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Nitric oxide–cyclic GMP pathway with some emphasis on cavernosal contractility

Abstract

Nitric oxide (NO) is formed from the conversion of L-arginine by nitric oxide synthase (NOS), which exists in three isoforms: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). nNOS is expressed in penile neurons innervating the corpus cavernosum, and eNOS protein expression has been identified primarily in both cavernosal smooth muscle and endothelium. NO is released from nerve endings and endothelial cells and stimulates the activity of soluble guanylate cyclase (sGC), leading to an increase in cyclic guanosine-3′,5′-monophosphate (cGMP) and, finally, to calcium depletion from the cytosolic space and cavernous smooth muscle relaxation. The effects of cGMP are mediated by cGMP dependent protein kinases, cGMP-gated ion channels, and cGMP-regulated phosphodiesterases (PDE). Thus, cGMP effect depends on the expression of a cell-specific cGMP-receptor protein in a given cell type. Numerous systemic vasculature diseases that cause erectile dysfunction (ED) are highly associated with endothelial dysfunction, 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. Phosphodiesterase 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.

Introduction

Nitric oxide (NO) was first described by Stuehr and Marletta (1985)1 as a product of activated murine macrophages. Also, the substance known as endothelium-derived relaxing factor (EDRF), described by Furchgott and Zawadzki (1980),2, 3 has been identified as NO.

Soluble guanylate cyclase (sGC), responsible for the enzymatic conversion of guanosine-5-triphosphate (GTP) to cyclic guanosine-3′,5′-monophosphate (cGMP), was first identified as a constituent of mammalian cells almost three decades ago.4

NO and cGMP together comprise an especially wide-ranging signal transduction system when one considers the many roles of cGMP in physiological regulation, including smooth muscle relaxation, visual transduction, intestinal ion transport, and platelet function.5

Erectile dysfunction (ED) is defined as the consistent inability to achieve or maintain an erection sufficient for satisfactory sexual performance, and is considered to be a natural process of aging.6 Studies have shown that ED is caused by inadequate relaxation of the corpus cavernosum with defect in NO production.7

It is clear that NO is the predominant neurotransmitter responsible for cavernasal smooth muscle relaxation and hence penile erection. Its action is mediated through the generation of the second messenger cGMP. Neurally derived NO has been established as a mediator of smooth muscle relaxation in the penis, and it is thought that constitutive forms of nitric oxide synthase (NOS) work to mediate the erection.8 Released NO activates sGC, which catalyzes the conversion of GTP to the intracellular second messenger cGMP in smooth muscle cells. An increase in cGMP modulates cellular events, such as relaxation of smooth muscle cells.9

This review will describe current knowledge of cellular events involved in cavernosal relaxation and the range of putative factors involved in NO-mediated relaxation.

Synthesis of NO

Recent observations suggest that the main site of NO biosynthesis in human corpus cavernosum is within the terminal branches of cavernosal nerves supplying the erectile tissue. It is strongly suggested that NO released from nonadrenergic–noncholinergic (NANC) neurons increases the production of cGMP, which in turn relaxes the cavernous smooth muscle.10, 11 Endothelial-derived NO plays a major role in the sustained erectile response and is a major source of NO in the penis.8 Some suggest that NO is highly labile, therefore, it cannot be stored as a preformed neurotransmitter.12 Alternatively, another neurotransmitter such as vasoactive intestinal polypeptide (VIP) may interact with either endothelial or smooth muscle cells in the corpus cavernosum to trigger the local formation of NO.13 Other proerectile mediators, such as acetylcholine, calcitonin gene-related peptide (CGRP) or substance P, act via endothelial cells by promoting the synthesis and release of NO by these cells.14 Bivalacqua et al (2001)15 found in their study that in vivo adenoviral gene transfer of CGRP can physiologically improve erectile function in the aged rat, while others reported that intracavernosal injections of CGRP in combination with adrenomedullin (ADM) or prostaglandin E1 (PGE1) induce penile erection by activating different receptors.16, 17

The combination of molecular oxygen and the amino acid arginine in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) and NO synthase (NOS) yields citrulline and NO, through a 5-electron oxidation of the guanidine nitrogen of L-arginine (Figure 1).18 L-citrulline can be converted by arginine synthase (AS) to form L-arginine, the precursor for NO. Each of these enzymes, cofactors, or transport systems could be an eventual target of pharmacologic intervention in the NO cascade.

