TABLE OF CONTENT
Title page – – – – – – – – – – -i
Certification page – – – – – – – – -ii
Dedication — – – – – – – – – -iii
Acknowledgement- – – – – – – – -iv
Table of contents – – – – – – – – – -v
Abstract – – – – – – – – – – -viii
CHAPTER ONE
Introduction – – – – – – – – – -1
1.1 Prevalence of erectile Dysfunction in women- – – – -2
1.2 Prevalence of erectile Dysfunction in men– – – – -3
1.3 Objective study and aims – – – – – – – -4
1.4 Nitric oxide-cyclic GMP pathways with some emphasis on
cavernosal contractility – – – – – – – – -4
1.5 Synthesis of Nitric oxide – – – – – – -6
1.6 Inactivation – – – – – – – – -10
1.7 The nitric oxide Receptor: soluble guanylate cyclase – – -10
1.8 Intracellular cyclic GMP receptor proteins – – -12
CHAPTER TWO
Literature review
2.1Normal pennies Anatomy – – – – – – -17
2.2How election occurs in males – – – – – -18
2.3How election sustained – – – – – – -18
2.4Causes of ED in males – – – – – – -18
2.5Physical causes ED in males – – – – – -19
2.6Psychological causes of ED in males – – – – -23
2.7Diagnosis of erectile dysfunction – – – – – -24
2.8Literature review on female erectile dysfunction – – -27
2.9Normal anatomy of the female external Gentialia – – -27
2.10 Causes of ED in females – – – – – -28
2.11 Psychological causes of ED in females – – – -31
2.12 Mechanisms of action burantashi – – – – -32
2.13 Taxonomy – – – – – – – – -35
vi
2.14 Cholesterol – – – – – – – – -36
2.15 Dietary source and effect of diet in cholesterol – – -38
2.16 Functions‟ of cholesterol in body – – – – -40
2.17 Lipoprotein metabolism – – – – – – -41
2.18 Reaction catalyzed by LCAT – – – – – -42
2.19 Very-low density lipoprotein (VLDL) – – – – -45
2.20 Metabolism of VLDL – – – – – – -45
2.21 Low density lipoproteins – – – – – – -45
2.22 Function of LDL – – – – – – – -47
2.23 High density lipoprotein – – – – – – -47
2.24 Clinical significant – – – – – – -48
2.25 Role of alpha adrenergic receptors in erectile function – -49
2.26 Role of alpha 1 and alpha 2 adrenergic receptors in human penile
erectile function – – – – – – – – -51
2.27Classification of alpha-adrenergic receptor subtypes in human corpus
cavernosum – – – – – – – – -52
2.28Idenification of alpha 1 adrenergic reception subtypes in human corpus
cavernosum – – – – – – – – -53
2.29Identification and characterisation of alpha-2 adrenergic receptor
substypes in penile corpus cavernosum – – – – -54
2.30Functional (psysiological) studies of alpha and alpha-2 adrenergic
receptor in erectile tissue. – – – – – – -55
2.31Molecular mechanism of alpha-1 adrenergic receptors in erectile tissue
corpus cavernosum – – – – – – – -56
2.32Molecular mechanism of action of alpha-2 adrenergic receptor in
erectile tissue corpus cavernosum – – – – -57
2.33Blockade of alpha 1 and alpha 2 adrenergic receptor activity by
selective and non-selective alpha 1 an 2 receptor antagonises -59
2.34Alpha 1 and alpha 2 selective antagonists – – – -60
2.35Alpha 1 selective antagonists – – – – – -61
2.36Alpha 2 selective antagonists – – – – – -62
2.37Physiological (Functional) antagonism of alpha and alpha 2 adrenergic
receptors activity by vasodilators: the balance between contraction
and Relaxtion – – – – – – – – -63
vii
CHAPTER THREE
Material and methods
3.1materials – – – – – – – – -68
3.2extract yield of ethanol extract and aqueous extract – – -75
3.3Phytochemical properties of extract – – – – -79
3.4Effect of extracts on serum glutamate oxaloacetate transferase (SGOT)
activity of Wistar rats – – – – – – —— -81
3.5Effect of extract on serum glutamate pyrurate transaminase (SGPT)
activity of Wistar rats – – – – – – – — 82
3.6Effect of extracts on alkaline Phosphatase (ALP) activity of wistar rats
– – – – – – – – – – – 83
3.7Effect of extracts on plasma glutamate transferase activity (IULV) of
wistar rats. – – – – – – – – -83
CHAPTER FOUR
4.1. Yield of the extracts – – – – – – – -82
4.2. Phytochemical data – – – – – – – -82
4.2. Effect of extracts on cholesterol level of rats – – – -83
4.3. Effect of extracts on LDL level of rats – – – – -84
4.4. Effects of extracts on HDL of rats – – – – -85
4.4. Effects of extracts on Triacylglycerol on diabetic rats – -86
CHAPTER FIVE
5.1Discussion and conclusion – – – – – – -87
References – – – – – – – – – -88
viii
ABSTRACT
This work was carried out to investigate the effects of Burrantashi
extracts on the lipoproteins Burantashi is a popular seasoning agent
to barbecued meat (Suya) in Nigeria. Found in the northern parts of
the country. Lipoproteins are the principal steroid or fat that is
synthesized in the liver or intestines of animals. Erectile
dysfunction (ED) is defined as the consistent or recurrent inability
of a man to attain or maintain penile erection, sufficient for sexual
activity (2nd international consultation on sexual dysfunction Paris,
June 28th
–July 1
st 2003). Following the discovery and introduction
of burantashi research on the mechanism underlying penile
erection, had an enormous boost and many preclinical and clinical
papers have been published in the last five years on the peripheral
regulation of, and the mediators involved in human penile erection.
The most widely accepted risk factors for e.g. are discussed. The
research is focused on human data and the safety and effectiveness
of burantashi stem as a phosphodiesterase – 5- inhibitors (PDEs)
used to treat erectile dysfunction.
1
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.
2
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.
3
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.
4
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
5
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.
6
This review will describe current knowledge of cellular events involved
in cavernosal relaxation and the range of putative factors involved in NOmediated relaxation.
1.5 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, calcitonin 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
7
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.
8
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.
9
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 NMonomethyl -L- arginine (L- NMMA), nitro-L- arginine methyl ester (LNAME). 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
10
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-bearingenzymes. 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
11
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 Cterminal 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.
12
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 Snitrosothiols. 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.
13
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.
14
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 enzyme‟s 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
15
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.
16
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.
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