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Review Article |
Department of Pharmacology, University of Washington, Seattle, Washington
Abstract
Abstract I. Introduction A. Definition of Phosphodiesterase Enzymes B. Early Studies C. Current Studies—Functional Pools of Cyclic Nucleotides Subserved by Specific Phosphodiesterases D. Why Phosphodiesterases Make Good Drug Targets E. Multiple Forms of Phosphodiesterases—Current Understanding F. Nomenclature G. Crystal Structures 1. Catalytic Domain Structures. 2. Glutamine Switch. 3. Regulatory Domain Structure. 4. Inhibitor Specificity. II. Phosphodiesterase Families A. Phosphodiesterase 1 Family 1. Overview. 2. Biochemistry/Structure. 3. Genetics/Splicing. 4. Localization. 5. Pharmacology/Function. B. Phosphodiesterase 2 Family 1. Overview. 2. Biochemistry/Structure. 3. Genetics/Splicing. 4. Localization. 5. Pharmacology/Function. C. Phosphodiesterase 3 Family 1. Overview. 2. Biochemistry/Structure. 3. Genetics/Splicing. 4. Localization. 5. Pharmacology/Function. D. Phosphodiesterase 4 Family 1. Overview. 2. Biochemistry/Structure. 3. Genetics/Splicing. 4. Localization. 5. Regulation by Phosphorylation. 6. Pharmacology/Function. E. Phosphodiesterase 5 Family 1. Overview. 2. Biochemistry/Structure. 3. Genetics/Splicing. 4. Localization. 5. Pharmacology/Function. F. Phosphodiesterase 6 Family 1. Overview. 2. Biochemistry/Structure. 3. Genetics/Splicing. 4. Localization. G. Phosphodiesterase 7 Family 1. Overview. 2. Biochemistry/Structure. 3. Genetics/Splicing. 4. Localization. 5. Pharmacology/Function. H. Phosphodiesterase 8 Family 1. Overview. 2. Biochemistry/Structure. 3. Genetics/Splicing. 4. Localization. 5. Pharmacology/Function. I. Phosphodiesterase 9 Family 1. Overview. 2. Biochemistry/Structure. 3. Genetics/Splicing. 4. Localization. 5. Pharmacology/Function. J. Phosphodiesterase 10 Family 1. Overview. 2. Biochemistry/Structure. 3. Localization. 4. Pharmacology/Function. K. Phosphodiesterase 11 Family 1. Overview. 2. Biochemistry/Structure. 3. Genetics/Splicing. 4. Localization. 5. Pharmacology/Function. III. Concluding Remarks: The Future of ''Phosphodiesterase Pharmacology''?
Cyclic nucleotide phosphodiesterases (PDEs) are enzymes that regulate the cellular levels of the second messengers, cAMP and cGMP, by controlling their rates of degradation. There are 11 different PDE families, with each family typically having several different isoforms and splice variants. These unique PDEs differ in their three-dimensional structure, kinetic properties, modes of regulation, intracellular localization, cellular expression, and inhibitor sensitivities. Current data suggest that individual isozymes modulate distinct regulatory pathways in the cell. These properties therefore offer the opportunity for selectively targeting specific PDEs for treatment of specific disease states. The feasibility of these enzymes as drug targets is exemplified by the commercial and clinical successes of the erectile dysfunction drugs, sildenafil (Viagra), tadalafil (Cialis), and vardenafil (Levitra). PDE inhibitors are also currently available or in development for treatment of a variety of other pathological conditions. In this review the basic biochemical properties, cellular regulation, expression patterns, and physiological functions of the different PDE isoforms will be discussed. How these properties relate to the current and future development of PDE inhibitors as pharmacological agents is especially considered. PDEs hold great promise as drug targets and recent research advances make this an exciting time for the field of PDE research.
A. Definition of Phosphodiesterase Enzymes
The cyclic nucleotide phosphodiesterases (PDEs1) described in this review are a family of related phosphohydrolases that selectively catalyze the hydrolysis of the 3' cyclic phosphate bonds of adenosine and/or guanosine 3',5' cyclic monophosphate. The structure of cAMP and the bond hydrolyzed is shown in Fig. 1. These enzymes are often referred to as class I cyclic nucleotide PDEs to differentiate them from class II enzymes. Class II enzymes are found in many species including mammals and will also catalyze the hydrolysis of the phosphodiester bond. However, in general, the Class II enzymes do not show the same substrate selectivity as the class I enzymes and much more is known about the class I enzymes.
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Almost immediately after the discovery of cAMP by Sutherland and colleagues, cyclic nucleotide PDE activity was described (Butcher and Sutherland, 1962
). With the subsequent discovery of cGMP, it was found that both cAMP and cGMP could be hydrolyzed by the same type of activity, i.e., hydrolysis of the 3' cyclic phosphate bond. On the basis of substrate competition studies, it was clear that at least some of these activities must have the same catalytic site. In fact, many of the early studies on cyclic nucleotides were directed toward understanding PDE activity since at that time it was much easier to measure PDE activity than either cAMP or cGMP themselves or the enzymes that catalyzed their synthesis. With the advent of assays using radioactive substrate, it became clear that there were likely to be multiple forms of PDEs with different kinetic and regulatory properties (Thompson et al., 1979; Beavo et al., 1982). However, it was not until higher resolution fractionation techniques, monoclonal antibodies, and molecular cloning and sequencing procedures were applied to the PDEs that the truly large number of different gene products was fully appreciated.
C. Current Studies—Functional Pools of Cyclic Nucleotides Subserved by Specific Phosphodiesterases
The complexity of the cyclic nucleotide PDE system has forced increasingly more sophisticated and complex approaches to be adopted to understand the roles of PDEs in regulation of cAMP and cGMP in the cell. It is now very clear that any single cell type can express several different PDEs and also that the nature and localization of these PDEs is likely to be a major regulator of the local concentration of cAMP or cGMP in the cell. PDEs are regulated not only at the genetic level but also by diverse biochemical mechanisms including phosphorylation/dephosphorylation, allosteric binding of cGMP or cAMP, binding of Ca+2/calmodulin, and various protein-protein interactions. The concept that a major role for many PDEs is to modulate the three-dimensional shape, the amplitude, and the temporal duration of "clouds" of cyclic nucleotide in the cell is developing. Some PDEs undoubtedly function just to keep this cloud from spreading to inappropriate areas of the cell. Others function to regulate local access to specific cAMP and cGMP receptors in the cell, which are often tethered to specific intracellular locales. It is also conceivable that not all PDEs function to control cyclic nucleotide hydrolysis but instead act as scaffolding proteins or use allosteric changes induced by binding of cyclic nucleotides to alter protein-protein interactions. In this case the hydrolytic step would then terminate the allosteric change. Although this latter concept is quite possible, no firm evidence for it has been demonstrated.