Figure 1
figure1

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), tetrahydrobiopterin (BH4), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). AS, NOS, cofactor, and L-arginine availability are all possible sites of pharmacologic intervention in this pathway.

Oral administration of L-arginine in high doses seems to cause significant subjective improvement in sexual function in men with organic ED only if they have decreased production of plasma and urine nitrite and nitrates, which are stable metabolites of NO.19

There are at least three isoforms of NOS (neuronal, endothelial, and macrophage)18, 20 (Table 1). A constitutive form of NOS is found in endothelium and neurons, and is calcium dependent.21 The constitutive NOS found in endothelial and smooth muscle cells has been named NOS-3, whereas the constitutive NOS found in neural and epithelial tissue has been named NOS-1. The latter has also been found in a number of cell types including skeletal and cardiac muscle.22 An inducible form of NOS, now designated iNOS, is calcium independent.23 It is induced within 4–24 h of the appropriate stimulus and can produce NO in a 100-fold greater amount than can constitutive NOS.

Table 1 Properties of the three isoforms of nitric oxide synthase (NOS)

Neuronal NOS has multiple regulator sites, including binding sites for nicotinamide adenine dinucleotide phosphate (NADPH), flavin adenine dinucleotide (FAD), and flavin monoucleotide (FMN). All of these are cofactors for the synthesis of NO.24 These cofactors bind to a reductase domain to process electron transfer. This is then linked to a heme and tetrahydrobiopterin (BH4)-containing catalytic oxygenase domain by calcium–calmodulin complex25 (Figure 2). The complete enzyme converts L-arginine to L-citrulline and NO in the presence of molecular oxygen. In addition to the various protein modules or domains of neuronal NOS, which are involved in electron transfer, substrate binding, oxygen activation, and calcium binding, a four-amino-acid motif (glycine–leucine–glycine–phenylalanine, GLGF) has been identified in amino terminal region of NOS-1. Although the function of this amino-acid motif in NOS-1 has not been established, studies on other proteins containing this motif indicates that it may serve to target proteins to specific sites in the cell.25 nNOS has a recognition site for calmodulin that is also present in eNOS and macrophage NOS. The constitutive isoforms are generally regulated by Ca2+–calmodulin, whereas inducible forms are not.23

Figure 2
figure2

Modular structure of neuronal nitric oxide synthase showing approximate locations of prosthetic groups and cofactors.

nNOS in the penis is expressed primarily as a variant of the brain form of nNOS and has been termed PnNOS. It has an additional 102-bp alternative exon located between exons 16 and 17. The function of this additional coding region is unknown. PnNOS is thought to be responsible for triggering the nitrergic mechanism responsible for cavernosal relaxation.26, 27 A similar variant, nNOSu, is present in the neuromuscular plates of skeletal muscles,28 including the perineal muscles involved in erectile rigidity and ejaculation in rats.29 The control of NO synthesis in the cavernosal nerve, whether due to sexual stimulation emanating centrally from the brain, or peripherally by means of the dorsal nerve spinal reflex, is assumed to be exerted through the activation of PnNOS activity.30 This mechanism occurs mainly by Ca2+ binding to calmodulin by means of a Ca2+ flux through the N-methyl-D-aspartate receptor (NMDAR). Both the NMDAR and inhibitors of nNOS activity, such as protein inhibitor of NOS (PIN)31, 32, 33 and carboxy-terminal PDZ ligand of nNOS (CAPON),34 also bind to nNOS. PIN, NMDAR subunits, and a truncated variant of the NMDAR subunit 1mRNA (NMDAR1-T) have been located in the peripheral penile nerves and pelvic ganglion.27

