Description
CHAPTER ONE
INTRODUCTION
Erectile dysfunction, ED, is a sexual dysfunction that affects the reproductive systems of both men and women. By definition according to National Institute of Health consensus Development Panel on impotence (1993), in Males, it is a sexual dysfunction characterized with the inability to develop or maintain an erection of the penis sufficient for satisfactory sexual performance. It is also known as Male impotence or Baby D syndrome, while in women, according to American Psychiatric Association (1994), it is characterized with the persistent or recurrent inability to attain, or maintain until completion of the sexual activity, an adequate Lubrication- Swelling response that otherwise is present during female sexual arousal and sexual activity is thus prevented. Hence, it is called Women impotence or female erectile dysfunction.
The word impotence may also be used to describe other problems that may interfere with sexual intercourse and reproduction, such as lack of Sexual Desire and problems with ejaculation or orgasm. Using the term “erectile dysfunction,” however makes it clear that those other problems are not involved (NIH, 2005).
An erection occurs as a hydraulic effect due to blood entering and being retained in sponge-like bodies within the penis and clitoris. The process is most often than not initiated as a result of sexual arousal, when signals are transmitted from the brain to nerves in the pelvis.
Erectile dysfunction is, therefore indicated when an erection is consistently difficult or impossible to produce, despite arousal (Laumann et al., 1999).
1.1 PREVALENCE OF ERECTILE DYSFUNCTION IN WOMEN
Erectile dysfunction which is known as Female erection dysfunction in women occurs in about 43% of American Women (NIH Consensus Conference, 1993). And this medical Condition is a persistent or recurrent inability to attain or maintain clitoral erection until completion of the sexual activity, an adequate Lubrication –Swelling response that is normally present during Female sexual arousal and sexual activity is therefore, absent. The individual having the condition is said to experience frigidity (American Psychiatric Association, 1994). Again, according to Otubu et al. (1998) about 8.7% of Women suffer from this very condition in the United States while between 35.3 – 40%, according to Adequnloye (2002) and Eze (1994) of Women in Nigeria suffer from this condition. Spector and Carey (1994) reported 5-10% in the United States.
In addition, Female erectile dysfunction occurs at any age but majorly in old age. Hence, the most significant age related change is menopause (Karen, 2000) and (Rod et al., 2005). However, erectile dysfunction may be caused by diabetes, atherosclerosis, hormonal imbalances, neurological problems etc. (Organic causes) or stress, depression etc.
Because treating the underlying causes (Organic or Psychological), the first line treatment of ED consists of a trial of PDES inhibitor (the first of which was Sildenafil or Viagra). In some cases, treatment can involve prostag-Landin tablets in the Urethra, intravenous injection with a fine needle into the penis or clitoris that causes swelling of Penis or Clitoris Pump or Vascular surgery, estrogen replacement therapy for the women etc.
Although there are various methods and techniques that are used to treat this very condition, however, for the purpose of this project, the treatment is restricted to Yohimbe, an extract from Pausinystalia yohimbe.
1.2 PREVALENCE OF ERECTILE DYSFUNCTION IN MEN
Erectile dysfunction, ED, varies in severity; some men have a total inability to achieve an erection, others have inconsistent ability to achieve an erection, and still others can sustain only brief erection. The variation in severity of erectile dysfunction makes estimating its frequency difficult.
Many men also are reluctant to discuss erectile dysfunction with their doctors, and thus, the condition is under-diagnosed. Nevertheless, experts have estimated that ED affects 30 million men in the United States. Again, according to the statistical research carried out by Adegunloye (2002) and Eze (1994) respectively in Nigeria, results shows that about 23-26.4% of men suffer from this condition while according to Spector and Carey (1999) discovered that about 4-9% of men suffer from the condition in the United States.
