Role of oestrogens in male erectile function 1

This study is therefore envisaged to explore the paracrine endpoints of oestrogens in male sexual function, in particular their effects on the normal physiological principles of penile e

Chapter 1 INTRODUCTION

1 INTRODUCTION

Erectile dysfunction (ED), defined by the National Institutes of Health (NIH, 1993) consensus development panel as the inability to achieve or maintain an erection sufficient for satisfactory sexual performance, has gained much clinical attention with the emergence and unprecedented patient compliance to the oral phosphodiesterase (PDE) 5 inhibitor, sildenafil citrate Arising from intensive worldwide research efforts into the pathophysiology of ED over the last three decades, this pharmaceutical breakthrough has incidentally lifted the taboo of seeking medical attention for this important quality of life concern However, ED is a symptom and not the disease per se and therefore the eventuality of successful pharmacotherapy with end-organ effectiveness should not deter

us from recognising and delineating various precipitating causes and pathophysiology of presenting clinical situations for better patient evaluation, management and permanent resolve From being relegated in the fifties as psychogenic in over 90% of ED cases (Wershub, 1959; Smith, 1981), the last two decades have ushered in a new era in the scientific outlook of this multifactorial disorder Erectile physiology is a complex interaction of multiple organ systems coordinating a number of neurologic, vascular, endocrine and cellular inputs with processes in the penile corpora cavernosa (CC) and ED may therefore arise from various risk factors acting on any of these numerous systems / pathways (Mobley and Baum, 1998) Another important premise at this stage is that ED

is progressively more prevalent in older men, because ageing is also associated with many of the risk factors and co-morbid conditions for ED including central and peripheral nervous system disorders, cardiovascular and peripheral vascular dysfunctions and concomitant medications (Korenman, 1998) As it is expected that the present number of men aged over 65 years will more than double by 2025 (Ponholzer et al.,

2002), this negative correlation of aging with ED is a global concern Furthermore, the aging process in man is also accompanied by a number of endocrine changes in addition

to systemic illnesses Several studies such as the Massachusetts Male Aging Study (Gray

et al., 1991; Feldman et al., 2002) have clearly demonstrated age-related changes in serum levels of total testosterone, free testosterone and sex hormone binding globulin (SHBG) Consequently, several of these symptoms including ED may have been associated with such age-related hormone changes Steroid hormone secretions in general, conform to health status in men and therefore understanding the dynamics of endocrine changes in man is also important because of their role in sexual and reproductive function Normal male reproductive function depends on the secretion of luteinizing and follicle stimulating hormones (LH and FSH) by the pituitary gland under the influence of hypothalamic gonadotropin releasing hormone (GnRH) Luteinizing hormone-induced testicular / Leydig cells secretion of testosterone is associated with a diurnal fluctuations and negative feedback control The present understanding is men produce a moderate amount of oestrogen along steroidogenesis of testosterone (T) (Berg, 1998) and 75 – 90% of this “female” hormone in the male is from a simple enzymatic conversion of testosterone called aromatization (Oettel, 2002a) While androgen and oestrogen constitute opposite sides of the same coin and their quantitative balance is what determines the physiological effect on target tissues (Sharpe, 1997), changes due to aging

at the hypothalamo-pituitary-testicular axis may lead to decline in circulating T levels (Cohen, 1998) As a result of this decline, the “ying yang” physiological balance between androgen and oestrogen in normal man may be disturbed Further compounding factors here are the significantly higher oestradiol (E2) levels in the aging male (Oettel, 2002a), changing E2/T ratio (Rubens et al., 1974) and the strong gonadotropin-suppressant effect

of E2 compared to testosterone Hence these factors will result in a state of secondary hypogonadism with decrements in LH and FSH release (Cohen, 1998) There sets in a vicious cycle with changes in E2/T ratio which may be further aggrevated by the continued peripheral conversion of the available testosterone to oestradiol through aromatization Thus, the negative / detrimental effects of T deficiency will continue with advancing age leading to progressive and undeterred functional deterioration of testosterone compared to the relative and absolute overproduction of oestrogen Considering this dual imbalance, it may be hypothesized that androgen deficiency is not the sole cause for the identified decrease in nocturnal penile tumescence (NPT) and spontaneous morning erections and loss of libido (Tserotas and Merino, 1998) and that E2may play a decisive role However, despite the concomitant elevation of oestrogen during this stage in the aging male, available scientific information on the physiological role of