D. Why Phosphodiesterases Make Good Drug Targets
Immediately after the discovery of PDE activity, it was found that caffeine was an effective inhibitor of PDE activity and a number of nonselective PDE inhibitors including the caffeine analog, theophylline, have been in use as therapeutic agents for many years now. Thus, the principle that inhibition of PDE activity could be a valid therapeutic target is now well accepted. However, most of the early PDE inhibitors had a very narrow therapeutic index, due at least in part to the fact that nearly all of the early inhibitors would inhibit most, if not all, PDE activity in every tissue.
One important general reason that PDEs have been pursued as therapeutic targets is related to the basic pharmacological principle that regulation of degradation of any ligand or second messenger can often make a more rapid and larger percentage change in concentration than comparable regulation of the rates of synthesis. This is true for either pharmacokinetic changes in drug levels or changes in amounts of an endogenous cellular regulatory molecule or metabolite. This intrinsic property is further enhanced if the machinery in the cell that alters degradation has an intrinsically higher Vmax value than the machinery for synthesis. In this case, it has been known for over 40 years that almost all tissues contain at least an order of magnitude higher maximal PDE activity than cyclase activity for either cAMP or cGMP. This, of course, is not the whole story as it is unlikely that most PDEs operate under Vmax substrate conditions in the cell. Nevertheless, this high potential activity is present, and the current idea is that in many compartments of the cell, substrate levels may be quite high.
It has been apparent for many years now that there is a rather extraordinarily large number of different forms of PDEs expressed in mammalian tissues, each of which can have a unique architecture at the active site. Moreover, there is increasing evidence that many of these PDEs are tightly connected to different physiological functions in the body and by inference also to different pathological conditions. Therefore, it has been widely believed that it should be possible to develop isoform selective inhibitors that can target specific functions and pathological conditions without a high likelihood of causing nonspecific side effects. The recent therapeutic and commercial success of agents such as sildenafil (Viagra), a selective PDE5 inhibitor, has validated the concept. These properties are discussed in more detail in later paragraphs.
Another reason PDEs are likely to be good drug targets relates to the concentrations of their substrates in the cell. It is commonly accepted that the levels of cAMP and cGMP in most cells are typically <1 to 10 µM. This means that a competitive inhibitor would not need to compete with very high levels of endogenous substrate to be effective. This fact has, for example, hindered the development of most protein kinase inhibitors, as they need to have high enough affinity to displace millimolar concentrations of ATP. At the same time, such an inhibitor must be selective among thousands of other enzymes that use ATP. However, the challenging development of protein kinase inhibitors is not impossible as selective inhibitors are beginning to appear. So the fact that PDEs are relatively unique in their substrate binding requirements and also that they use a substrate that is 100 to 1000 times lower than ATP makes them an intrinsically more attractive pharmacological target than many other enzymes that use more abundant substrates. In fact, as discussed later, recent evidence from many different companies suggests that it is relatively straightforward and feasible to identify and develop small molecules with substantially different affinities for most of the different PDE families.
E. Multiple Forms of Phosphodiesterases—Current Understanding
One of the most important reasons that the PDEs are recognized as being good drug targets is the fact that there are so many different isoforms. Only since the completion of the human and mouse genome projects has the extent of this enzyme diversity begun to be fully appreciated. Originally, PDEs were classified on the basis of their substrate specificity, modes of regulation, and elution order from DEAE ion exchange columns. As soon as primary amino acid and nucleotide sequences started to become available, they were further classified according to family relationships based on homologies in primary sequence.
Currently, it is widely accepted that there are 11 different families of PDE comprising 21 different gene products2 (Tables 1, 2, 3; Fig. 2). However, there are many more than 21 mRNA and protein products transcribed from these genes because of the use of alternative transcriptional start sites and alternative splicing of mRNA precursor molecules. Current estimates are for >100 different mRNA products, most of which can be translated into different proteins. However, given the present difficulty of predicting transcriptional start sites and splice variants from primary genomic sequence data, it is still not known with any certainty for any species how many different PDE mRNAs are transcribed. Furthermore, it also is not yet clear whether all transcript variants are present in all species.
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Given the complexity of the PDE family, the question often arises about the physiological relevance of multiple isoforms. Is this complexity just a manifestation of functional redundancy, or does it have regulatory significance in the organism? It is probably fair to say that most experts in the field are coming to the common viewpoint that although there may be some redundancy among isozymes, many, if not most, of the different PDE variants play specific physiological roles. That is, there is functional relevance to the different mRNA and protein sequences that are transcribed, and there is functional relevance to the use of alternative start sites having different regulatory promoter regions. In the sections to follow we will try to emphasize cases for which such examples have already been shown or are strongly implicated as well as point out many other cases for which specific functions are likely, but firm supporting data have not yet been obtained.
Given the large number of different PDEs present in mammals, it is not surprising that there have been and probably will continue to be issues of nomenclature. Several years ago a number of investigators in the field arrived at a consensus system that has largely been followed by most authors since then (Beavo et al., 1994). A typical name would be as shown for the following example: HsPDE1A2. The Hs signifies the species of origin, Homo sapiens; PDE denotes a 3',5' cyclic nucleotide phosphodiesterase; the Arabic numeral 1 signifies that it is a member of the PDE1 gene family; the capital A signifies it is the A gene (often but not always the first member of the family reported in GenBank3); and finally the number 2 signifies that it is the second variant reported in the databases. Recently, a slightly modified version of this nomenclature has been promoted largely by the mouse genomic community (http://www.informatics.jax.org/mgihome/nomen/gene.shtml). In this nomenclature, the same PDE would be written mostly in lower case letters in italics as Pde1a_v2 when referring to the nucleotide and in nonitalicized capital letters as PDE1A_V2 when referring to the protein. The letter V or v stands for variant. Given the similarity in concept, it would seem that either system should be acceptable and in fact presently both are used, depending on journal preferences.
