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PDE5 is a dimeric enzyme that is composed of two identical 100 kD proteins. There are four known isoforms of PDE5 ( PDE5A 1-4 ) ACC . No : NM _ 03343I : these isoforms are products of a single gene and are formed by alternative splicing of mRNA . The enzymatic characteris- tics of the PDE5A1-3 appear to be quite similar although there may be some selectivity in their tissue distributions ; PDE5A3 is largely expressed in smooth muscle. The enzymatic properties of PDE5A4 have not been character- ized . Each of the monomers in PDE5 is a chimeric protein that contains two major functional domains that are approximately equal in size , i . e ., a catalytic domain ( C domain ) located in the more C-terminal portion of the protein and a regulatory domain ( R domain ) located in the more N-terminal portion. The single catalytic site located in the C domain is the target for sildenafil. The R domain of PDE5 contains allosteric cGMP-binding sites that contribute importantly to regulation of enzyme functions and to potency of these PDE5 inhibitors . However , the allosteric cGMP-binding sites in PDE5 are evolutionarily and biochemically distinct from that of the catalytic site . These sites are highly specific for cGMP : and do not interact with sildenafil. The catalytic site binds cGMP in a shallow pocket along the sur- face of the enzyme . When cGMP occupies this site , the cyclic phosphate bond of cGMP is brought into proximity with the catalytic machinery of the enzyme , which involves an array of amino acids and divalent cations including Zn2 +. This arrangement provides for the rapid hydrolysis of the cyclic phosphate bond of cGMP to form 5 ‘ -GMP , which has low affinity for the enzyme and rapidly dissociates from PDE5 . 5 ‘ -GMP is inactive in the cellular cGMP-signaling pathway . Other cellular phosphohydrolases do not hydrolyze the novel cyclic phosphate bond of eGMP or cAMP . Because the structure of sildenafil resembles that of eGMP , it can occupy the PDE5 catalytic site , thus blocking access to eGMP . In addition , sildenafil is stable and is not inactivated by the catalytic machinery ; nor is it metabolized significantly in the smooth muscle cell . For these reasons , occupation of the PDE5 catalytic site by sildenafil competitively inhibits eGMP breakdown since eGMP cannot gain access to the catalytic machinery . In the face of ongoing synthesis of eGMP in any tissue containing PDE5 , this will cause eGMP to accumulate and to increase cGMP signaling through PKG . In the penile corpora cavernosa , this contributes to improved erectile function . Although the C domain of PDE5 is the direct target of PDE5 inhibitors, functions of the R domain enhance the PDE5 inhibitor actions on the enzyme . Allosteric cGMP-binding is provided by sites in the R domain ; whether one or two eGMP molecules are bound per subunit is still unclear . In addition , there is a single consensus phosphorylation site for PKG or PKA near the N-terminus . Phosphorylation of this site activates PDE5 catalytic function and thereby provides for negative feedback regulation of cGMP levels. Phosphorylation of this serine is tightly controlled by cGMP levels since occupation of the PDE5 allosteric cGMP-binding sites is required for phosphorylation to occur , and the site is preferentially phosphorylated by PKG compared to PKA . Therefore , it is likely that the site is only phosphorylated when cGMP is elevated in the cell . When cGMP binds to the allosteric sites , cGMP is not degraded as it is in the catalytic site , but PDE5 enzyme functions are altered . Cyclic GMP binding to the allosteric sites in the R domain pro- duces a conformational change that exposes the serine allowing it to be rapid- ly phosphorylated , thereby increasing PDE5 catalytic activity. Cyclic GMP occupation of the allosteric sites also increases the affinity of the catalytic site for cGMP , thereby further activating PDE5 catalytic site func- tions. However , in the presence of a PDE5 inhibitor and ongoing syn- thesis of cGMP , cellular cGMP is elevated , which fosters increased binding of cGMP to the allosteric sites and phosphorylation of the serine by activated PKG ; as a result , the affinity with which PDE5 inhibitors bind at the catalytic site is increased . Therefore , due to its molecular mechanism , the potency of sildenafil is actually greater than would occur in the absence of the R domain . This property of the enzyme translates into greater clinical efficacy and poten- cy of sildenafil and other PDE5 inhibitors . Following ingestion of a PDE5 inhibitor tablet , any elevation of cGMP in smooth muscle cells should increase the avidity with which the PDE5 catalytic site binds that inhibitor . That is , the PDE5 inhibitor , by fostering increased binding of cGMP to PDE5 allosteric sites , stimulates its own efficacy and potency.