The nitrergic activation of penile erection is not restricted to peripheral nerves of the corpora cavernosa but is also dependent on central nervous system control. Copulatory behavior and ejaculation are also central nervous system (CNS) regulated.35 Erectile stimuli originate from the medial preoptic area and paraventricular nucleus of the hypothalamus through the L6–S1 lumbosacral level of the spinal cord containing the sacral parasympathetic nucleus (SPN).36 Pudendal nerve motor neurons innervating the perineal striated muscles that induce maximum rigidity at the time of ejaculation are located in Onuf's nucleus of the ventral sacral spinal cord and the dorsomedial and the dorsolateral nuclei of the lower lumbosacral spinal cord.29 Electrical stimulation of the medial preoptic area or microinjection of oxytocin, glutamate, or apomorphine into the medial preoptic area and paraventricular nucleus induces noncontact erections37 and increased intracavernosal pressure,38 by means of an NO-mediated process within the CNS.39 Nitrergic and oxytocinergic neurons have been located in the SPN and in Onuf's nucleus.40 Retrograde tracing from the corpora cavernosa has identified neurons in the spinal SPN, in parvocellular region of the paraventricular nucleus, and medial preoptic area.35

It was found that PnNOS, the brain-type nNOS, and PIN, were expressed in the hypothalamus.41 In contrast, NMDAR1-T was expressed only in the penis, whereas the brain-type NMDAR1 was present in the brain and sacral spinal cord and not in the penis. PnNOS was found in the media preoptic area, posterior magnocellular, and the parvocellular regions of the paraventricular nucleus, supraoptic nucleus, septohypothalamic nucleus, medial septum, 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 staining was present in neurons of the medial preoptic area, paraventricular nucleus, medial septum, and cortex, but not in the supraoptic nucleus, septohypothalamic nucleus, or organum vasculosum of the lamina terminalis.41

Inhibitors of NOS are substrate analogues of L-arginine, such as N-monomethyl-L-arginine (L-NMMA), N-nitro-L-arginine methyl ester (L-NAME), and N-amino-L-arginine.42, 43

Among the NOS inactivating L-arginine derivatives, vinyl-L-NIO (N5-(1-imino-3-butenyl)-L-ornithine; L-VNIO) is a potent, mechanism-based inhibitor that attacks the heme cofactor of NOS with a marked selectivity for nNOS.44, 45 The use of the selective nNOS inhibitor, 7-nitroindazole (7-NI), has shown that inhibition of this isoform reduces the erectile response and that if higher doses are used (which inhibit eNOS as well) the whole erectile response to cavernous nerve stimulation is reduced.46

Drugs that inhibit the dephosphorylation of eNOS might alleviate ED. eNOS abnormalities may play a role in diabetic ED. Hyperglycemia decreases NO production by eNOS via O-linked glycosylation of eNOS at the Akt target S1177 in hyperglycemic cell culture conditions and in animal models of diabetes.47 ED in diabetes is associated with peripheral nerve damage but may involve diminished endothelial production of NO as well.48 Numerous systemic vasculature diseases (hypertension, atherosclerosis, hypercholesterolemia, diabetes mellitus, etc) that cause ED are highly associated with endothelial dysfunction, which has been shown to contribute to decreased erectile function in men and a number of animal models of penile erection.8 Deposition of advanced glycation end products (eg pentosidine, pyrraline), which can quench NO both in animal models and cell cultures, was associated directly and indirectly with declines in eNOS activity in the corpora cavernosa of diabetic ED patients compared with their nondiabetic counterparts.49

The activity of nNOS is controlled by a number of mechanisms. A balance of various inhibitory and stimulatory transcription factors determines gene transcription of the enzyme. Enzyme activity can be halted by phosphorylation by a cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) or cGMP-dependent protein kinase (PKG), providing a negative feedback loop.50 The enzyme is activated by increased intracellular calcium, which binds to calmodulin to form the essential cofactor.