Regardless of the method used, a problem that is still apparent in either system has been how to unambiguously assign what designation should be used to identify any new variant (i.e., alternative start site variant or splice variant). In the original system it depended (in theory at least) on its order of appearance in GenBank. Unfortunately, it is not uncommon for a series of PDEs to be cloned and entered into GenBank by one investigator working on one species without knowing that another investigator working in another species has cloned, characterized, and started the deposition process of the same variant gene product. Therefore, the naming of splice or start variants has not always been consistent, particularly among species. In some ways this is inevitable and will require periodic updates of the nomenclature. The data in the tables in this review for each enzyme family are provided as an attempt to temporarily reconcile much of this discrepancy, but the reader is reminded that there will undoubtedly continue to be inconsistencies. For the foreseeable future, therefore, it is probably best for authors to refer to unique GenBank sequence identifiers when referring to a specific PDE isozyme at least once in their manuscripts. Given the nature of science and scientists, it is to be expected that inconsistencies such as this will continue to crop up as more variants are characterized in the coming years, and some method of reconciliation will need to be agreed upon and applied.
Within the last 4 years crystal structures for the catalytic domains from representative members of seven different PDE families have been solved. Several excellent reviews (Card et al., 2004; Zhang et al., 2004b; Jeon et al., 2005) have appeared describing the details of the structure-function relationships discovered from these studies so only a few of the highlights will be discussed here. The solved structures include those for PDE1B (Zhang et al., 2004b), PDE3B (Scapin et al., 2004), PDE4B (Xu et al., 2000, 2004; Card et al., 2004, 2005), PDE4D (Lee et al., 2002a; Huai et al., 2003a,b, 2004a; Card et al., 2004a, 2005), PDE5A (Sung et al., 2003; Card et al., 2004; Huai et al., 2004a; Zhang et al., 2004b), PDE7A (Wang et al., 2005), and PDE9A (Huai et al., 2004b). In addition, the structure of the regulatory tandem GAF domains of PDE2A has also been published (Martinez et al., 2002). However, no high-resolution structure for any PDE holoenzyme has been reported, so we know little about the molecular details of how the regulatory domains influence catalysis. Low-resolution structures determined by electron microscopy to 
29 Å resolution have been reported for PDE5A and PDE6 holoenzymes.
1. Catalytic Domain Structures. Remarkably, the overall folds and functional structural elements for each of the catalytic domains are very similar despite the fact that sequence from any one catalytic domain family exhibits only approximately 25 to 35% amino acid sequence identity with any other. Some important general lessons have been learned from the crystal structures of the catalytic domains. First, all of these catalytic domains contain three subdomains composed largely of 16 helices (Fig. 3A). The active site is formed at the junction of the helices by residues that are highly conserved among all the PDEs. At the bottom of the substrate binding pocket are two divalent metal binding sites. The metals, zinc or magnesium, are coordinated by residues located on each of the three different domains. The metal binding site that binds zinc has two histidine and two aspartic acid residues that are absolutely conserved among all PDEs studied to date. These residues form part of the signature recognition sequence for cyclic nucleotide PDEs, which is itself a subset of the larger HD (histidine/aspartate) domain structure that is found in a superfamily of enzymes with a predicted or known phosphohydrolase activity. These phosphohydrolase enzymes are all thought to be involved in nucleic acid metabolism, signal transduction, and possibly other functions in bacteria, archaea, and eukaryotes. As the highly conserved residues in the HD superfamily are histidines or aspartates, it is thought that they all can act to coordinate divalent cations and are therefore necessary for the activity of these proteins. This is certainly true for the phosphodiesterases. The HD domain is found in >3000 different proteins now in the SMART database (http://smart.embl-heidelberg.de/).
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2. Glutamine Switch.
One of the more interesting ideas to come from the structural studies is a proposal for the molecular mechanism of cyclic nucleotide specificity. In each of the PDEs for which a structure has been solved, there seems to be an invariant glutamine that stabilizes the binding of the purine ring in the binding pocket (Fig. 3B) (Zhang et al., 2004b). For appropriate hydrogen bonds to form to both cAMP and cGMP, this glutamine must be able to rotate freely. At the resolution now available, it seems that for all the PDEs that hydrolyze both cyclic nucleotides with relatively high affinity this free rotation is possible. For PDEs that are highly selective for cAMP at low substrate levels, this glutamine is constrained by neighboring residues into the favored orientation for cAMP binding. Conversely, for those PDEs that prefer cGMP, the glutamine is constrained into the other cGMP favoring position. One possible exception to this tenet has been noted recently for PDE5, which may have additional interactions that are important for specificity (Zoraghi et al., 2006). So, for those PDEs that hydrolyze both cAMP and cGMP, the glutamine is free to rotate, and this "glutamine switch" hypothesis seems to form the molecular basis for much of the substrate selectivity noted between different phosphodiesterases.
3. Regulatory Domain Structure.
Only one structure for a regulatory domain of a class I PDE has appeared. This is the parallel tandem GAF domain of PDE2A (Fig. 3C) (Martinez et al., 2002). This PDE is "activated" by binding of cGMP to an allosteric binding site somewhere on the N-terminal half of the PDE. These binding domains are now known to be part of a much larger group of small molecule binding domains called GAF domains that are found in nearly all phyla. The acronym GAF originates from the first three such domains that were identified (mammalian cGMP binding PDEs, Anabaena adenylyl cyclases, and plant FhlA transcription factors). Several PDE family proteins other than PDE2 also contain N-terminal GAF domains. With the PDE2A GAF domain crystal structure we now know that the GAF-B and not the GAF-A domain contains bound cGMP. Interestingly, the signature NKxxFDxxE sequence found in all of the mammalian GAF domains is not actually in the cyclic nucleotide binding pocket but rather seems to be important for closing a helix across the mouth of the open pocket. Recently, the structure of a cAMP binding tandem GAF domain in the Anabaena adenylyl cyclase was also solved (Martinez et al., 2005). Whereas the structure of the mammalian PDE GAF domains is a parallel dimer, the Anabaena cyclase GAF domain is an antiparallel dimer. Despite this difference, it is known that the mammalian GAF domains can substitute for those in the Anabaena adenylyl cyclase (Kanacher et al., 2002). With these new structures, it may now be possible to begin to use structure-aided drug design techniques to target these allosteric regulatory nucleotide-binding sites on the five families of PDEs that contain them.