While erectile dysfunction can occur at any age, it is uncommon among young men and more common in the elderly. By the age of 45, most men have experienced erectile dysfunction at least some of the time. According to the Massachusetts Male Aging Study, complete impotence increases from 5% among Men 40 years of age to 15% among Men 70 years and older. Population studies conducted in the Netherlands found out that some degree of ED occurred in 20% of Men between 50 – 54 and in 50% of men between ages 70 – 78. In 1998, the National Ambulatory Medical care Survey counted 1,520,000 Doctor Offices visited for ED.
1.3 OBJECTIVE STUDY AND AIMS
This project focuses to give a clear picture of the effect on erectile tissues of the Penis, Clitoris of both Men and Women.
1.4 NITRIC OXIDE-CYCLIC GMP PATHWAY WITH SOME EMPHASIS ON CAVERNOSAL CONTRACTILITY
Nitric Oxide (NO) is formed from the conversion of L- arginine by nitric oxide synthase (NOS), 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 Calcuim depletion from the cytosolic space and Cavernous Smooth muscle relaxation. The effect of cGMP are mediated by cGMP dependent Protein Kinase, 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 decrease 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.
﴾International Journal of impotence Research (2004)﴿. Nitric oxide (NO) was first described by Stuehr and Marletta (1985) as a product of activated murine machrophages. Also, the substance known as endothelium- derived relaxing factor (EDRF), described by Furchgott and Zawadzki (1980), 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. No and cGMP together comprise an especially wide-ranging signals 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.
Erectile dysfunction (ED) is defined as the constituent inability to achieve or maintain an erection sufficient for satisfactory sexual performance and is considered to be a natural process of ageing. Studies have shown that ED is caused by inadequate relaxing of the corpus cavernosum with defeat in NO production.
It is clear that NO is the predominant neurotransmitter responsible for cavernasal Smooth muscle relaxation and hence penile erection. Its action is medicated through the generation of the second messenger cGMP. Neutrally, 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 convesion 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.
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 Nitric Oxide (NO).
Recent observation suggest that the main site of NO biosythesis 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. Endothelial –derived NO plays a major role in the penis. Some suggest that NO is highly labile, therefore it cannot be stored as a preformed neurotransmitter. Other proerectile mediators, such as acetylcholine, calci-tonin gene related peptide (CGRP) or substance P, act via endothelialcells by prompting the synthesis and release of NO by these cells, ﴾Bivalacqua et al., 2001). Found in their study that in vivo adenoviral gene transfer of CGRP in combination with adrenomedullin (ADM) or prostaglandin E1(PGEI) induce penile erection by activating different receptors.
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 citruline nitrogen of L- arginine. L- citrulline can be converted by arginine synthase (AS) to form L-arginine, the precursor for NO. Each of these enzymes, co-factors, or transport systems could be an eventual target of pharmacologic intervention in the NO cascade.
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. There are at least three isoform of NOS (neuronal, endothelial, and macrophage). A constitutive form of NOS is found in endothelial and neurons, and is calcium dependent. The constitutive NOS-3, whereas the constitute NOS found in neutral and epithelial tissue has been named NOS-1. An inducible form of NOS, now designated iNOS, is calcium independent. It is induced within 4-24h of the appropriate stimulus and can produce NO in a 100-fold greater amount than can constitutive NOS.
Neutral 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 (O factors for the synthesis of NO. these cofactors bind to a reductase domain to process election transfer. This is then linked to heme and tetrahydrobiopterin (BH4) – containing catalytic oxygenenase domain by calcium-calmodulin complex (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-2 has not been established, a study on other proteins containing this motif indicates that it may serve to target proteins to specific sites in the cell. nNOS has a recognition site for calmodulin that is also present in eNOS and macrophages NOS. The constitutive isoforms are generally regulated by Ca2+ -calmodulin, whereas inducible forms are not.