E2 on erectile function is too scant to correlate it with the aetiology of ED in these cases The existing knowledge is that therapeutically, oestrogen was as effective as antiandrogens or GnRH analogues in countering the hypersexuality of paraphilias (Levitsky and Owens, 1999) and oestrogen supplementation prior to gender reassignment reportedly feminized male transsexuals (Goh, 1999) Indeed, months of oestrogen therapy

is the mainstay for the development of breast and other female traits in these patients In the process of such preoperative treatment, prospective male to female transsexuals have observed loss of NPT and morning erections and a gradual reduction in erectile and ejaculatory capacity (Adaikan, 1998) Hence, it appears that E2 plays a distinct negative physiological role in male erectile function

Together with the identification of oestrogen receptors alpha and beta (ERα and ERβ) at some sites of the male reproductive tract (Merchenthaler and Shughrue, 2000; Diagram

1), several reports from animal studies have indicated the positive role of oestrogen receptor activation in sperm production and fertility (Hess et al., 1997; Sharpe, 1997 and Berg, 1998)

Diagram 1: Oestrogen Receptor Distribution (from Merchenthaler and Shughrue, 2000)

Confirmatory changes are seen in the testes of the experimental ERα knock-out mice (ERαKO) model; an ineffectual fluid reabsorptive function of oestrogen is implicated in testicular atrophy from intraluminal fluid back pressure (Hess et al., 1997) Furthermore, this ER gene mutation model also shows that some components of male typical aggressive and sexual behaviours may be oestrogen-dependent (Ogawa et al., 1997;

females, there were a statistically significant decrease in intromission and ejaculatory failure; in open-field behavioural studies, this animal model was docile Taken together, these findings indicate a mediatory role for ER-alpha in the expression of some parameters of sexual and violent behaviours in male rodents

Underlying these reports is the hypothesis for this thesis in establishing the new identity for oestrogenic activity and its relationship to sexual dysfunction in males It could be that some of the physiological effects once considered androgenic might instead arise after aromatization of androgen leading to abnormal or imbalanced oestrogen level This will make the pathophysiological effects of excessive endogenous oestrogen clinically more significant in the male Besides its occurrence in physiological aging, other precipitating conditions of such hyperestrogenism include aromatase hyperactivity (Elias

et al., 1990), non-insulin dependent diabetes mellitus (Oettel, 2002a), rapid weight gain, obesity, hypercholesterolemia, cholelithiasis and chronic liver diseases (Thomas, 1993), idiopathic haemochromatosis (Stremmel et al., 1988), Klinefelter’s disease (Heinig et al., 2002), as well as tumours of male breast (Thomas, 1993; Heinig et al., 2002), Leydig cells (Mineur et al., 1987) and adrenal cortex (Veldhuis et al., 1985) Similar hypothesis

of great health concern which is scientifically yet to be qualified/quantified is the oestrogenic activity of compounds of environmental and plant origin, structurally related

to the endogenous oestrogen (Adlercreutz and Mazur, 1997) These oestrogen mimetics are hazardous to human reproductive health as they are also anti-androgenic (Danzo, 1998); indirect exposure of the foetus during sexual development affects differentiation and growth of the male reproductive tract (Sohoni and Sumpter, 1998) and is implicated

in low sperm counts, cryptorchidism and increased risk for testicular cancer (Cheek and McLachlan, 1998) Recently, a population study suggested that these environmental

agents could also be antierectile (Oliva et al., 2002) Thus, an excessive intake (phytoestrogens) / exposure to these chemical substances (xenoestrogens) on a daily basis are likely to affect not only the fertility but also the sexual profile of the male population Oestrogenic activity of these exogenous substances is also seen by their binding affinity for endogenous oestrogen receptors and extracellular proteins such as SHBG, α fetoprotein and albumin fractions (Stephens, 1997); this indicates that such actions arising from modulations on the endogenous hormone levels may be more complex Thus

in light of these reports, the hypothetic basis of this study is the possibility of oestrogens compromising endogenous androgen milieu and erectile function A systematic delineation of the effects of natural oestrogen mimetics on erectile parameters is aimed to provide a rational understanding to bridge the scientific void in this area This study is therefore envisaged to explore the paracrine endpoints of oestrogens in male sexual function, in particular their effects on the normal physiological principles of penile erection and to propose a basis for such possible dysfunctional changes of hormone modulations in specific clinical presentations

1.1 Erectile Physiology

This component reviews the conceptual developments in the physiological control of penile erection, which include peripheral neuroanatomy, signal transduction, central pathways and coordinating vascular and humoral inputs based on directives from basic animal models and clinical epidemiologic designs These parameters will be used as a rational framework for the investigations, understanding and discussions of the pathophysiological changes in erectile function secondary to the proposed experimental hyperostrogenism