4. Inhibitor Specificity.
Several of the crystal structures published have contained bound PDE inhibitors. A third particularly interesting general concept to appear from these structural studies is the idea that although PDE inhibitors bind at the active site, three different modes of binding were found to occur (Card et al., 2004; Jeon et al., 2005). Crystal structures for PDE4B, PDE4D, and PDE5A with inhibitors revealed that the compounds interact with the enzymes either through hydrogen bonds with residues involved in nucleotide binding, through interactions with hydrophobic residues lining the active site channel, or with metal ions mediated through water (Card et al., 2004). This variety of mechanisms of inhibitor binding has encouraged medicinal chemists in their quest for increasingly selective drugs. Elucidation of more PDE structures in the absence or presence of inhibitors should facilitate the discovery of more potent and selective PDE inhibitors in the future.
II. Phosphodiesterase Families
1. Overview.
Calcium- and calmodulin-dependent phosphodiesterases, now known as PDE1s, were one of the first families to be identified (Cheung, 1970) and have been extensively studied. The name derives from both the fact this family had one of the first modes of regulation identified and also the fact that in several tissues members of this family elute from chromatographic separation columns as the first peak of PDE activity. Several recent reviews on PDE1s have appeared (Sonnenburg et al., 1998; Kakkar et al., 1999; Goraya and Cooper, 2005), and the reader is directed to these manuscripts for more detailed information than could be included in this review. The distinguishing feature of this PDE family is their regulation by Ca+2/calmodulin (CaM). The binding of one Ca+2/CaM complex per monomer to binding sites near the N terminus stimulates cyclic nucleotide hydrolysis. The three PDE1 isoforms, PDE1A, PDE1B, and PDE1C, are usually expressed in different cell types within a tissue or regions within a cell and therefore can help to differentially regulate a diverse number of cyclic nucleotide-dependent physiological processes in a calcium-dependent manner.
2. Biochemistry/Structure.
The different PDE1 isoforms all have the same overall structural arrangement. As shown in Fig. 4, the PDE1s consist of two N-terminal CaM binding domains that span an inhibitory sequence. These domains are followed by a conserved C-terminal catalytic domain. Ca+2/CaM stimulates activity by causing an increase in the Vmax with little effect on the Km (Kincaid et al., 1985). All PDE1 enzymes can hydrolyze both cAMP and cGMP, although the affinity for each nucleotide varies by isoform (Table 1). The PDE1As are highly specific for cGMP as substrate with a much lower Km for cGMP (5 µM) than for cAMP (112 µM). However, at higher substrate levels, hydrolysis rates are much closer (the Vmax for cAMP and cGMP are similar). The PDE1B enzymes also prefer cGMP (Km 2.4 µM) to cAMP (Km 24 µM) as substrate, whereas the PDE1Cs hydrolyze the two cyclic nucleotides equally well. The absolute Vmax values for the different PDE1 gene products vary, with the highest being >200 µmol/min/mg with cGMP as substrate for PDE1A (Hansen et al., 1988). Values of 20 to 30 µmol/min/mg have been reported for PDE1B (Sharma and Wang, 1986; Hashimoto et al., 1989), whereas a reliable value for pure PDE1C has not been reported. Generally the Vmax values for cAMP and cGMP are similar for any one isoform with the Vmax ratios for cGMP/cAMP being typically between 0.5 and 2.
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In the presence of Ca+2/CaM, the PDE1s are thought to be tetrameric, consisting of two catalytic monomers and two molecules of calmodulin. There is as yet no crystal structure of any PDE1 holoenzyme either with or without calmodulin in complex, so the orientation of how the calmodulin molecules are bound, how the inhibitory region interacts with the catalytic region, or how the binding of calmodulin activates the enzyme is not known with any precision. However, the crystal structure of the PDE1B catalytic domain has been solved (Zhang et al., 2004b), and in general it is very similar to the other five PDE catalytic domain structures (Card et al., 2004; Ke, 2004). Biochemical data suggest that binding of Ca+2/CaM relieves an inhibition of activity (Sonnenburg et al., 1995). Myosin light chain kinase is an example of another CaM-activated enzyme for which this occurs and has been demonstrated to contain an autoinhibitory substrate-like sequence (Hoeflich and Ikura, 2002).
Significant differences in affinity for Ca+2/CaM exist between different PDE1 enzymes. This is a consequence of different amino-terminal sequences that exist for many of the different variants. In general, the PDE1 enzymes have a fairly high affinity for Ca+2/CaM. The EC50 for activation by calcium has been found to vary from 0.27 (PDE1A1) to 3.02 µM (PDE1C1). The affinity for Ca+2/CaM can also be affected by phosphorylation. Phosphorylation of PDE1A2 by PKA increases the EC50 for activation by CaM from 0.51 to 9.1 nM (Sharma and Wang, 1985). Similarly, phosphorylation of PDE1B by CaM kinase II reduces the affinity of PDE1B for CaM by 6-fold (Hashimoto et al., 1989). In both cases phosphorylation can be reversed by the phosphatase, calcineurin. PDE1C also has been reported to be a target for phosphorylation, and its activity is inhibited by PKA (Ang and Antoni, 2002).
3. Genetics/Splicing.
The three PDE1 isoforms are products of separate genes and all three have unique variants produced by alternative splicing or alternative transcriptional start sites. Thus, genetic regulation results in the production of a multitude of diverse PDE1 proteins. The different protein products are diagrammed in Fig. 4. The PDE1A isoform has the largest number of variants. Only the human proteins are diagrammed. Some of the unique amino-terminal sequences have been found to confer different functional properties on the variants. As discussed above, differences have been noted in CaM affinity as the unique N-terminal sequences are in or near the CaM binding domains. Significant differences in other kinetic properties among the variants of each isoform have not been reported. Hypothetically, an alternative reason for the existence of unique mRNA products is to allow for more finely tuned regulation of expression. To date only the PDE1B gene has been disrupted (Reed et al., 2002) (see next section).