It is also likely that co-transmitters influence nNOS activity, perhaps by altering calcium concentration by activation of prejunctional receptors. VIP is a probable stimulatory co-transmitter, while noradrenaline acting on α-2 adrenoceptors inhibits NO formation.13

Inactivation

NO is inactivated by heme and the free radical, superoxide. Thus, scavengers of superoxide anion such as superoxide dismutase (SOD) may protect NO, enhancing its potency and prolonging its duration of action. Conversely, interaction of NO with superoxide may generate the potent tissue-damaging moiety, peroxynitrite (ONOO), which has a high affinity for sulfhydryl groups and thus inactivates several key sulfhydryl-bearing-enzymes. This effect of peroxynitrite is regulated by the cellular content of glutathione. Since glutathione is the major intracellular soluble sulfhydryl-containing compound, factors that regulate the biosynthesis and decomposition of glutathions may have important consequences.18

Glutathione also interacts with NO under physiologic conditions to generate S-nitrosoglutathione, a more stable form of NO. Nitrosoglutathione may serve as an endogenous long-lived adduct or carrier of NO. Vascular glutathione is decreased in diabetes mellitus and atherosclerosis, and this may account for the increased incidence of cardiovascular complications and ED in these conditions.18, 51

Khan et al (2001) found that NO—and electrical field-stimulated (EFS)—mediated cavernosal smooth muscle relaxation is impaired in a rabbit model of diabetes but SOD significantly reversed the impaired relaxation. Therefore, in diabetes, the generation of reactive oxygen species may play an important role in the development of ED.52

Production of the superoxide radicals in rabbit cavernous tissues increases during the state of hypercholesterolemia, which may lead to functional impairment of cavernous smooth muscle relaxation in response to endothelium-mediated stimuli.53

ED associated with aging is related in part to an increase in cavernosal superoxide anion formation. Gene-transfer of extracellular (EC)-superoxide dismutase (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 α1 and β1 respectively. Further subunits termed α2 and β2 have also been identified from the human foetal brain (82 kDa) and rat kidney (76 kDa), respectively. Recently, GUCIA2, the gene coding for the α2-subunit, has been localized to position q21–q22 on the human chromosome 11.55 Subunits isolated from the adult human brain, termed α3 and β3 (81 kDa and 70 kDa, respectively), may represent additional isoforms; however, their close homology to the bovine α1 and β1 subunits suggests they may simply be species variants of existing subtypes.56 Reverse transcriptase-polymerase chain reaction (RT-PCR) has shown the existence of mRNA coding for both α3 and β3 subunits in vascular smooth muscle and endothelial cells in culture and in freshly isolated human vascular tissue.57

Soluble GC is a heterodimer with at least three functional domains for each subunit (Figure 3). These domains are a heme-binding domain, dimerization domain, and catalytic domain. The N-terminal portion of each subunit constitutes a heme-binding domain and represents the least conserved region of the protein; it is the heme moiety that confers the NO-sensitivity of the enzyme.58 Heme-reconstituted sGC can be activated nearly 100-fold by NO.58 Soluble GC may contain 1 mole of heme bound per monomer, depending on the purification protocol. The latter determination appears to be considerably more NO sensitive than an equivalent protein containing 1 mole heme per dimmer.59

Figure 3
figure3

Structure of soluble guanylate cyclase heterodimer. The N-terminal constitutes a heme-binding domain with His105 providing the axial ligand to the fifth coordinate of the heme-iron. The central portion mediates dimerization of the monomers, a prerequisite for catalytic activity. The C-terminal region forms the catalytic domains for conversion of GTP to cGMP.

