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Siegel GJ, Agranoff BW, Albers RW, et al., editors. Basic Neurochemistry: Molecular, Cellular and Medical Aspects. 6th edition. Philadelphia: Lippincott-Raven; 1999.

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Basic Neurochemistry: Molecular, Cellular and Medical Aspects. 6th edition.

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Cyclic Nucleotide Phosphodiesterases

and .

Correspondence to Ronald S. Duman, Laboratory of Molecular Psychiatry, Department of Psychiatry, Yale University School of Medicine and Connecticut Mental Health Center, 34 Park Street, New Haven, Connecticut 06508.

Given the significant role of cyclic nucleotides in signal-transduction pathways, it is not surprising that their metabolism and synthesis is highly regulated. Such metabolism is achieved by a large number of enzymes, the phosphodiesterases (PDE), which catalyze the conversion of cAMP and cGMP into 5′-AMP and 5′-GMP, respectively, via hydrolysis of the 3′-phosphoester bonds (Fig. 22-2).

There are multiple forms of phosphodiesterase in brain

Seven major families of PDE, listed in Table 22-1, have been delineated based primarily on two criteria: (i) the kinetic properties of each enzyme for hydrolyzing cAMP and cGMP and (ii) the mechanisms for regulation of PDE activity [32,33] (Fig. 22-6). The kinetic properties are characterized according to the affinity of each enzyme for cAMP or cGMP. The affinity is derived from the Km values: a low Km signifies that the enzyme has a high affinity for substrate. For example, a low-Km PDE typically has a Km of <5 μM.

Table 22-1. Classification and Selected Properties of Cyclic Nucleotide Phosphodiesterases.

Table 22-1

Classification and Selected Properties of Cyclic Nucleotide Phosphodiesterases.

Figure 22-6. Schematic illustration of the overall structure and regulatory sites of representative phosphodiesterase (PDE) subtypes.

Figure 22-6

Schematic illustration of the overall structure and regulatory sites of representative phosphodiesterase (PDE) subtypes. The catalytic domain of the PDEs is relatively conserved, and the preferred substrate(s) for each type is shown. The regulatory domains (more...)

The PDE1 family consists of several soluble subtypes in the brain and peripheral tissues, which are stimulated by Ca2+/calmodulin. Two isozymes of PDE1, with Mr of 61,000 and 63,000, account for more than 90% of total brain PDE activity. Separate genes for each of these isozymes have been identified and are referred to as PDE1A (61,000) and PDE1B (63,000). Both enzymes exhibit relatively low affinity for cGMP and cAMP. PDE1A is expressed at highest concentrations in cerebral cortex and hippocampus and at moderate concentrations in amygdala [34]. Expression of PDE1B is high in brain regions innervated by dopamine, including striatum, nucleus accumbens and olfactory tubercle, with moderate expression in hippocampus and cerebral cortex [35]. An additional isozyme, PDE1C, with an Mr of 73,000, also encoded by a distinct gene, has been identified in brain and testes. Interestingly, PDE1C has a high affinity for cAMP, in contrast to the low affinity that PDE1A and PDE1B exhibit for cyclic nucleotides.

The activity of PDE1 isozymes can be regulated under physiological conditions by those extracellular signals that influence intracellular Ca2+ levels. This may be particularly relevant, in a physiological sense, for PDE1C, given its high affinity for cAMP. There is also evidence that these enzymes are regulated by protein phosphorylation [36,37]. The 61,000 and 63,000 isozymes are good substrates for cAMP-dependent and Ca2+/calmodulin-dependent protein kinase, respectively. Such phosphorylation decreases the functional capacity of the enzymes by decreasing their affinity for Ca2+/calmodulin complexes.

There are two PDE families that are regulated by cGMP: PDE2 is stimulated and PDE3 is inhibited. Both of these PDE types are found in soluble and membrane-associated forms. cGMP-stimulated PDE2 has low affinity for cAMP and cGMP. There are at least two cyclic nucleotide-binding sites on PDE2 isozymes: one is presumably the catalytic site and the other is possibly the high-affinity site which allosterically regulates the catalytic activity of the enzyme. PDE2 can be activated 10- to 50-fold by the concentrations of cGMP found in cells. However, stimulation is transient since cGMP is also a substrate for the enzyme and, therefore, is metabolized rapidly. Localization studies demonstrate that PDE2 expression is restricted largely to the brain and adrenal gland [38]. Within the brain, highest concentrations of the enzyme are seen in the cerebral cortex, striatum and hippocampus. PDE2 may be a primary effector for the physiological actions of cGMP in cells that do not express appreciable amounts of cGMP-dependent protein kinase. In contrast to PDE2, there is currently no evidence for the presence of cGMP-inhibited PDE3 in the brain. Instead, PDE3, which has a low Km for cAMP, is enriched in heart and vascular tissues, where it regulates cardiac and smooth muscle contraction.