4. Localization.
PDE1 expression is highly regulated, and individual isoforms and variants are localized to specific tissues and cell types. The localization of PDE1s in the central and peripheral nervous systems exemplifies this principle. All three PDE1 isoforms are expressed in the brain and many peripheral neurons but to greatly differing degrees depending on region. For example, PDE1C2 is highly localized to olfactory epithelium where it is thought to play an important role in rapid regulation of cAMP responses to odorants (Yan et al., 1995). PDE1 expression is differentially localized not only to different regions, but even to individual neurons of the same type within a region. For example, PDE1B is highly expressed in some but not all Purkinje neurons (Shimizu-Albergine et al., 2003). Different PDE1 isoforms are differentially localized in testis and sperm of the reproductive system (Yan et al., 2001), heart and vessels of the cardiovascular system, and macrophages and T lymphocytes of the immune system (Essayan, 2001). Most PDE1 isoforms are reported to be cytosolic. However, there are instances of PDE1s being localized to subcellular regions. For example, in human and mouse sperm, most of the activity is found in the midpiece of the tail and fractionates with the particulate portion of the cell (Vasta et al., 2005). However, little is known about the molecular mechanisms responsible for subcellular localization of PDE1s. It is likely that the unique N-terminal or C-terminal regions of the various isoforms allow the different proteins to be targeted to specific subcellular domains.
5. Pharmacology/Function.
Given their in vitro regulation by Ca2+/CaM, PDE1s are presumed to all function at least in part as a mechanism for integrating cell signaling pathways mediated by cAMP and cGMP with pathways that regulate intracellular calcium levels. However, this very reasonable hypothesis has been hard to prove experimentally in most instances. One reason for this is the lack of truly specific cell-permeable inhibitors for this PDE family. Although several molecules that show some selectivity toward the PDE1s in vitro have been described, few if any show enough selectivity or permeability to be useful for distinguishing these PDEs from other families in intact cells. Therefore, few definitive studies on the functional roles of the various PDE1 isoenzymes exist. In addition, mice with gene disruptions are available only for the PDE1B isozyme in this family. Nevertheless, PDE1 has been implicated to play a role in several physiological and pathological processes. PDE1A mRNA is induced in several cell types upon chronic agonist stimulation, suggesting that it performs important feedback regulatory functions in these cells. For instance, PDE1A is up-regulated in rat aorta in response to chronic nitroglycerin treatment (Kim et al., 2001).
PDE1B knockout mice have increased locomotor activity and in some paradigms decreased learning and memory (Reed et al., 2002). Using these mice, a role for PDE1B in dopaminergic signaling also has been suggested. PDE1B may also contribute to other neuronal functions as it is expressed in multiple CNS regions. PDE1B is induced in several types of activated immune cells (Essayan, 2001). It is hoped that exactly how the up-regulation of these PDE1 variants contributes to the function of the cells will be elucidated in the near future.
PDE1C has been demonstrated to be a major regulator of smooth muscle proliferation, at least in human smooth muscle. In humans PDE1C is absent in quiescent smooth muscle but is expressed in proliferating smooth muscle in culture and in smooth muscle cells isolated from atherosclerotic lesions (Rybalkin et al., 2003b). The importance of PDE1C in the proliferative phenotype was underscored by the finding that antisense oligonucleotide treatment against PDE1C halted proliferation (Rybalkin et al., 2003b). It should be noted that this isozyme is not expressed in the smooth muscle of most other animal models, underscoring the species selectivity of some PDE isozymes and functions subserved by them. Another likely role for PDE1C is in olfaction. The very high expression of PDE1C2 in olfactory sensory cilia strongly suggests that this is the isozyme that modulates the amplitude and duration of the cAMP signal in this tissue in response to odorant stimulation (Yan et al., 1995).
1. Overview. PDE2 is a dual substrate enzyme that has both a high Vmax and low Km for hydrolysis of both cAMP and cGMP. The distinguishing feature of this PDE is that it is allosterically stimulated by cGMP binding to one of its GAF domains (note: GAF domains are small molecule binding motifs present in many regulatory enzymes and discussed in more detail below). PDE2 is expressed in a wide variety of tissues and cell types including brain, heart, platelets, endothelial cells, adrenal glomerulosa cells, and macrophages. Given that PDE2 is expressed in such a diverse number of cell types, has a complex biochemical regulation, and has the ability to hydrolyze both cAMP and cGMP with high activity, it is likely that it will be involved in regulating a variety of different processes and its function(s) therefore are not easily generalized.
2. Biochemistry/Structure.
PDE2 hydrolyzes both cAMP and cGMP and displays positive cooperativity for both nucleotides (Martins et al., 1982). This cooperativity that arises from binding of nucleotides to the allosteric GAF-B domain causes a conformational change in the protein and increases enzyme activity. It is well documented that cGMP binding stimulates cAMP hydrolysis. However, there are as yet no known in vivo examples for the reverse, cAMP stimulation of cGMP hydrolysis, although this has not been thoroughly investigated. Given the 30-100-fold lower affinity for cAMP at the allosteric GAF-B domain site (Wu et al., 2004) and current ideas about levels of cAMP in cells, it is presumed that activation of cGMP hydrolysis by cAMP does not happen in vivo. However, it is possible that localized pools of cAMP and cGMP may be present in cells that allow such activation in some subcellular compartments.
The crystal structure of the PDE2A tandem GAF-A/GAF-B domain has been determined (Martinez et al., 2002) and more recently compared with that of the tandem GAF-A/GAF-B domains of the Anabaena adenylyl cyclase, CyaB2 (Martinez et al., 2005). The PDE2 GAF-B domain binds cGMP with high affinity and selectivity whereas in the cyclase both GAF-A and GAF-B bind cyclic nucleotide with a strong preference for cAMP. Many of the important contacts between the nucleotide and the protein are made via the peptide backbone and not side chains. Others contacts are on the 
4 helix that seems to fold over the cGMP, holding it firmly in place. Interestingly, when the GAF domains of PDE2 are used to replace the GAF domains of the cyclase using molecular biological techniques, the chimeric protein is now stimulated by cGMP (Kanacher et al., 2002). This finding strongly suggests that the GAF domains operate as a molecular switch that upon cyclic nucleotide binding can regulate the enzyme activity of adjoining catalytic domains. Moreover, this basic switch has been conserved for >2 billion years of evolution across organisms. Rather unexpectedly, it was also found that the PDE GAF domains exist in a parallel dimeric structure whereas the cyclase GAF domains are antiparallel. Despite the different dimeric configurations, the PDE GAF domains can be joined to the cyclase catalytic domain and still act to regulate cyclase activity. As might be expected, both the PDE2 and cyclase GAF domains have a very similar architecture in the binding region for cyclic nucleotide. As detailed studies on the structure and binding selectivity have been published recently (Wu et al., 2004; Francis et al., 2005), we will not further address these issues in this review. Current work is now focused on obtaining structural information on the PDE2A holoenzyme so that the molecular mechanisms by which activation occurs can be modeled.