Oxidation of the heme group to a ferric state results in loss of the enzyme activity; thus, reducing agents such as thiols or ascorbate enhance enzyme activation, and thereby facilitating the reaction between NO and (ferrous) heme. On the other hand, oxidizing agents such as methylene blue inhibit enzyme activation (thiols may also facilitate enzyme activation by forming S-nitrosothiols with NO released from nitrovasodilator drugs).60

The heme moiety is bound to the enzyme protein via an imidazole axial ligand, shown by point mutation to be provided by His 105 in the β1-subunit.61 At the C-terminus of each subunit is a catalytic domain that exhibits a high degree of homology, both between sGC monomers and the C-terminal regions of particulate GC and AC (adenylate cyclase).62 Intervening between the heme-binding and catalytic regions is a dimerization domain that is thought to mediate the subunit association to form heterodimers, which is obligatory for catalytic activity.

Binding of NO to the heme-iron of sGC results in the formation of a pentacoordinate nitrosyl–heme complex, which breaks the bond to the axial histidine and activates the enzyme.4, 63 NO is the only nitrogen monoxide capable of stimulating sGC activity.64 Cofactors, including Mn2+ and Mg2+, are required for the catalytic conversion of GTP to cGMP by sGC. The Mg2+ is thought to act as the most physiologically important cofactor. In the presence of Mg2+, stimulation of the enzyme by NO results in a marked increase in Vmax (>200-fold) and a decrease in Km for the substrate GTP from 100 to 50 μM.65

In addition to iron, sGC possesses a second metal ion, copper, which is also thought to function as a cofactor for enzyme activity.66 Free copper ions inhibit purified sGC activity by reducing Vmax, although the potency of NO-stimulation is unaffected.

Activation of sGC can be achieved satisfactorily with NO donors, such as glyceryl trinitrate, nitroprusside, or S-nitrosothiols. Agents like methylene blue and LY83583 (6-anilinoqinoline-5,8-quinone) can be utilized for inhibition of the enzyme.4 Both compounds have been shown to release superoxide in aqueous solution and a significant component of their activity may therefore be via inactivation of NO. Further, such compounds can directly inhibit NOS activity.67 N-methyl hydroxylamine may represent a rather more selective sGC inhibitor.68

ODQ {1H-[1,2,4] oxadiazolol [4,3-a] quinoxaline-1-one} has been demonstrated to block NO-dependent smooth muscle relaxation in the respiratory and urogenital tracts69 and vasculature and inhibit the NO-mediated reduction in platelet reactivity.70 The mechanism of inhibition is thought to occur via modulation of the heme-iron.70

YC-1 [3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole] has provided a non-NO-based activator of sGC that is effective in a biological milieu.71, 72 YC-1 can evoke erectile responses when given intracavernously and it enhances erections induced by cavernous nerve stimulation and apomorphine when given systemically.73 Activation of sGC by YC-1 may provide an alternative means for enhancing the activity of neurally derived NO during sexual stimulation in the corpus cavernosum, representing a novel approach for the treatment of ED.73, 74

Due to the ubiquitous nature of the NO–sGC–cGMP pathway, signal transduction by sGC also has profound pathophysiological significance. For example, septic shock and migraine may be due to overactivity of the pathway, and impotence, hypertension, and asthma as a result of underactivity.

Intracellular cyclic GMP receptor proteins

Cyclic GMP interacts with three types of intracellular receptor proteins: cGMP-dependent protein kinases (PKGs), cGMP-regulated ion channels, and cGMP-regulated cyclic nucleotide phosphodiesterases (PDEs). This means that cGMP can alter cell function through protein phosphorylation or through mechanisms not directly related to protein phosphorylation.