1.2 Historical Milestones and Current Thoughts

1.2.1 Autonomic Control of Penile Erection

The innervation of the penis has somatic, sympathetic and parasympathetic components (Langley and Anderson, 1895) The somatic sensory nerve is carried in the pudendal nerve and divides into the inferior hemorrhoidal nerve, perineal nerve and the dorsal nerve of the penis The sympathetic supply to the genitalia is derived from the twelfth thoracic and upper lumbar segments of the spinal cord The parasympathetic innervation

is from the sacral outflow at the second, third and fourth segments of the sacral cord and constitutes the preganglionic fibres to form the pelvic nerve or nervi erigente (nerve of erection) Autonomic innervation of the CC is comprised of the parenchymal and perivascular nerves (Christ et al., 1997) Studies at the cellular level demonstrate adrenergic nerve fibres in the penile tissues obtained from several animal species; these include fibres identified in the cavernosa of mice (Bock and Gorgas, 1977), rabbits (Klinge and Penttila, 1969; Fujimoto and Takeshige, 1975) and dogs (Bell, 1972) In human penile tissues, catecholamine fluorescent fibres and terminals were demonstrated

in the cavernosa as well as the spongiosum (Benson et al., 1980) These fibres wind through the trabeculae, approach the walls of the cavernous spaces and extend into the spongiosum In addition, the blood vessels of the cavernosum and spongiosum demonstrate adrenergic varicosities Pharmacological demonstration of α- (contractile) and β- (relaxant) adrenoceptors in the human penis (Adaikan, 1979; Adaikan and Karim, 1981) concurred with these histological findings Similarly, acetylcholinesterase positive nerve fibres have been identified in the penile tissues from rats (Dail and Hamill, 1989), rabbits (Klinge and Penttila, 1969) and primates (Steers et al., 1984) Ultrastructural examination of human CC reveals that these cholinergic terminals are located in close

proximity to cavernous blood vessels and smooth muscle (Benson et al., 1980) and coexist with adrenergic and nonadrenergic, noncholinergic (NANC / nitrergic) fibres (Sathananthan et al., 1991) Furthermore, pathways for vasoactive intestinal polypeptide (VIP) and calcitonin gene related peptide (CGRP) have been implicated in penile erection Nerve fibres immunoreactive to VIP have been identified in the CC and around the helicine arteries of human penis (Polak et al., 1981; Steers et al., 1984) and in the penis of various mammalian species (Alm et al., 1977; Larsson, 1977) Furthermore, VIP has been shown to relax cavernosal strips from dog (Carati et al., 1985) and man (Adaikan et al., 1984), in a concentration-dependent manner This VIP-induced relaxation was inhibited by the nitric oxide (NO) synthesis blocker N-ω-nitro-L-arginine (Kim et al., 1995) In view of the colocalization of acetylcholine (ACh), VIP and neuronal nitric oxide synthase (nNOS) in parasympathetic neurons (Hedlund et al., 1999), it is believed that they may act synergistically through inhibition of α1 adrenergic activity by ACh and release of NO by VIP (Lue, 2002) CGRP has been immunohistochemically identified in cavernous smooth muscle, nerve endings and within the arterial walls of the human CC (Su et al., 1986; Stief et al., 1990) Both VIPergic and CGRP positive nerves may thus, play a supportive role in modulating penile erection Other candidates include a relaxant factor acting through potassium (K+) channels (Okamura et al., 1998), non-NO dependent pathway (Reilly et al., 1997a) or involving cyclic adenosine monophosphate (cAMP) (Angulo et al., 2000)

1.2.2 Penile Erectile Process

Corpus cavernosum smooth muscle (CCSM) contributes predominantly to the control of tumescence and erection (Adaikan et al., 1999) Its contraction mediates detumescence / flaccidity and relaxation determines tumescence or erection The excitatory (adrenergic)

neurotransmission elicits contraction and the inhibitory (NANC- nitrergic) component mediates relaxation (Adaikan and Karim, 1978; Adaikan, 1979; Adaikan et al., 1991a) Dual (contractile and relaxant) effectiveness have been demonstrated in the human CC for adrenoceptors (Adaikan and Karim, 1981), cholinoceptors (Adaikan et al., 1983), and histaminergic receptors (Adaikan and Karim, 1976) in addition to various prostaglandins (Adaikan, 1979; Hedlund and Andersson, 1985) and their receptors (Angulo et al., 2002; Moreland et al., 2003)

(pro-of stimuli including through appropriate nerve stimulation

3 Intracavernous injections of putative neurotransmitter and modulator substances into the flaccid and erect penis have been used to demonstrate their effectiveness in animals and man