3. Genetics/Splicing.
PDE2 protein has been found in a wide variety of tissues and cell types. PDE2 activity and protein were originally purified from heart, liver, adrenal gland, and platelets (Martins et al., 1982; Yamamoto et al., 1983) and are also found in brain, endothelial cells, and macrophages (Tenor and Schudt, 1996; Juilfs et al., 1999; Bender et al., 2004). As with other PDEs, the expression of PDE2A can be regulated, and once produced, PDE2 can be localized to discrete regions or specific cell types within tissues. For instance, in endothelial cells PDE2A is expressed under basal conditions only in smaller vessels and capillaries, but not in larger vessels (Sadhu et al., 1999). In addition, although PDE2A is widely expressed in the brain, its highest expression seems to be localized to specific regions and cell types (Juilfs et al., 1999). PDE2A cellular localization will be discussed more in the context of function in a later section. On the subcellular level, PDE2 activity has been purified from both supernatant and particulate fractions as there are both soluble (PDE2A1) and membrane-associated (PDE2A2/3) variants of this PDE. The different localization of PDE2A2/3 is probably mediated by a unique N-terminal sequence that is absent from PDE2A1 (Fig. 5).
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). PDE2A1 and PDE2A2 seem to be splice variants. PDE2A1 has a 62-nucleotide insert that encodes a frame shift. This allows initiation of translation at a methionine downstream of the PDE2A2 start site. PDE2A2 has so far only been found in rat. PDE2A3 lacks the insert of PDE2A1 and shares 19 amino acids in common with PDE2A2 that are not present in PDE2A1. PDE2A3 also has 25 amino acids at the N terminus that are unique. There are no known differences in kinetic behavior between the PDE2A variants. However, as mentioned before the different N-terminal sequences likely mediate different localizations of the proteins.
5. Pharmacology/Function.
PDE2 is involved in a variety of physiological processes. The availability of PDE2 selective inhibitors has greatly aided in the elucidation of PDE2 functions (Table 4). However, to date, PDE2 inhibitors have served primarily as research tools and have not entered clinical usage. Interestingly, the enzyme has been found to regulate either cAMP or cGMP, depending on the cell type in which it is expressed. It is thought that the cooperative kinetic behavior seen for cAMP and cGMP hydrolysis in vitro is important physiologically for the ability of cGMP to stimulate cAMP hydrolysis. Thus, a unique property of PDE2 is its ability to mediate negative "cross talk" between the cGMP and cAMP pathways. This is perhaps best exemplified in adrenal glomerulosa cells of several species in which PDE2 is highly expressed and is thought to mediate the inhibitory effects of atrial natriuretic peptide (ANP) on aldosterone secretion (MacFarland et al., 1991; Nikolaev et al., 2005). In these cells, elevation of cGMP by ANP activates PDE2 that in turn lowers cAMP. Functionally, the PDE2 activation by ANP decreases cAMP that has been stimulated by adrenocorticotropin and results in decreased aldosterone secretion. This cross-talk between the cGMP and cAMP pathways probably occurs in other cellular settings as well. For example, in platelets it is thought that high levels of NO elicit high cGMP accumulation that activates PDE2 and reduces cAMP (Dickinson et al., 1997; Dunkern and Hatzelmann, 2005). However, at lower cGMP levels PDE2 inhibitors alone have little effect on platelet aggregation in comparison to PDE3 inhibitors as PDE3 seems to play a more prominent role in cAMP regulation under these conditions. These results probably reflect a complicated interplay between cGMP-mediated inhibition of PDE3 and stimulation of PDE2. Together the two PDEs mediate opposing regulation of cAMP hydrolysis.
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Cardiac myocytes are another cell type in which PDE2 is stimulated by cGMP and interplay between PDE2 and PDE3 may occur. The role of PDE2 in regulating cardiac function was well reviewed in a recent report (Fischmeister et al., 2005). In human cardiac myocytes, PDE2 has been shown to be a regulator of cardiac L-type Ca+2 current. L-type Ca+2 channels are classically recognized as targets for activation by 
-adrenergic receptor-stimulated cAMP and PKA, and alterations in channel activity have both chronotropic and ionotropic effects. Several studies have demonstrated that cGMP can oppose cAMP in myocytes by activating PDE2, thus reducing cAMP and affecting cardiac function (Mery et al., 1995; Vandecasteele et al., 2001; Fischmeister et al., 2005). Using real-time imaging PDE2 in cardiac cells was found to be compartmentalized and shaped the cAMP response to catecholamine stimulation (Mongillo et al., 2006). PDE2 not only regulates cAMP and Ca+2 current but also seems to mediate the ability of NO and cGMP to affect cAMP. As in platelets, the activation of PDE2 by cGMP is opposed by the inhibition of PDE3, and the balance of the two effects can be different depending on the tissue and species (Fischmeister et al., 2005). In addition, cGMP may directly inactivate the channel via cGMP-dependent protein kinase (PKG) phosphorylation.
As PDE2 also has a high activity toward cGMP, in some cells it may serve simply as a regulator of cGMP. This seems to be especially true in the brain in which PDE2 is highly expressed in several discrete regions and also in olfactory neurons (Juilfs et al., 1997). For instance, in cultured neurons and hippocampal slices PDE2 serves to regulate cGMP, and functionally PDE2 inhibitor treatment of rats enhanced long-term potentiation and memory without affecting basal synaptic transmission (Boess et al., 2004). PDE2 has also been proposed to regulate cGMP elevated by N-methyl-D-aspartate in rat neurons from cortex and hippocampus (Suvarna and O'Donnell, 2002), NO elevated cGMP in rat striatal cells (Wykes et al., 2002), and cGMP in rat olfactory sensory neurons (Meyer et al., 2000).
PDE2 has been found to be up-regulated upon monocyte to macrophage differentiation (Bender, 2003) and is highly expressed in rat (Witwicka et al., 2002) and mouse glycolate-elicited peritoneal macrophages (Tenor and Schudt, 1996). In these cells PDE2 activity represents much of the cGMP PDE activity and may serve as the primary regulator of cGMP. However, these cells also respond to ANP, raising the possibility that PDE2 may also serve to regulate cAMP in response to cGMP in macrophages. It is hoped that future experiments will identify what role is played by PDE2 in macrophages.