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 trigging the nitregic mechanism responsible for cavernosal relaxation. A similar variant, nNO-SU is present in the neuromuscular plates of skeletal muscles, including the perineal muscles involved in erectile rigidity and ejaculation in rats. 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. This mechanism occurs mainly by Ca2+ binding to calmodulin by means of Ca2+ flux through the N-methyl-D-aspartate receptor (NMDAR). Both the NMDAR and inhibitors of nNOS activity, such as protein inhibitors of nNOS activity, such as protein inhibitors of NOS(PIN) and carboxy terminal POZ Ligand of nNOS (CAPON), also bind to nNOS .
The nitrognic activation of penile erection is not restricted to peripheral nerves of the corpora cavernosa but is also dependent on central nervous system (CNS) regulated.
It was found that PnNOS, the brain type nNOS, and PIN were expressed in the hypothalamus in contrast, NMDAR1-T was expressed only in the penis, whereas the brain –type- NMDARI 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 paraventriccular nucleus, Supraoptic nucleus, septohypothalamic nucleus, medial septum, Cortex, and in some of the nNOS staining neurone through the brain. It was absent in organum vasculosum of the lamina terminalis.
PIN staining was present in neurons of the medial septum and cortex, but not in the supraoptic nucleus septohypothalamic nucleus or organum vasculosym of the Laminal terminals.
Inhibitors of NOS are substrate analogues of L-arginine, such as N-Monomethyl -L- arginine (L- NMMA), nitro-L- arginine methyl ester (L-NAME). and N-amino –L- arginine.
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 glycossylation of eNOS at the targets S1177 in hyperglycemic cell culture conditions and in animal models of diabetes. ED in diabetes is associated with peripheral nerve damage but may involve diminished endothelial-production of NO as well. Numerous systemic vasculature, diseases (hypertension, atherosclerosis, hyperoholesterolemia, 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.
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 feed back loop. 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 x-2 adrenoceptors inhibits NO formation.
1.6 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 prolong its duration of action. Conversely, interaction of NO with super oxide may generate the potent tissue damaging moiety, peroxynitrite (ON00-1), which has a high affinity for sulfhydryl groups and thus inactives several key sulphydryl-bearing-enzymes. This effect of perotynitrite is regulated by the cellular content of glutathione.
Khan et al., (2001) found that NO- and electrical field stimulated (EFS) – mediated cavernosal smooth muscle relaxation is impaired in a rabbit of diabetes but SOD significantly reversed the impaired relaxation. Manipulation of physiological NO concentration is unlikely to give physiological benefits 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.
1.7 The NO receptor: Soluble guanylate cyclase
Soluble GC is a heme- containing protein found in the cytosolic fraction of virualy all mammalian cells. With the highest concentrations found in the lungs and brain. Several isoforms of sGC have been Cloned and characterized. Originally sGC was purified (to apparent homogeiniety) from bovine and rat lung and shown to exist as a heterodimer, consisting of 82 Koa (rat) or 73Koa (bovine) and 70Koasubunits, termed x, and β1respectively. Further subunits termed x1 and β2 have also been identified from the human foetal brain (82Koa) and rat kidney (76Koa), respectively, GUCIA2; the gene coding for the x2-Subunit,has been localized to position q21-q22 on the human chromosome 11.
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 domainand represents the least conserved region of the protein; it is the heme moiety that confers the NO-sensitivity of the enzyme. Heme- reconstituted more NO sensitive than an equivalent protein containing 1 mole heme per dimmer.
Oxidation of the heme group to a ferric state results in less of the enzyme activity; thus reducing agents such as thiols or ascorbate enhances 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).
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 B1-Subunit. 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 (udenylate cyclase). Interveining 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 pentacordinate nitrosyl- heme complex, which breaks the breaks the bond to the bond to the axial histidine and activate the enzyme. In addition to iron, sGC possesses a second metal ion, copper which is also thought to function as a cofactor for enzyme activity. Free copper ions inhibit purified sGC activity by reducing Vmax, although the potency of NO-stimulation is unaffected. Activation of sGC can be achieved satisfactory with NO donors, such as glycerol trinitrate nitroprusside, or S-nitrosothiols. Agents like methylene blue and LY83583 (6-anilinoginoline – 5,8-quinoline) can be utilized for inhibition of the enzyme. Both compounds have been shown to release superoxide in aqueos solution and a significant component of their activity may therefore be via inactivation of NO.