The existence of non-adrenergic non-cholinergic / NANC erectile neurotransmission in human penis has been documented (Adaikan, 1979; Adaikan and Karim, 1978) which was eventually identified as nitric oxide (Rajfer et al., 1992) Scientific evidence suggests that NO from NANC nerve endings is indeed the important modulator of penile erection (Adaikan et al., 1991a) and it can also be physiologically derived from the endothelial cells (Furchgott and Zawadzki, 1980; Bredt and Snyder, 1992) Produced enzymatically

from L-arginine and molecular oxygen (Palmer et al., 1988a and 1988b), this chemical substance mediates nitrergic transmission in the human CC Neural fibres immunoreactive for nitric oxide synthase (NOS), the enzyme(s) that catalyses this reaction, have been identified in the penis of various mammalian species through use of specific antiserum These fibres are concentrated in the trabeculae of corpora cavernosa and around the deep cavernosal and dorsal penile arteries (Burnett et al., 1992) Furthermore, use of NADPH-diaphorase, a specific marker for this enzyme, has provided information on the distribution of these fibres in human CC (Gopalakrishnakone et al., 1994) Intracavernous injection of NO donors such as S-nitrosocysteine and sodium nitroprusside has also confirmed this proerectile effect through studies in primates and man (Hellstrom et al., 1994)

In vivo animal studies have established the definitive role of neural NO in cavernosal relaxation after selective chemical ablation of the endothelium (Trigo-Rocha et al., 1993) This has led to the hypothesis that neural NO may be more important than the endothelium dependent nitric oxide (EDNO) under physiologic conditions (Ignarro et al., 1990; Vanhatalo et al., 1996) However, since compounds like ACh act through EDNO, the latter may still have a moderate supportive role in the myogenic activity of the CC and erection Regardless of its origin whether neuronal, endothelial or cavernosal smooth muscle (Kakiailatu, 2000), the NO once released, diffuses into the smooth muscle cell and causes relaxation of the corpora cavernosa through the guanylate cyclase and cyclic guanosine monophosphate (cGMP) pathway (Rajfer et al., 1992) Phosphodiesterase5 being the major hydrolyzing enzyme of cGMP is an important regulator of this relaxation (Kim, 2003)

Thus, the neuropharmacological processes that modulate the erectile activity are:

1 alpha adrenoceptor mediated motor neurotransmission that determines detumescence

2 relaxant neurotransmission through the chemical mediator nitric oxide and cGMP and

3 endogenous autacoids enhancing erectile activity through appropriate effects on the contractile or relaxant components (some of these release cAMP as the second messenger) (Adaikan et al., 1991b; 1996 and 1999), (Diagram 2)

VIP + ACh + Histamine

Papaverine / α-Blockers

Maintenance of rugosity

NANC/Nitrergic

cGMP cAMP

Neurotransmitter(s)

of non-erectile state

Modulators of erection

Neurotransmitter(s)

of erection

Diagram 2: Physiopharmacology of Human Penile Erection

(from Adaikan et al., 1999)

In recent years, studies involving nitrergic pathways leading to cellular cGMP release have contributed to the successful end organ therapy for impotence using oral PDE inhibitors such as sildenafil (Boolell et al., 1996), tadalafil (Porst, 2002) and vardenafil (Hellstrom 2003) Endogenous modulators such as PGE1 have been successfully shown

to relax the human cavernosum (Adaikan, 1979) and effective intracavernously in treating ED (Adaikan et al., 1986b; Virag and Adaikan, 1987)

1.2.2.2 Penile Detumescence

Electrical stimulation of CC invariably produced a contractile response which was antagonised by the alpha adrenergic receptor blockers like phentolamine (Adaikan and Karim, 1978; Adaikan, 1979) This indicates that the motor neurotransmission in erectile physiology is adrenergic Suppression of this adrenergic neurotransmission then unmasks the inhibitory response which is NANC (Adaikan and Karim, 1978) or nitrergic (Adaikan

et al., 1991a) in nature Clinically, intracorporal injection of phentolamine is a successful method of diagnosing erectile dysfunction Other compounds such as trazodone, ketanserin and yohimbine also antagonise the noradrenaline (NA) induced contraction of human CCSM and facilitate erection through their alpha receptor blocking effects (Adaikan et al., 1990) As expected, pharmacologically precipitated priapism or prolonged penile erection may be treated with intracavernosal alpha adrenergic agonists These results indicate that NA is the most dominant excitatory (contractile, anti-erectile) neurotransmitter of erectile function (Adaikan et al., 1991a) Further in vitro studies have demonstrated that the contractile response to be mediated through a heterogeneous population of alpha adrenoceptor subtypes; α1A, α1B and α1Ccorrespond to three distinct genes, all three being expressed in the human CC (Traish et al., 1995)

Beta adrenoceptors comprise only one tenth of the total adrenoceptors in the penile tissues (Levin and Wein, 1980) While these receptors do not significantly contribute to the relaxation (Adaikan, 1979; Adaikan and Karim, 1981), they may play a definitive role

in balancing the alpha receptor-mediated adrenergic tone Thus, the response to alpha adrenergic agonists is significantly potentiated when these proerectile beta adrenoceptors

are blocked by propranolol (Adaikan and Ratnam, 1988) Intracavernosal injection of beta agonist, salbutamol, induces penile tumescence and relaxes penile arterial strips that are precontracted with NA (Simonsen et al., 1997)