PDE2 may play a role in regulation of fluid and cell extravasation during inflammatory conditions as PDE2 is localized to microvessels, especially venous capillary and endothelial cells, but apparently not to larger vessels (Sadhu et al., 1999). One recent observation that is likely to be of considerable importance is the finding that in endothelial cells, PDE2A mRNA and activity are highly induced in response to tumor necrosis factor- stimulation in vitro (Seybold et al., 2005). Moreover, blockade of PDE2 activity with the PDE2 selective inhibitor 9-(6-phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6-one (PDP) seems to greatly alter the barrier function of endothelial cells. This suggests that PDE2 is likely to play an important role in regulating fluid and protein integrity of the circulatory system under pathological conditions. Therefore, PDE2 may be a good pharmacological target for pathological states such as sepsis or in more localized inflammatory responses.
The availability of PDE2 selective inhibitors has greatly facilitated the elucidation of PDE2 function in a variety of tissues as evidenced in the previous paragraphs. One of the first PDE2 selective inhibitors, erythro-9-(2-hydroxy-3-nonyl)adenine, was found to have an IC50 for PDE2 in the high nanomolar to low micromolar range and an at least 50-fold selectivity over other PDEs (Podzuweit et al., 1995). Although this compound also potently inhibits adenosine deaminase, with the proper controls it has been successfully used as a tool to probe PDE2 function. Recently, Bayer has developed several newer inhibitors with increased potency and improved selectivity (Boess et al., 2004; Seybold et al., 2005). For example, one of these compounds, PDP, has an IC50 of 0.6 nM and a >1000-fold selectivity (Seybold et al., 2005). This compound was found to inhibit thrombin-induced edema formation in mouse lung (Seybold et al., 2005). Another PDE2-selective inhibitor, BAY 60-7750, has been reported to improve memory in animal models. Although studies of these compounds in humans are lacking, they may hold promise in treating disorders of endothelial permeability or learning and memory.
1. Overview. The PDE3 family isoforms have been extensively studied, especially in regard to their physiological functions and their usefulness as drug targets. One distinguishing feature of the PDE3 family is their biochemical property of being able to hydrolyze both cAMP and cGMP, but in a manner suggesting that in vivo the hydrolysis of cAMP is inhibited by cGMP. Thus, they have earned the title "the cGMP-inhibited PDE". They also are distinguished by their ability to be activated by several phosphorylation pathways including the PKA and PI3K/PKB pathways. Two PDE3 genes, PDE3A and PDE3B, have been identified, but splice/start variants have been conclusively demonstrated only for the PDE3A isoform.
2. Biochemistry/Structure.
PDE3s were initially purified and described as enzymes that hydrolyze both cAMP and cGMP with relatively high affinities (KmcAMP <0.4 µM; KmcGMP <0.3 µM). However, the Vmax for hydrolysis of cAMP is nearly 10-fold higher than the Vmax for cGMP. Therefore, in vitro cGMP can act as an inhibitor of cAMP hydrolysis with an apparent Ki of 0.6 µM. This inhibition also occurs in intact cells as was first demonstrated in platelets (Maurice and Haslam, 1990) and is now thought to occur in most other cells containing PDE3 (see section II.C.5.). It may also be that under some conditions, e.g., low cGMP levels, that this family of PDEs also is important for controlling the levels of cGMP in the cell since it does have a very high affinity for this nucleotide.
The PDE3A and PDE3B isoforms have a high degree of amino acid identity (>80% for much of the catalytic region) and very similar kinetic properties. Both PDE3 isoforms contain an insert in the catalytic domain that is not present in other PDEs. Currently the function of the insert is unknown. Recently a crystal structure of the PDE3B catalytic domain in complex with the inhibitor, cilostamide, was published and should enhance our understanding of the molecular nature of PDE3 catalysis and aid in inhibitor design as well (Scapin et al., 2004).
Both PDE3A and PDE3B activity are regulated by phosphorylation in response to hormonal stimulation in several cell types. In platelets and perhaps also in heart, prostaglandins and epinephrine feedback through PKA to activate PDE3A (Shakur et al., 2001). PDE3B is also a substrate for PKA and is activated by it (Shakur et al., 2001). Similarly, first-messenger signals such as insulin, IGF1, and leptin acting through the PI3K/PKB pathway can induce a phosphorylation of PDE3B and probably also PDE3A to stimulate activity (Shakur et al., 2001). It is thought that these phosphorylation events are important to many of the physiological processes controlled by these hormones. It is likely that in cell types expressing large amounts of PDE3, phosphorylation by PKA and activation of PDE3 can mediate part or all of the tachyphylaxis seen in response to continued agonist stimulation (over the minute to hour time scale). Several of the phosphorylation sites have been identified (Fig. 6).
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; Wechsler et al., 2002). The PDE3A2 and PDE3A3 variants are alternate start site truncations of PDE3A1. PDE3A2 is a shortened version of PDE3A1 due to a separate downstream transcriptional start site. PDE3A3 is a truncated version of PDE3A2 that is thought to be a product of the PDE3A2 mRNA in which translation begins at a downstream ATG (Wechsler et al., 2002). Although PDE3B proteins of multiple sizes have been reported, no PDE3B splice or alternative start variants have been identified. It is thought that the multiple protein sizes reported for PDE3B isolated from tissues are likely to be due to proteolysis. However, the possibility that isoforms of different lengths might be generated by an as yet unidentified alternative start sites in some tissues has not yet been fully ruled out. The genetic regulation of PDE3 expression is relevant as PDE3 expression is altered with adipocyte differentiation (Rahn Landstrom et al., 2000) and heart failure (Ding et al., 2005) and has been noted to change in vascular smooth muscle (Maurice et al., 2003). In addition, down-regulation of PDE3A expression is observed in human failing hearts or mouse hearts subjected to chronic pressure overload (Ding et al., 2005). Decreased PDE3A activity is associated with increased apoptosis of myocytes. This was demonstrated in isolated cardiomyocytes subjected to pharmacological inhibition of PDE3 or adenovirus-delivered antisense PDE3A (Ding et al., 2005).