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, hypertention, and asthma as a result of underactivity.
1.8 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 alter cell function through mechanism not directly related to protein phosphorylation.
Two general classes of cGMP kinases exist in vertebrate cells: a type 1 and a type 11 form. The type 1 cGMP kinase is more abundant and widely distributed and has been isolated from vascular and other tissues while the ype 11 form has been detected in vertebrate intestinal epithelial cells.
Cyclic GMP kinase are found in a number of different cells but are most abundant in three cell types in vertebrates smooth muscle, platelet and cerebellum. 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. (Chitaley et al., 2001) found that Rho-kinase antagonism stimulates rat penile erection independently of NO (Mills et al., 2002) 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.
The mechanisms by which cGMP kinase act are 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+] in smooth muscle cells activated with contractile agonists or by depolarization. The generation of PKGs by cGMP leads to a number of events that decrease [Ca2+]. It has been shown to phosphorylate and therefore inhibit the inositol 1,4,5- triphosphate [IPs] receptor on the sarcopasmic 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.
nNOS and eNOS are activated by calcium entry into the cell, binding to calmodium associated with the enzymes. Whereas physiologic penile erection lasts several minutes, the calcium dependent activation of nNOS or eNOS is quite transient. Recently, several groups showed that the phosphotidylinositol 3-kinase (P13- kinase) pathway that activates the serine/threonine protein kinase (also known as PKB) causes direct phosporylation of eNOS, reducing the enzymes calcium requirement and causing increased production of NO. This pathway is responsible for both shear stress and growth –factor enhancement of blood flow that can last for hours. Finding of Hurt et al., support a model in which rapid brief activation of neuronal NOS initiates of Ca2+-ATPase by the stimulation of phosphatidylinositol -4-phosphate (PIP) formation by cGMP kinase and phosphorylation of 240-KDA protein that mediates the activation of Ca2+ -ATPase by cGMP kinase. PKG may catalyze the phosphorylation of phisphatidylinositol 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.
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 (PDE2-PDE11) and their isoforms,which are distinguished by their substrate specificities and tssue concentration.
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 humans cavernous tissue was shown by means of RT-PCR and Nothan blot analysis. The expression of the following genes were detected in human cavernous tissue: PDE1A, PDE1B, PDE2A, and PDE10A, which hydrolyze both cAMP and cGMP; the cAMP specific PDES, PDE3A, PDE4A-D, PDE7A, and PDE8A, and the cGMP-specific PDEs and PDE5A and PDE9A. The molecular identification of PDE isoenzymes was paralled by efforts to detect and characterize the hydrolyzing activities of PDE proteins expressed in human penile erectile tissue. Based on the result s of organ bat studies on the effects of various PDE inhibitors (papaverine, guazinone, mitrinone, rolipram, and zaprinast) in the adrenergic tension of isolated human corpous cavernosum, street and co-workers concluded that cavernous smooth muscle tone is mainly regulated by cAMP and that cGMP –inhibited PDEsis of major importance in the control of cAMP turnover, while others postulated that cGMP-specific PDEs 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. Accordingly, drugs that inhibit PDEs can enhance and prolong the smooth muscle relaxant effects of the NO-cGMP cascade in the corpus cavernosum, thereby potentiating penile erection. The prototype of this now therapeutic class of PDEs inhibitors is sildenafil, which was approved for treatment of ED in 1998. Tadalafil and vardenafil are new agents in this class.
Silidenafil is more selective for PDEs than for other PDEs: >80-fold more than for PDE1:> 1000- fold more than for PDE2 to PDE4i and about 10-fold more than for PDE6, an enzyme found in the retina. 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. In vitro studies with tadalafil have demonstrated a 710000-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. Vardenafil is also selective for PDE5 in vitro and more selective for PDE5 than for PDE1 to PDE4.
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.