Sildenafil is a competitive and reversible inhibitor of cGMP hydrolysis by the catalytic site of PDE5 . Sildenafil is an analog of cGMP , the PDE5 substrate , but PDE5 has - 1000-fold higher affinity for sildenafil than for cGMP. The purine ring of cGMP is mimicked by similar components in sildenafil , and this undoubtedly provides in part for the specificity of the com- pound for the PDE5 catalytic site. However , the novel components of the sildenafil structure provide for the higher affinity , and these additional ele- ments in the sildenafil structure most likely exploit interactions in and around the PDE5 catalytic site that are not utilized in interacting with cGMP . Furthermore , these distinctive structural components most likely contribute to the specificity of the compound for the PDE5 catalytic site since the structure cannot be accommodated in other cGMP-binding sites such as those found in PKG and cGMP-gated cation channels , in the catalytic sites of other PDEs , or in the allosteric cGMP-binding sites of PDE5. The characteristics of the novel interactions between sildenafil and the catalytic site of PDE5 are empha- sized by the fact that the potency of sildenafil can be significantly altered by minor changes in its molecular structure . Although the molecular differences in sildenafil and vardenafil are apparently modest , vardenafil is -10-40 times more potent in inhibiting PDE5. Among several possibilities , this could be due to the different positions of nitrogen atoms in the imidazotriazinone ring of the two compounds , which might engender differences in electron dis- tribution .

In all cells , the level of cGMP is primarily determined by the relative rates of cGMP synthesis by the GCs and its breakdown by PDEs. An imbal- ance between the synthesis ( e . g ., decreased release of nitric oxide due to penile nerve deterioration or endothelial cell damage ) and breakdown of cGMP can compromise the accumulation of cGMP in cells . Cyclic GMP-dependent pro- tein kinase ( PKG ), the main mediator of smooth muscle relaxation , may become deficient , or intracellular Cat could become excessive or overly active . Finally , among other insufficiencies , the contractile machinery itself may not be amenable to adequate relaxation . Under these scenarios , the cGMP- signaling pathway may not be sufficiently activated to elicit adequate vasodila- tion to foster penile erection . For example , the cGMP level achieved by sexual arousal may be insufficient to activate PKG and bring about proper relaxation of the penile vascular smooth muscle ; this can result in MED . Since PDE5 is the major cGMP-hydrolyzing PDE in the penile corpora cavernosa , it is a log- ical target for pharmacological intervention to enhance cGMP accumulation by blocking the cGMP hydrolytic activity of this enzyme. Sildenafil alone has no effect on vascular smooth muscle tone , and in the absence of sexual arousal , it has no effect to facilitate penile erection . The need for the driving force of the nitric oxide agonist in order for sildenafil to act suggests that basal GC activity is low in the penile vasculature . However , when PDE5 catalytic activity is blocked by an inhibitor such as sildenafil , even a modest elevation of GC activity could produce a robust elevation of intracellular cGMP thereby improving erectile function . This is consistent with the action of nitric oxide and sildenafil on separate steps within the same nitric oxide / cGMP signaling pathway , with nitric oxide increasing cGMP formation by GC and sildenafil efficiently blocking cGMP breakdown. This synergistic effect amplifies cGMP accumulation and provides at least in part for targeting of the sildenafil effect to the penis . This also the reason that concomitant use of nitrovasodilators ( which release nitric oxide ) and sildenafil is strongly con- traindicated since severe systemic hypotension may result.