4. Localization.
PDE3A is relatively highly expressed in platelets, as well as in vascular smooth muscle, cardiac myocytes, and oocytes (Shakur et al., 2001). PDE3B is a major PDE in adipose tissue, liver, and pancreas, as well as in several cardiovascular tissues (Shakur et al., 2001). Both PDE3A and PDE3B contain a large and a small hydrophobic domain at their N termini (Fig. 6). The large domain is 195 amino acids and is predicted to form six transmembrane helices that allow association of PDE3 with membranes (Shakur et al., 2001). However, it is not yet well established how many, or if any, of these helices actually transect the membrane. The second smaller domain of roughly 50 amino acids is thought to be involved in targeting of the enzyme and forms weaker interactions (Shakur et al., 2001). Full-length PDE3A and PDE3B expressed in Sf9 cells are both predominantly particulate. However, truncations eliminating the first hydrophobic region resulted in 50% of the enzyme becoming cytosolic, whereas truncating both of the domains results in 100% of the enzyme becoming cytosolic (Kenan et al., 2000). In tissues, PDE3B is almost always found to be particulate, whereas PDE3A has been found to be both cytosolic and particulate. PDE3A and PDE3B are expressed in a variety of tissues and have distinct but overlapping localizations (Table 2). For instance, both PDE3A and PDE3B are found in vascular smooth muscle cells (Palmer and Maurice, 2000). In contrast, PDE3A is distinctly expressed in platelets and oocytes, whereas PDE3B is unique to T lymphocytes, macrophages, cells, and adipocytes (Shakur et al., 2001).
5. Pharmacology/Function.
There are a relatively large number of PDE3 selective inhibitors (Table 4). Many of them are commercially available and pharmaceutical firms have also developed a significant number of proprietary compounds. PDE3 selective inhibitors include amrinone, milrinone, cilostamide, and cilostazol. The most potent is trequinsin. Amrinone was the first recognized but possesses only modest affinity and selectivity. To date there have been no inhibitors described that clearly distinguish between PDE3A and PDE3B, although at least one, OPC-33450, has been reported to show some selectivity (Sudo et al., 2000). The plethora of available PDE3 specific inhibitors and the use of genetic techniques have allowed elucidation of PDE3 functions in multiple physiological processes. PDE3 inhibitors antagonize platelet aggregation, block oocyte maturation, increase myocardial contractility, and enhance vascular and airway smooth muscle relaxation.
In platelets, aggregation is highly regulated by cyclic nucleotides. PDE3A is a regulator of this process and PDE3 inhibitors effectively prevent aggregation (Shakur et al., 2001). In fact one drug, cilastazol (Pletal), is approved for treatment of intermittent claudication. Its mechanism of action is thought to involve inhibition of platelet aggregation along with inhibition of smooth muscle proliferation and vasodilation. There is also substantial evidence that cGMP, acting as a competitive inhibitor of PDE3A, exerts most of its antiplatelet effects by increasing cAMP via inhibition of PDE3A (Maurice and Haslam, 1990).
PDE3A has been found to be important not only for platelet function, but also for oocyte maturation. It has been demonstrated that inhibition of PDE3A prevents oocyte maturation in vitro and in vivo (Conti et al., 2002). In more recent gene disruption studies it was found that PDE3A-/- mice are viable and ovulate a normal number of oocytes but are completely infertile as their oocytes contain higher levels of cAMP and fail to undergo spontaneous maturation (Masciarelli et al., 2004). Further studies showed that this occurred because ovulated oocytes were arrested at the germinal vesicle stage. Male PDE3A-/- mice are fertile and to date no obvious phenotypes as a result of the disruption have been reported.
PDE3 enzymes are also involved in regulation of cardiac contractility and vascular smooth muscle (Maurice et al., 2003). PDE3 inhibitors were initially investigated for the treatment of heart failure, but their use for this indication has fallen out of favor because of untoward arrhythmic side effects. Nonetheless, in intravenous form the PDE3 inhibitor milrinone (Primacor) is approved for use in heart failure. Recently it has been reported that PI3K
can be associated with PDE3B, and this interaction controls PDE3B activity (Patrucco et al., 2004). The PDE3B interaction with PI3K is thought to be important for the regulation of cardiac contractility and was found to affect cardiac hypertrophy in a mouse model of chronic pressure overload (Patrucco et al., 2004). Interestingly, both PDE3A and PDE3B are expressed in vascular smooth muscle cells and are likely to modulate contraction. Their expression in vascular smooth muscle cells is altered under several conditions such as elevated cAMP, the switch from contractile to proliferative phenotype, and hypoxia (Dunkerley et al., 2002; Murray et al., 2002; Maurice et al., 2003).
The most intensely studied roles for PDE3B have been in the areas of insulin, IGF1, and leptin signaling. Activation of PDE3B is thought to be important for the antilipolytic and antiglycogenolytic actions of insulin (Shakur et al., 2001), as well as for IGF1 and leptin inhibition of glucagon-like peptide-1-stimulated insulin release from pancreatic islets (Zhao et al., 1997, 1998). This idea has now been expanded to include at least part of the effects of leptin on food intake and body weight (Zhao et al., 2002). At the molecular level it is thought that leptin, IGF1, and insulin activation of PI3K in turn stimulates PKB phosphorylation of PDE3B triggering activation of the enzyme (Zhao et al., 1998; Rondinone et al., 2000; Shakur et al., 2001) and its possible association with 14-3-3 proteins (Onuma et al., 2002). Opposing the phosphorylation by PKB is thought to be protein phosphatase 2A (Shakur et al., 2001). The involvement of PDE3B in regulation of these important metabolic pathways has encouraged researchers to begin exploring the possible roles of this enzyme in disorders such as obesity and diabetes.
1. Overview. Almost immediately after assays able to detect PDE activity at low substrate levels were developed, what we now recognize as PDE4 was described. That is, a "low Km, cAMP-specific PDE activity." This activity was initially characterized by the fact that it could be selectively inhibited by the drug rolipram and the enzymes were once named RoI-PDEs (rolipram-inhibited PDEs) based on this property. One consequence of the early discovery of PDE4 is that it has been one of the most studied PDEs, and a great deal is known about its biochemistry, genetics, and physiological function. It is now recognized that there are four genes (PDE4A-PDE4D) that make up this family. Moreover, each gene has multiple variants. Currently, >20 have been described (Fig. 7). PDE4 is expressed in a plethora of tissues and cell types and plays a role in a large number of physiological processes.
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