Apart from noradrenaline, it was found that endothelin (Holmquist et al., 1990; Lau et al., 1991; Zhao and Christ, 1995; Whittingham et al., 1996) along with other endothelium derived constrictor factors such as thromboxane A2, leukotrienes or PGF2α acted as an important modulator of penile flaccidity and detumescence (Hedlund and Andersson, 1985) While endothelin 1 (ET-1) induced cavernosal contraction is identified to be mediated through stimulation of the guanosine triphosphate - GTPase RhoA and activation of Rho-kinase (and abolished by Rho-kinase inhibitor Y-27632) (Chang et al., 2003), the role of constrictor prostaglandins in penile tumescence has not been explored further

1.2.2.3 Penile Tumescence

Cholinergic fibres were initially implicated as the primary effectors of penile erection However, Adaikan and associates showed that ACh had a dual effect on human CC strips (Adaikan et al., 1983) and cultured CCSM cells contracted on exposure to carbachol (Costa et al., 1993) Furthermore, Benson and colleagues using acetylcholinesterase localisation techniques showed that cholinergic nerve fibres, in contrast to the abundant adrenergic fibres, were seen infrequently in human CC and spongiosum (Benson et al., 1980)

Although stimulation of the parasympathetic sacral nerve fibres in rabbits, cats and dogs was erectogenic (Langley and Anderson, 1895; 1896; Seamans and Langworthy, 1938), there was no evidence that acetylcholine was the mediating neurotransmitter (Eckhardt, 1863; Henderson and Roepke, 1933; Dorr and Brody, 1967) The limited involvement of

cholinergic system is further supported by the fact that although physostigmine intensified the erectile effect of pelvic nerve stimulation in the dog, atropine failed to ablate it fully (Henderson and Roepke, 1933; Dorr and Brody, 1967) Thus ACh is unlikely to be the direct mediator of erection A more likely explanation for the role of the cholinergic component is that it may contribute to the suppression of adrenergic tone through presynaptic modulation of NA release (Tejada et al., 1988) and enhance the action of EDNO (Tejada et al., 1989) This is further supported by the fact that EDNO mediates both the ACh (Simonsen et al., 2002) and bradykinin induced relaxation of the

CC strips (Kimoto et al., 1990) and sildenafil enhanced the relaxant effect of ACh on the precontracted CC (Aydin et al., 2001)

Agents contributing to the penile relaxation (tumescence) and contraction (detumescence) are expected to facilitate erection and antierection respectively.

1.2.3 Cellular Erectile Physiology

Erectile physiology fluctuates between two functionally opposite responses namely contraction and relaxation (Figure 1) At the cellular level, an integrated autonomic myoneural stimulation results in an appropriate response through gap junctions forming syncytial tissue triads (Christ, 1997) Resulting action currents act on the proximal smooth muscle cell bundles and the distal cells through these gap junctions (Christ et al., 1997)

1.2.3.1 Cavernous Myoneural Junction

In general, the neurotransmission is controlled by opposing sympathetic and parasympathetic nerve terminals However at the end organ level, the major input for erection is NANC-mediated as discussed earlier The emerging comprehensive concept

of penile autonomic neurotransmission includes the release of substances from more than

one source as co-transmitters, neuromodulators and even primary transmitters (Mandrek,

1994) Since a variety of chemical substances contribute to the ultimate mediation of

erection, ED may stem from defects in any of this multitude of factors Hence, the

following is the review of the studies on cellular pharmacology of penile erection

Figure 1: Process of Human Penile Erection

(from Maggi et al., 2000)

1.2.3.2 Cavernous Smooth Muscle Contraction

Selective excitatory stimulation of the CCSM membrane receptors promotes contraction

or flaccidity It has been shown that the receptor activation facilitates binding of

guanosine triphosphate (GTP) to G protein present on the cytoplasmic membrane and

amplification of the original signal (Christ et al., 1997) This is followed by activation of

phospholipase C that hydrolyses the phospholipid fraction phosphatidyl inositol 4, 5

biphosphate This compound is converted into two second messengers namely diacyl

glycerol and inositol 1, 4, 5 triphosphate (Ferris and Snyder, 1992) The first compound

activates protein kinase C on the membrane The second active metabolite diffuses

through the cytoplasm to initiate the release of calcium from internal storage vesicles

