from cytosolic supernatants PKG promote rapid and efficient phosphorylation of prepared from human cavernous smooth muscle,95 substrates in response to signals such as NO. whereas others reported the presence of PDEs 2, 3, and 5.96 Based on the results of organ bath studies on the effects of various PDE inhibitors (papaverine, Conclusions , , , and ) on the adrenergic tension of isolated human corpous cavernosum, Stief and co-workers94 concluded that The NO–sGC–cGMP pathway plays a crucial role in cavernous smooth muscle tone is mainly regulated the initiation and maintenance of cavernosal relaxa- by cAMP and that cGMP-inhibited PDE3 is of major tion. It would appear to achieve this predominantly importance in the control of cAMP turnover, while through the actions of PKGs. Cyclic GMP can alter others postulated that cGMP-specific PDE5 is the cell function through protein phosphorylation or predominant isoenzyme in the degradation of cyclic through mechanisms not directly related to protein nucleotide monophosphate (cNMP) in the corpus phosphorylation. It is also becoming clear that the cavernosum. Nevertheless, both conclusions are localization of these cGMP receptor proteins in the supported by the efficacy of intracavernous milri- cell is an important factor in the regulation of cell none and orally administered sildenafil to induce function by cGMP. Soluble GC plays an important penile erection sufficient for sexual intercourse.97 role in the transduction of inter- and intracellular PDE5 is the predominant degrading cGMP signals conveyed by NO. To fulfill this role, sGC has in the corpus cavernosum.94,98 Accordingly, drugs evolved a unique heme-coordination, which custo-

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