Two general classes of cGMP kinases exist in vertebrate cells: a type I and a type II form. The type I cGMP kinase is more abundant and widely distributed and has been isolated from vascular and other tissues while the type II form has been detected in vertebrate intestinal epithelial cells.75

Cyclic GMP kinases are found in a number of different cells but are most abundant in three cell types in vertebrates: smooth muscle, platelet, and cerebellum.76 Appreciable amounts are also found in cardiac muscle, and lesser amounts are found in leukocytes,77, 78 skeletal muscle, hepatocytes, vascular endothelial cells, and renal tubular epithelium.76, 79

It is currently thought that at least one mechanism of cGMP-induced relaxation is the reduction of intracellular-free Ca2+ levels ([Ca2+]i), since [Ca2+]i is the signal for activation of myosin light-chain kinase (MLCK) and contraction in smooth muscle. This enzyme phosphorylates myosin regulatory light chains (MRLC), which then form cross bridges with actin thin filaments and so generate force. Cross bridges are dephosphorylated by myosin light-chain phosphatase (MLCP) resulting in relaxation (Figure 4).50, 80 Calcium channel blockers predictably cause vasodilation because they reduce intracellular Ca2+. Substances that increase cAMP like β2 agonist, may cause relaxation in smooth muscle by accelerating the inactivation of MLCK and facilitating the expulsion of calcium from the cell.81 Theoretically, vasodilators should not cause ED; however, patients with severe atherosclerosis may require higher blood pressure to deliver sufficient flow to the penis, and the lowering of blood pressure by these agents may result in partial erection.

Figure 4
figure4

Control of smooth muscle contraction and site of action of calcium channel-blocking drugs. Contraction is triggered by influx of calcium through transmembrane calcium channels. The calcium combines with calmodulin to form a complex that converts myosin light-chain kinase to its active form (MLCK*). The latter phosphorylates the myosin light chains, thereby initiating the interaction of myosin with actin. Activation of RhoA leads to the activation of Rho-kinase (ROK), which in turn phosphorylates the regulatory myosin-binding subunit of myosin phosphatase (MLCP), which results in the inhibition of the enzyme. β2 agonists (and other substances that increase cAMP) may cause relaxation in smooth muscle by accelerating the inactivation of MLCK and facilitating the expulsion of calcium from the cell. MLC (myosin light chain).

The calcium-sensitizing Rho-A/Rho-kinase pathway may play a synergistic role in cavernosal vasoconstriction to maintain penile flaccidity. Rho-kinase is known to inhibit MLCP, and to directly phosphorylate myosin light-chain (in solution), altogether resulting in a net increase in activated myosin and the promotion of cellular contraction. Although Rho-kinase protein and mRNA have been detected in cavernosal tissue, the role of Rho-kinase in the regulation of cavernosal tone is unknown.82 Chitaley et al (2001)82 found that Rho-kinase antagonism stimulates rat penile erection independently of NO. Mills et al (2002)83 in their study support the hypothesis that NO inhibits Rho-kinase-induced cavernosal vasoconstriction during erection. These initial findings introduce a novel potential therapeutic approach for the treatment of ED.84

The mechanism by which cGMP kinase acts is still not understood. Findings from several laboratories have indicated that one effect of cGMP kinase is stimulation of a Ca2+-pumping ATPase, an action that would be predicted to lower [Ca2+]i in smooth muscle cells activated with contractile agonists or by depolarization.85 The generation of PKGs by cGMP leads to a number of events that decrease [Ca2+]i. It has been shown to phosphorylate and therefore inhibit the inositol 1,4,5-triphophate (IP3) receptor on the sarcoplasmic reticulum, thus preventing calcium release from the store. In addition, PKG increases activity of plasma and sarcolemmal (mediated via the regulatory protein, phospholamban) cation-ATPase pumps encouraging sequestration of calcium into stores and out of the cell.86, 87

nNOS and eNOS are activated by calcium entry into the cell, binding to calmodulin associated with the enzymes.88 Whereas physiologic penile erection lasts several minutes, the calcium-dependent activation of nNOS or eNOS is quite transient. Recently, several groups showed that the phosphatidylinositol 3-kinase (PI3-kinase) pathway that activates the serine/threonine protein kinase Akt (also known as PKB) causes direct phosphorylation of eNOS, reducing the enzyme's calcium requirement and causing increased production of NO.89 This pathway is responsible for both shear stress and growth-factor 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 vasorelaxant drug papaverine cause rapid increases in phosphorylated (activated) Akt and eNOS. Phosphorylation 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