Intracellular calcium ions play a major role in erectile physiology with increase in calcium maintaining flaccidity and reduction facilitating relaxation (Noack and Noack, 1997) The physiological response on the erectile tissue by the endogenous and exogenous substances may be said to be mediated through changes in the intracellular calcium levels (Melman and Gingell, 1999) Contraction of CCSM is facilitated by calcium entry through the L type calcium channels (Noack and Noack, 1997) with a 2 - 3 fold increase in its intracellular levels i.e from 120-270 nm to 550-700 nm (Stief et al., 1997) This transmembranic calcium ion transfer is triggered by various contractile stimuli mentioned earlier and follows the concentration gradient to result in increase of cytosolic calcium This step initiates the binding of calcium to calmodulin and the resultant calcium calmodulin complex phosphorylates light chain of myosin through myosin light chain kinase This promotes an interaction between actin and myosin and the subsequent phosphorylation and contraction of the smooth muscle (Chacko and Longhurst, 1994) The next important step to initiate cavernosal smooth muscle relaxation is seen as dephosphorylation and dissociation of the calcium-calmodulin complex Furthermore, channels for potassium (Christ et al., 1993a; Insuk et al., 2003) and chloride ions (Christ, 1997)have been identified in the cavernosum, their individual roles in spontaneous electrical activity and interdependence to calcium have been described (Karkanis et al., 2003)

1.2.3.3 Cavernous Smooth Muscle Relaxation

Cyclic adenosine monophosphate (cAMP) and cGMP modulate intracellular calcium stores (Lincoln and Cornwell, 1991)to initiate and sustain cavernosal relaxation cAMP exerts its effects by stimulating cAMP dependent protein kinase A and cGMP causes the relaxation through protein kinase G; these events result in dephosphorylation of myosin

light chains mentioned above.Studies indicate a complementary interplay between the two signaling mechanisms (Lincoln et al., 1990; Adaikan et al., 1991b; Jiang et al., 1992) Second messengers (cAMP and cGMP) mediated activation of potassium channel

is the likely key factor for potassium efflux and secondary reduction in calcium influx This decrease in calcium current through voltage dependent calcium channels promotes cavernosal relaxation (Stief et al., 1997; Christ et al., 1997)

1.2.3.4 Ion Transfers at Gap Junctions

Action potentials were not identified by electrophysiological investigations in the cultured cells of the human CC (Andersson and Wagner, 1995) Instead, cavernous smooth muscle tone was a function of inter-cellular transport of various ions and second messengers through intercellular spaces referred to as gap junctions (Christ et al., 1993b)

At these junctions, the communication was established by hexagonal arrays of protein units called connexons (Bennett et al., 1991); each unit was in the form of a channel that extended to the next in the adjacent smooth muscle cell These gap junctions led to the formation what is identified now as the syncytial tissue triad in regulation of cavernosal smooth muscle contraction or relaxation This system permits rapid transfer of physiological activity from cell to cell and exchange of various chemical messengers (Lerner et al., 1993) For instance, the diameter of each channel is regulated by the amount of intracellular calcium whose influx causes the subunits to merge resulting in contraction / flaccidity (Christ et al., 1997) A connexon isomer which is identified in the human CCSM cell membrane is named connexin 43 (Brink et al., 1996; Christ et al., 1997)

At the cellular level, a physiological antagonism of the intracellular calcium, effected by changes in the concentration of the second messengers, cAMP or cGMP causes

relaxation of the cavernosal smooth muscle (Christ et al., 1997) Clinically useful intracavernous agents like PGE1 (Adaikan et al., 1986b; Virag and Adaikan, 1987) act through increases in the levels of cAMP The relaxant effect of NO of both neural and endothelial origin is mediated through the elevations of cGMP through which PDE inhibitors work (Boolell et al., 1996; Porst, 2002; Hellstrom 2003) As described earlier, secondary to these increases is the activation of protein kinase A or G from cAMP and cGMP pathway links, respectively (Jiang et al., 1992; Cornwell et al., 1994; Figure 2)

Figure 2: Molecular Mechanism of Penile Erection

(from Lue, 2002)

1.2.4 Central Pathways

The role of supraspinal mechanisms in erectile function have been studied in various species using selective lesioning and electrical and chemical stimulation techniques Studies in primates and rodents have identified the role of hypothalamic and limbic pathways in erection The medial preoptic anterior hypothalamic region functions as an integrating centre in modulating responses (De Groat and Steers, 1988; Sachs and Meisel, 1988) Efferent pathways from this area enter the medial forebrain bundle and then pass caudally into the midbrain tegmental region near the lateral part of substantia nigra Distal to the midbrain, the efferent pathways traverse the ventrolateral part of pons and medulla before integrating with the autonomic centres in the spinal cord (see Holstege, 1987) Impulses may then exit the spinal cord through the thoracolumbar sympathetic or the sacral parasympathetic fibres In contrast to the role of hypothalamus in sexual responses, the function of other higher centres in modulating penile erection is less clear This is because a variety of stimuli can elicit erectile responses through interlinking of different anatomical substrates to the hypothalamus (Andersson, 2003) These include 1 visual and somatosensory signals from the thalamic nuclei 2 olfactory inputs through the rhinencephalon including the cingulate gyrus, septum and the hippocampus 3 emotion and memory associated through the limbic structures comprising temporal and frontal cortical lobes and the hippocampus This indicates that several neural mechanisms elicit penile erection and may thus be separately or collectively affected in patients suffering from ED

