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.

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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.

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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 1520000 Doctor Offices visited for ED.

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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

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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.

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This review will describe current knowledge of cellular events involved

in cavernosal relaxation and the range of putative factors involved in NO-

mediated 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 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

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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

(BH

4

) - 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 Ca

2+

-calmodulin whereas inducible forms are not.

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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 Ca

2+

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.

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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

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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

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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-Subunithas 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.

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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 –

58-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.

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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 Ca

2+

- pumping ATPase

an action that would be predicted to lower [Ca

2+]

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 [Ca

2+

]. It has been shown to phosphorylate and therefore

inhibit the inositol 145- 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.

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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

Ca

2+-

ATPase by the stimulation of phosphatidylinositol -4-phosphate

(PIP) formation by cGMP kinase and phosphorylation of 240-KDA

protein that mediates the activation of Ca

2+

-ATPase by cGMP kinase.

PKG may catalyze the phosphorylation of phisphatidylinositol kinase.

Leading to the formation of PIP and the activation of Ca

2+

-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 Ca

2+

-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 isoformswhich are distinguished by

their substrate specificities and tssue concentration.

To date five of these 11 isoenzymes (PDE1234 and 5) have

been proven to be of pharmacological relevance. Currently the

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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.

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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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