Other mechanisms for the lowering of [Ca2+]i by cGMP kinase include activation of Ca2+-ATPase by the stimulation of phosphatidylinositol-4-phosphate (PIP) formation by cGMP kinase77 and phosphorylation of 240-kDa protein that mediates the activation of Ca2+-ATPase by cGMP kinase.78 PKG may catalyze the phosphorylation of phosphatidylinositol kinase, leading to the formation of PIP and the activation of Ca2+-ATPase by the lipid. The role of the 240-kDa protein is unknown. It is possible that this protein is a component of the cytoskeleton that is involved in the recruitment of additional Ca2+-ATPase molecules from internal stores to the plasma membrane. Other mechanisms have been proposed for the effects of cGMP, and these include inhibition of G protein function and inhibition of phospholipase C (PLC) activation.50 These effects could contribute to [Ca2+]i reduction in agonist-stimulated smooth muscle, but they would have little effect on depolarized cells or tissues because PLC activation is not involved in the mobilization of [Ca2+]i in this instance.81 There is evidence that increases in cGMP also lower the Ca2+-sensitivity of cross bridge phosphorylation. This would contribute to a decline in force through actions on the myosin light-chain kinase/phosphatase system.77 In addition, increased cGMP-dependent protein kinases decrease activity of plasma membrane calcium channels by phosphorylation (L-type channels) and by membrane hyperpolarization secondary to potassium (K+) channel activation (voltage-gated channels). There may also be a feedback loop within smooth muscle cells whereby a rise in [Ca2+]i stimulates endogenous NO production, which will then lower [Ca2+]i through the generation of cGMP.50, 91

Vasodilatation and relaxation of cavernosal smooth muscle cells (SMC) engorges the corpora cavernosa with blood at arterial pressure. The subcellular mechanism by which tumescence occurs involves NO-induced activation of sGC, increased cGMP levels, and activation of PKG. This phosphorylates numerous ion channels and pumps, each promoting a reduction in cytosolic calcium. In particular, PKG activates high-conductance Ca2+-sensitive K+ (BKCa) channels, which hyperpolarize the arterial and cavernosal SMC membranes, causing relaxation. This mechanism appears to be compromised with age and vascular disease, leading to ED. Thus, increasing cavernosal NOS expression, cGMP levels and/or BKCa channel expression is an effective therapy for experimental ED. Future therapies may involve augmenting K+ channel expression by gene transfer or increasing channel function through the use of Type 5 phosphodiesterase (PDE5) inhibitors or phosphatase inhibitors.92

Regulation of phosphodiesterase (PDE) activity is an important component of control of cGMP concentration and hence activity of the NO–cGMP pathway. Mammalian PDEs comprise 11 identified families (PDE1–PDE11) and their isoforms, which are distinguished by their substrate specificities and tissue concentrations.93