1.2.5 Spinal Pathways

Electrophysiological experiments in the rat have provided insight into the pathways and mechanisms involved in reflexogenic erections (Steers et al., 1988) Electrical stimulation

of dorsal nerve elicits the reflex arc with discharges from the major pelvic ganglion into the penile nerves The horseradish peroxidase tracing studies in the rat and monkey have demonstrated termination of the pudendal afferents in the dorsal commissure and medial dorsal horn of the spinal cord (Nunez et al., 1986; Roppolo et al., 1985) Interneurons in these regions are activated by tactile stimulation of the afferents in the penis and are involved in transmitting sensation to the brain, in addition to activating the sacral preganglionic neurons that initiate erection The parasympathetic preganglionic neurons are located in the intermediolateral nucleus and send dendritic projections to the same regions that receive the penile afferent inputs These neural components form the anatomical substrates for reflexogenic erections

Similar spinal reflex mechanism or pathway is present in man and it becomes evident following spinal cord lesions For example, in patients with complete upper level spinal cord injury, reflexogenic erections can occur, although not as sustained or spontaneous as desired Sensory stimuli elicited in the glans and perigenital areas are eventually carried

by the dorsal penile nerves to the sacral spinal cord through the pudendal nerves (Andersson and Wagner, 1995) The efferent limb of the pudendal nerve which is contained in the sacral parasympathetic outflow supplies the ischiocavernosus and the bulbospongiosus muscles A stimulation of this motor innervation does not appear to elicit appreciable increase in intracavernous pressure (ICP) Nevertheless, the resultant contraction of these striated muscles contributes to additional rigidity at coitus by pressure effects on the glans and proximal part of the penis and may also play a role in nocturnal penile tumescence (Giuliano et al., 1995) In view of the fact that the efferent limb of the pudendal nerve terminates at the ischiocavernosus and bulbospongiosus muscles, it is conceivable that during the reflexogenic loop a trigger of the

parasympathetic outflow via “nitrergic pathway” mediated through the afferent sensation

to the brain or by other mechanism is likely in man

1.2.6 Vascular Factors

The neurophysiological mechanisms are integrated with the vascular supply to produce and maintain penile erection All levels of central, spinal and peripheral systems participate in the neural control of the vascular events

1.2.6.1 Cavernosal Blood Flow

Internal pudendal artery, a branch from the anterior division of the hypogastric or internal iliac artery, is the source for penile arteries Cavernous tissue is primarily supplied by the deep penile or cavernous artery which is one of the terminal branches of the common penile artery The dorsal penile artery may sometimes supply the cavernous tissue with multiple branches along the shaft of penis, as a normal variant The cavernous artery penetrates the tunica albuginea to enter the crura of corpora cavernosa along with the cavernous vein and nerve and divide into multiple terminal helicine branches These arteries open directly into the cavernous spaces (Lue, 2002) The cavernous spaces drain into a system of venules that coalesce on the outer surface of cavernosa just beneath the tunica albuginea These venules form a number of veins traversing the tunica, called the emissary veins which drain into the circumflex veins These in turn drain into the deep dorsal vein of the penis, in the dorsal midline of the penile shaft between the dorsal arteries The proximal emissary veins form the cavernous vein which then drains into the internal pudendal vein (Andersson and Wagner, 1995; Lue, 1998; 2002)

1.2.6.2 Intracavernous Pressure

Functionally, in the flaccid state, there is a high resistance, low flow arterial system regulated by the contracted cavernosal smooth muscle tissue (Oates et al., 1995) With

the initiation of erection, the cavernous and arterial smooth muscles relax, resulting in a low resistance system that entails arterial inflow (Wegner et al., 1995)

At full tumescence, the blood flow increases from 5ml/min (5-8mm Hg) in the flaccid state to 50-80ml/min with the ICP then paralleling mean systolic blood pressure (Christ et al., 1997) Further increments to this pressure change are from venous outflow resistance Compression of subtunical venular plexus between the expanding sinuses and the non-compliant tunica further helps to sustain erection through impeded venous drainage (Aboseif et al., 1989) This is further augmented by pressure occlusion of the emissary veins (Fournier et al., 1987)