Sharron H . Francis and Jackie D . Corkin

Light Hall Room 702 , Department of Molecular Physiology & Biophysics , Vanderbilt University School of Medicine , Nashville , TN 37232-0615 , ( ISA Introduction The roles of cAMP ( cyclic AMP ) and cGMP ( cyclic GMP ) in regulating myr- iad physiological and pathophysiological processes are well established , and our understanding of the actions of these nucleotides continues to rapidly expand . Cyclic AMP was discovered in the late 1950s by Dr . Earl Sutherland ; the discovery of this novel compound derived from his studies on the regula- tion of glycogen metabolism . The vast number of biological systems affected by cAMP action was quickly appreciated. Cyclic GMP in biological tissues was discovered somewhat later in 1963. However , aside from the role of cGMP in visual transduction , scientists had difficulty in establishing a defini- tive role for cGMP in physiological processes . The importance of cGMP as a second messenger in relaxation of vascular smooth muscle emerged in the 1970s , and today we realize the impact of this molecule in many other systems. The role of cGMP in smooth muscle is the basis for the efficacious actions of nitrovasodilators such as nitroglycerin in the treatment of angina pectoris or cyclic nucleotide phosphodiesterase ( PDE ) inhibitors in the treat- ment of male erectile dysfunction ( MED ). Dr . Earl Sutherland and his colleagues were the first to identify and charac- terize the adenylyl and guanylyl cyclases that synthesize cAMP and cGMP and the PDEs that degrade these compounds. The phosphohydrolase activ- ity associated with PDEs is highly specific for action against the novel six- member cyclic phosphate ring , which is the distinguishing feature of cyclic nucleotides ; cAMP and cGMP are largely resistant to other phosphohydrolases. While there are numerous adaptive processes for reducing the synthesis of cAMP and cGMP , the role of PDE action is absolutely critical in controlling cellular levels of these highly active signaling molecules because the catalytic action of PDEs provides essentially the only way for a cell to rapidly and effi- ciently lower its cyclic nucleotide content. Furthermore , the catalytic activities of these enzymes are rapidly regulated by a variety of mechanisms in response to the changing intracellular milieu . A number of cells have been improving erectile function in a large percentage of men with erectile dys- function , and its safety profile is excellent. The overall safety of Viagra ‘ M in a variety of patient populations has been validated in more than 120 clinical trials , and despite more than five years of prescribing to more than 20 million patients worldwide , reported side effects are minimal . Whether the two other PDE5 inhibitors , i . e ., vardenafil ( LevitraFM ) and tadalafil ( Cialis ‘ M ), that have recently entered the marketplace will have the same safety and effi- cacy profile as sildenafil remains to be seen . Like sildenafil , tadalafil and var- denafil are highly selective and potent for the catalytic site of PDE5 . Since its introduction , sildenafil ( Viagra ‘ M ) has dominated the market for treatment of MED, and new prospects for its use in the treatment of other maladies are emerging. The remarkable therapeutic success and safety of sildenafil has focused attention on better defining the properties of this and other PDE5 inhibitors as well as on the properties of PDE5 that con- tribute to the high affinity of these inhibitors for interaction with the PDE5 cat- alytic site . Reports indicate that sildenafil may be therapeutically efficacious in the clinical management of other disease processes including improved reg- ulation of pulmonary artery pressure associated with pulmonary hypertension , relief of symptoms associated with Raynaud ‘ s phenomenon , improved recov- ery from stroke , and reduced tone in the lower esophageal sphincter of patients with achalasia. Sildenafil may also be neuroprotective. Features of PDE5 that contribute to efficacy and potency of sildenafil and the pharmacokinetics of sildenafil will be discussed in this Chapter . The clinical success and safety profile of sildenafil ( ViagraTM ) is now very well documented. The high potency and selectivity of sildenafil for PDE5 are certainly major factors in its efficacy and safety, but other factors also contribute to its success ; this includes the limited tissue distribu- tion of PDE5 and the stringent specificity of PDE5 for cGMP as a substrate . The tissue distribution of PDE5 is restricted compared to other PDE families . PDE5 is highly abundant in platelets , some neuronal cells , and notably in smooth muscle throughout the body including the vascular smooth muscle of the systemic vasculature and that of specialized vascular beds such as that of the penile corpora cavernosa. Because of this , pharmaco- logical intervention that targets PDE5 is less likely to have broad effects involving many tissues . As will be discussed below , specific targeting by PDE5 inhibitors is also achieved in part by the synergistic action of these drugs with sexual arousal . Importantly , PDE5 is not abundant in cardiac tissue , but it is undoubtedly present in the smooth muscle of the blood vessels traversing the heart muscle . Since PDE5 is highly specific for hydrolyzing cGMP , a PDE5 inhibitor is unlikely to elicit major problems in cAMP-signaling pathways . In some tissues containing the dual-specificity PDE3 , blocking PDE5 action can also cause an elevation of cAMP; this is because cGMP and cAMP are both hydrolyzed by PDE3 , and they compete for the PDE3 catalytic site . Selective elevation of either of the