To date, five of these 11 isoenzymes (PDE1, 2, 3, 4, and 5) have been proven to be of pharmacological relevance. Currently, the presence of mRNAs specific for 14 different human phosphodiesterase isoforms in human cavernous tissue was shown by means of RT-PCR and Nothern blot analysis.94 The expression of the following genes were detected in human cavernous tissue: PDE1A, PDE1B, PDE1C, PDE2A, and PDE10A, which hydrolyze both cAMP and cGMP; the cAMP-specific PDEs PDE3A, PDE4A-D, PDE7A and PDE8A, and the cGMP-specific PDEs PDE5A and PDE9A. The molecular identification of PDE isoenzymes was paralleled by efforts to detect and characterize the hydrolizing activities of PDE proteins expressed in human penile erectile tissue. In the early 1990s, Stief and co-workers reported the separation of hydrolytic activities of PDE isoenzymes 3, 4, and 5 from cytosolic supernatants prepared from human cavernous smooth muscle,95 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, quazinone, milrinone, rolipram, and zaprinast) on the adrenergic tension of isolated human corpous cavernosum, Stief and co-workers94 concluded that cavernous smooth muscle tone is mainly regulated by cAMP and that cGMP-inhibited PDE3 is of major importance in the control of cAMP turnover, while others postulated that cGMP-specific PDE5 is the predominant isoenzyme in the degradation of cyclic nucleotide monophosphate (cNMP) in the corpus cavernosum. Nevertheless, both conclusions are supported by the efficacy of intracavernous milrinone and orally administered sildenafil to induce penile erection sufficient for sexual intercourse.97 PDE5 is the predominant isozyme degrading cGMP in the corpus cavernosum.94, 98 Accordingly, drugs that inhibit PDE5 can enhance and prolong the smooth muscle relaxant effects of the NO–cGMP cascade in the corpus cavernosum, thereby potentiating penile erection.93 The prototype of this new therapeutic class of PDE5 inhibitors is sildenafil, which was approved for treatment of ED in 1998. Tadalafil and vardenafil are new agents in this class.93

Sildenafil is more selective for PDE599 than for other PDEs: >80-fold more than for PDE1; >1000-fold more than for PDE2 to PDE4; and about 10-fold more than for PDE6, an enzyme found in the retina.99 The lower selectivity of sildenafil for PDE5 over photoreceptor PDE6 may account for the color visual disturbances observed with increasing frequency with larger doses or higher plasma levels of sildenafil.99 A potent, selective, reversible PDE5 inhibitor, tadalafil is under regulatory review in Europe and North America as an oral therapy for mild-to-severe ED.100 Tadalafil is highly selective for PDE5 (IC50=0.94 nmol/l) over other isozymes.101 In vitro studies with tadalafil have demonstrated a >10 000-fold greater selectivity for PDE5 versus PDE1 to PDE4 and PDE7 to PDE10, as well as approximately 700-fold greater selectivity for PDE5 than for PDE6.93 Vardenafil is also selective for PDE5 in vitro and more selective for PDE5 than for PDE1 to PDE4. It is >15-fold more selective for PDE5 than for PDE6.102 Vardenafil was described to be more potent and selective than sildenafil on its inhibitory activity on PDE5.102 PDE5 inhibitors augment the response of exogenously applied nitrates, resulting in profound hypotension. For this reason, it is contraindicated in patients using nitroglycerin or other nitrate-based medications, which are NO donors.103, 104

It appears that no single mechanism explains all the effects of cGMP on relaxation in the variety of systems examined. The advantage for intracellular signaling is that elevation in cGMP and activation of PKG promote rapid and efficient phosphorylation of substrates in response to signals such as NO.

Conclusions

The NO–sGC–cGMP pathway plays a crucial role in the initiation and maintenance of cavernosal relaxation. It would appear to achieve this predominantly through the actions of PKGs. Cyclic GMP can alter cell function through protein phosphorylation or through mechanisms not directly related to protein phosphorylation. It is also becoming clear that the localization of these cGMP receptor proteins in the cell is an important factor in the regulation of cell function by cGMP. Soluble GC plays an important role in the transduction of inter- and intracellular signals conveyed by NO. To fulfill this role, sGC has evolved a unique heme-coordination, which customizes it for NO sensitivity and its product, cGMP, regulates a plethora of biological functions depending on location and cell type. It is crucial that the NO–sGC–cGMP transduction system is understood in its entirety, to provide the greatest opportunity for the design and development of therapeutics.

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Ghalayini, I. Nitric oxide–cyclic GMP pathway with some emphasis on cavernosal contractility. Int J Impot Res 16, 459–469 (2004). https://doi.org/10.1038/sj.ijir.3901256

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Keywords

  • nitric oxide
  • cGMP
  • soluble guanylate cyclase
  • phosphodiesterase
  • corpus cavernosum

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