1.2.7 Hormonal Factors

1.2.7.1 Effect of Prolactin

Both primary and secondary forms of hyperprolactinemia may be associated with loss of libido and ED The underlying mechanism is two-fold Centrally, excessive prolactin inhibits the secretion of GnRH and in the periphery, it is shown to interfere with the activity of 5α reductase, an enzyme required for the formation of dihydrotestosterone (DHT) in target tissues (Hsueh, 1988)

1.2.7.2 Effect of Oxytocin

Oxytocin plays a role in the hypothalamic regulation of erection and is released into circulation during sexual activity in humans (Carmichael et al., 1987) and animals (Stoneham et al., 1985); an intracerebroventricular or hippocampal administration induces penile erection in rats (Argiolas and Melis, 1995) The erectogenic effect of oxytocin which is calcium-dependent is probably mediated through the activation of neurons in the paraventricular nuclei (Argiolas et al., 1990)

1.2.7.3 Effect of Testosterone

1.2.7.3.1 Testosterone Physiology

Pulsatile releases of GnRH from the hypothalamus together with the secretion of pituitary gonadotropins, LH and FSH initiate and maintain testicular testosterone (Griffin and Wilson, 1992) Both LH and FSH contribute to testicular growth, spermatogenesis and steroidogenesis At the cellular level, their functions are mediated through cAMP formation; LH stimulates interstitial (Leydig) cells of the testis to increase the synthesis

of cAMP This is followed by the conversion of cholesterol through enzyme mediated steps to steroid hormones, an administration of T to intact animal suppressed LH release thereby causing Leydig cell atrophy (Winters, 1994) The FSH plays a facilitatory role in LH-mediated testosterone synthesis (Wilson, 1996)

In man, testicular Leydig cells account for synthesis of 95% of the testosterone Once formed, testosterone serves as the precursor (prohormone) for the formation of two other steroidsthat also mediate many of the systemic effects of testosterone For instance, T is 5α-reduced to DHT and oestrogen(s) is formed by aromatase enzyme complex in the peripheral tissues viz., fat, muscle, kidney and liver Oestrogens thus produced in males may function as simple active metabolites of androgens or have independent as well as opposing physiological functions to androgens (Griffin and Wilson, 2001) Thus a specific tissue effect of testosterone may be the net result of combined effects of testosterone itself and its active androgen as well as oestrogen metabolites (Diagram 3)

Diagram 3: Metabolites of Testosterone

(from Snyder, 2001)

Testis is the main source for testosterone with the overall daily production amounting to approximately 6mg About 85% of the circulating concentrations of DHT and oestradiol are derived from the periphery and the rest from testis (Griffin and Wilson, 2001) All steroid hormones exist either in a free (unbound) state or in combination with serum proteins; approximately 38% of testosterone is bound to albumin and 60% to sex hormone binding globulin (SHBG), also called testosterone binding globulin (TeBG) in man Testosterone bound to SHBG is biologically unavailable due to its relatively tight binding (Tenover, 1998) In comparison, circulating oestradiol is loosely bound to albumin or unbound and to a lesser extent to SHBG because of its low affinity compared

to testosterone (Winters, 1994)

Physiologically, in man, DHT mediates body hair growth, acne, scalp hair loss and prostate growth Unbound T promotes positive changes in muscle mass, skeletal growth, spermatogenesis and sexual function and oestradiol is shown to stimulate bone formation and breast tissue proliferation (Velazquez and Arata, 1998)

1.2.7.3.2 Testosterone in Male Sexual Function

The role of circulating androgens with regard to erectile physiology and sexual behaviour

is incompletely understood (Schiavi et al., 1991) since in men with normal gonadal function there is no correlation between testosterone levels and measures of sexual interest, activity or erectile function (Andersson and Wagner, 1995)

In lower animals, certain well-defined changes in sexual activity are precipitated following castration Among the different parameters of the copulatory pattern, the ability

to ejaculate disappears early, followed by intromission and mounting (Hart, 1967) Hormonal replacement restores all aspects of mating in these animals and it is interesting

to note that the amount of circulating testosterone required to reinstate such behaviour is considerably less than the normal range for the animal species (Davidson et al., 1978; Gray et al., 1980) Mechanism of T-induced sexual activity is mainly central This can be demonstrated by the fact that exogenous T restores apomorphine induced erections in castrated rats (Scaletta and Hull, 1990; Heaton and Varrin, 1994) Peripherally in cavernosa, it mediates nitrergic neurotransmission, accentuates NOS activity, promotes

NO release (Weiner et al., 1994; Chamness et al., 1995; Zvara et al., 1995) and increases ICP (Traish et al., 2003) Together, it implies that testosterone has a dual physiological role in animals, centrally on the libido and peripherally as a co-modulator of the mechanisms of penile erection