nucleotides can decrease hydrolysis of the other . However , since activation of either the cAMP- and cGMP-signaling pathways brings about smooth muscle relaxation , the overall effect of the PDE5 inhibitor would not be impaired in that tissue . PDE3 is much more widely distributed than is PDE5 . The likelihood of such ” cross-talk ” involving PDE5 in myocardial contractile cells is also diminished by the presence of low levels of PDE5 in these cells . Therefore , it is unlikely that PDE5 inhibitors would affect heart contractility by this mechanism . Role of ‘ cGMP signaling in effects of PDE5 inhibitors The role of cGMP in modulating function in smooth muscle throughout the body including that of the penile vasculature is well established ; all share a common pathway for cGMP signaling through activation of cGMP-dependent protein kinase ( PKG ) . Nitric oxide is released as a neurotrans- mitter from nerves innervating vascular structures in the penis , and it is also released as a paracrine agonist from endothelial cells lining blood vessel walls ; nitric oxide initiates the cGMP-signaling cascade by diffusing into the vascu- lar smooth muscle cell where it forms a high-affinity complex with a heme prosthetic group in the nitric oxide-sensitive guanylyl cyclase ( GC ). When nitric oxide binds to the heme group , GC is activated , and production of cGMP from GTP is dramatically increased. One of the main intracellular targets for cGMP is PKG ; cGMP binds to allosteric cGMP-binding sites on PKG and activates this kinase. The activated PKG then phosphorylates a number of proteins to cause diminished intracellular Cat ‘ effects due to increased extrusion of Cat +, increased sequestration of Ca 2 + in intracellular structures , and decreased sensitivity of target proteins to Ca 2 +. This results in decreased vasomotor tone and smooth muscle relaxation in all known smooth muscle cells . This process occurs selectively in the penile corpora cav- ernosa following sexual arousal due to increased local release of the neurotransmitter nitric oxide from the non-adrenergic , non-cholinergic nerve endings in the penis and from the arterial endothelial cells of the penile vasculature. As a result , blood flow into the penis increases , the capacitance of the cor- poreal sinusoids is enhanced , and blood accumulates within these structures . This provides for increased tumescence and an erectile response .

Viagra is a recent invention , but sex is not . And for as long as humans have engaged in sexual activity , there has been impotence . The word impotence first appeared in the fifteenth century , but today it has been abandoned in politically correct circles in favor of the term erectile dysfunction , or more colloquially , ED . Incredibly , it took a panel of experts to meet and decide on the name change . In 1993 , at the Consensus Meeting of the National Institutes of Health , participants agreed that the failure of the penis to achieve and sustain adequate rigidity for sexual intercourse was equivalent to other types of organ failures , such as hepatic dys- function for liver disease . Moreover , the experts recognized that the word impotence had negative connotations that should not be invoked just because a man ‘ s penis did not work properly . To be impotent , after all , means that someone lacks power , force , or effec- tiveness . Since we would not attribute such qualities to someone whose liver did not work well , the meeting attendees agreed that the term impotence should not be applied to men with erectile dysfunction . Nevertheless , unlike men with liver disease , kidney disease , or rhythm disturbances of the heart , men with erectile dysfunction do in fact feel impotent , in the full sense of the word . For this reason , I continue to use the word interchangeably with erectile dysfunc- tion . Depression , embarrassment , a diminished sense of masculin- ity-these are some of the feelings men experience when erectile dysfunction occurs . Women are often surprised at the depth of despair men exhibit when the erection fails . Of course , it can be disappointing at the moment for both partners . But when it first happens to a couple , many women describe an ” oh , well ” attitude about it , with the expectation that there will be other opportunities later . Only rarely do men have such a response . Typically , they expe- rience a frenzy of anxiety and despair when they go soft during a sexual encounter . The experience is mocked in the first of the Austin Powers movies , when the comic sleuth Austin temporarily loses his ” mojo .” The cocky , sexually aggressive aspect of his per- sona disappears until he gets it back . On a much more serious note , many a young man fantasizes about being a great lover-a Casanova or Don Juan-but if his penis will not cooperate enough even to get started , then what hope of sexual greatness can he entertain ? A torrent of questions rushes in on him when he expe- riences erectile dysfunction for the first time : ” What is happening ?” ” Why ?” ” Why now ?” ” Does this mean that my penis is broken for good ?” ” Will I never be able to have sex again ?” ” Will she laugh at me ?” ” Will she leave me ?” ” Will she tell everyone and shame me ?” Since a normal erection occurs as if by magic , the failure of the penis to respond appropriately in a sexual situation can be an unpleasant mystery for the affected man . An erection is one of the few things in life that is not improved by greater determination and willpower . A woman may roll over and say nonchalantly , ” Let ‘ s just sleep on it , and I bet things will be great in the morning .” The man will probably not sleep a wink .