The PDE4 isozymes are cAMP-specific, high-affinity PDEs. Members of this family are found in many tissues in both soluble and membrane-associated forms and are abundant in the central nervous system. PDE4A and PDE4B are expressed at relatively high concentrations in hippocampus, cerebral cortex and striatum and represent the majority of the membrane-bound form of PDE4 in these brain regions [39]. PDE4D represents the majority of the soluble PDE4 in the brain. Expression of PDE4C is relatively low in nervous tissue. Multiple isozymes have been identified, and at least four separate genes exist, which are highly conserved across several mammalian species [32]. The PDE4 isozymes are regulated by phosphorylation and by binding cAMP. In addition, expression of certain PDE4 genes is regulated significantly by activation of the cAMP intracellular pathway. Indeed, this is considered a primary mechanism for enhancing the function of these isozymes in some cell types.

Soluble PDE isozymes that hydolyze cGMP with high affinity are grouped into two families: PDE5 is expressed in smooth muscle, including lung and penis, and PDE6 in retina. The PDE5 and PDE6 isozymes are referred to also as the cGMP-specific PDEs, which display a 50-fold selectivity for cGMP relative to cAMP. Although both of these PDE types have high affinity for cGMP, they are distinct structurally. There are several members of the PDE6 family, all of which are light-activated photoreceptor isozymes. Activation of photoreceptor PDE6 in rod and cone outer segments is mediated by transducin, a G protein specific to retina (see Chaps. 20 and 47). These PDEs are multimeric enzymes, composed of α, β and γ subunits. They are inactive in the dark; light results in their activation via a complex biochemical cascade analogous to mechanisms for activation of G protein-coupled receptors. Briefly, light induces a conformational change in the retinal protein rhodopsin, which is structurally and functionally homologous to a G protein-coupled receptor. This leads to activation of transducin, which is structurally and functionally homologous to Gαs. Free transducin α subunits then directly bind to and activate PDE6. The rapid resulting hydrolysis of cGMP leads to changes in specific ionic currents in the photoreceptors and thereby mediates the physiological effects of light.

A seventh type of PDE has been cloned based on homology screening. This PDE, like PDE4 isozymes, hydrolyzes cAMP with high affinity. However, it is not inhibited by rolipram, a specific inhibitor of PDE4. PDE7 is expressed at high levels in skeletal muscle. Although relatively low levels of PDE7 are expressed in whole brain, it is possible that certain regions express higher levels of this or a related form of PDE.

Phosphodiesterases show a distinctive molecular structure

Members of each of the seven PDE families have been characterized by molecular cloning techniques [32,33]. All of the enzymes thus far identified in mammalian tissues contain a highly conserved region of approximately 300 amino acids toward the carboxy terminus. Deletion studies have confirmed that this region represents the catalytic domain. Within this region, there are a number of conserved histidine residues that appear to play a role in folding of the proteins and are required for their catalytic activity, based on mutational analyses. The histidine residues may bind zinc, given the structural similarities of the PDEs to zinc hydrolases.

The regions outside the catalytic domain, particularly the amino terminus, are much more variable across the different PDE families. These regions contain many of the regulatory sites that control PDE activity. For example, the amino terminus contains the Ca2+ calmodulin-binding site on PDE1. Similarly, those PDEs that possess high-affinity cGMP-binding sites that apparently serve an allosteric function, such as PDE2 and PDE6, share a distinct conserved site within this region. There are also several phosphorylation sites within this region in PDE1, PDE3, PDE4 and PDE6. There is evidence for an additional phosphorylation site in the carboxy terminus of certain PDE4 isozymes.

The association of PDE isozymes with the cell membrane is mediated by a conserved, hydrophobic sequence in the amino terminus of the proteins. This has been demonstrated most convincingly for PDE4A: when the amino terminus is removed, PDE4A is no longer localized to the membrane fraction. A similar amino-terminal sequence is found in the membrane-bound forms of PDE2, and it may mediate the membrane association of certain PDE3 isozymes.

In addition to the numbers of distinct genes that encode the seven described families of PDEs, there is evidence that multiple protein products can be derived from individual genes. This has been established for the PDE1, PDE2 and PDE4 gene families. These multiple forms provide yet another mechanism to increase the diversity of PDEs expressed in different tissues or under different biological conditions. For example, the four PDE4 genes give rise to at least 17 variants as a result of alternative splicing or the presence of multiple promoter start sites within the gene [32]. Many of these variants are conserved across species, which suggests that they may have functional importance.

While the physiological significance of most of these PDE4 variants has not been appreciated fully, certain variants have been shown to differ dramatically with respect to their regulatory properties. Short and long forms of PDE4D are generated by alternative splicing. Expression of the short forms, D1 and D2 (67–72 kDa), results from activation of an intronic promoter, whereas expression of the long form, D3 (93 kDa), results from activation of another promoter located further upstream. The long form, but not the short forms, contains a site for phosphorylation and is regulated by cAMP-dependent protein kinase [40]. Although the short forms are not regulated by phosphorylation, their expression is increased at the transcriptional level by cAMP [41]. The PDE4A—C genes also appear to be capable of giving rise to short and long forms that may be regulated in a similar manner. The activity of the long, but not the short, form is enhanced by phosphatidic acid and phosphatidylserine.

Phosphorylation is a primary mechanism for regulation of phosphodiesterase activity

PDE1 is phosphorylated by Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II), which results in decreased affinity of this enzyme for Ca2+/calmodulin and an increase in the concentration of Ca2+ needed for its activation. PDE1 also is phosphorylated by cAMP-dependent protein kinase, which also decreases the binding of Ca2+/calmodulin. The inhibition of PDE1 by cAMP-dependent protein kinase could sustain intracellular cAMP concentrations under certain physiological conditions.

cAMP-dependent protein kinase also phosphorylates PDE2, PDE3 and PDE4, although the effects of phosphorylation are different for each of these enzymes. Only the particulate form of PDE2 is phosphorylated by the protein kinase, but this does not influence enzyme activity. Phosphorylation of PDE3 by cAMP-dependent protein kinase in rat adipocytes stimulates the catalytic activity of the enzyme. PDE3 in these cells also is phosphorylated and activated by an insulin-activated kinase, which has not yet been identified with certainty.

PDE4 is activated similarly upon phosphorylation by cAMP-dependent protein kinase. As discussed above, only the long form of PDE4 is phosphorylated; the short forms lack the amino terminus that contains the phosphorylation site. This provides a mechanism for a rapid and readily reversible activation of PDE4 by phosphorylation, as well as more long-term and sustained regulation of PDE4 by gene expression. Certain PDE4 isozymes are phosphorylated by PKC, by mitogen-activated protein kinase (MAP-kinase) and in response to insulin. These phosphorylation reactions are characterized incompletely and their functional consequences are not yet established.

Phosphodiesterase inhibitors show promise as pharmacotherapeutic agents

This may not be surprising given the widespread role of cyclic nucleotides in the regulation of cell function [32,33]. The best examples of drugs that influence PDEs are the methylxanthines; these drugs are used therapeutically in the treatment of obstructive pulmonary disease and are the mild stimulants present in coffee, tea and related substances. Inhibition of PDE contributes to some of the clinical effects of these drugs.

Other examples of PDE inhibitors with possible clinical usefulness are inhibitors of PDE3 or PDE4 (Table 22-1). Based on the localization of PDE3 to heart and vascular tissue and the role of cAMP in mediating heart muscle contraction and smooth muscle relaxation, a large number of PDE3 inhibitors have been developed for possible clinical applications to cardiovascular medicine. Sildenafil, an inhibitor of PDE5, which is enriched in vascular smooth muscle, has been found to be effective for the treatment of male impotence.

Inhibitors of PDE4 have been developed as possible antidepressants. The rationale for this application comes from the observation that many types of antidepressant treatment appear to increase cAMP function in the brain. Persistent increases in cAMP function may lead to some of the long-term adaptive changes in the brain thought to underlie the antidepressant effects of these agents [42]. Although early clinical trials demonstrated that PDE4-specific inhibitors have antidepressant efficacy, these drugs also produce unwanted side effects that have limited their use. The presence of multiple PDE4 subtypes in the brain raises the possibility that more selective inhibitors may be developed that retain antidepressant actions without unwanted side effects.

Further evidence for the importance of PDE4 isozymes in neuronal function comes from the dunce mutation in Drosophila. This mutation, which results in learning and memory deficits, involves loss of function of a cAMP-specific PDE that is functionally homologous to PDE4. Of course, this observation would appear primarily to highlight the importance of cAMP in neuronal function, as opposed to any specific role for PDE in the nervous system.

Image ch22f2

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 1999, American Society for Neurochemistry.
Bookshelf ID: NBK27996

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