Phosphodiesterases and Cyclic Nucleotide Signaling in Endocrine Cells

Marco Conti

Division of Reproductive Biology Department of Gynecology and Obstetrics Stanford University School of Medicine Stanford, California 94305-5317


    INTRODUCTION
 TOP
 INTRODUCTION
 MODULAR STRUCTURE OF PDEs
 MECHANISMS OF REGULATION OF...
 SIGNALING CASCADES INVOLVING...
 HOW PDE PHARMACOLOGY MAY...
 REFERENCES
 
The discovery of cyclic nucleotides as second messengers has paved the way to much of what we know about signal transduction and the mechanisms of hormone action. Even though other signaling pathways activated by growth factors are the focus of much attention, cyclic nucleotides remain among the most important players in hormone action. When envisioned as a linear cascade, the steps involved in cyclic nucleotide signaling are well defined. Hormones bind to receptors that are coupled via G proteins to cyclases, which synthesize cAMP/cGMP. Cyclic nucleotides, in turn, bind and activate protein kinases that phosphorylate enzymes and transcription factors. Changes in gene expression and cell metabolism are the final outcome. This reductionist approach has been most effective in elucidating the steps involved in cyclic nucleotide signaling as well as the downstream targets. However, we must realize that several major questions have remained unanswered. Why does an identical cAMP signal induce replication in one case and withdrawal from the cell cycle and differentiation in another? How do the myriad of feedback regulations, which are being discovered at a steady pace, impact cyclic nucleotide signaling? How does one explain the redundancy of the components of the cyclic nucleotide cascade?

Over the years, new dimensions have added complexity to cyclic nucleotide signaling. It is now established that protein kinase As (PKAs) are not the only intracellular effectors of cAMP. Cyclic nucleotide gated channels (1) and cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs or EPACs) (2, 3) allow branching of the cyclic nucleotide signals. Compartmentalization of the different components of the signaling cascade is an important determinant of the signal outcome (4), and feedback mechanisms control practically every step of the cyclic nucleotide pathway (5). Therefore, a holistic approach to signaling may provide a better understanding of how cyclic nucleotides function in the cell. Signaling pathways, including cyclic nucleotides, are organized in a nonlinear fashion (6). When an extracellular stimulus reaches the plasma membrane, it is distributed into an array of signals that involves most transduction systems present in a cell, and each component of the signaling cascade is a node of inputs and outputs connecting different signaling pathways. Combinatorial signaling, coincidental detection, signal cross-talk, and signal channeling are buzzwords used to describe this intracellular network. In this context, some steps in the signaling cascade may have new and unexpected functions.

In the cyclic nucleotide cascade, phosphodiesterases (PDEs) are the enzymes that hydrolyze cAMP and cGMP, inactivating these second messengers. Together with phosphatases, PDEs are negative steps in the signaling pathway, and signal termination was thought to be their only function. However, they may have a much broader role in signaling when the whole intracellular network is considered. In view of the presence of multiple intracellular effectors of cyclic nucleotides, PDEs may play a role in distributing the cyclic nucleotide signal among PKAs, cyclic nucleotide-gated channels, and cAMP-GEFs. Because they are regulated by multiple second messengers and kinases, PDEs also integrate the cyclic nucleotide cascade with other signaling pathways. Finally, PDEs may contribute to signal compartmentalization by controlling the diffusion of the second messenger to different cellular compartments. Here, I will review the most recent advances concerning the structure of PDEs and their role in endocrine cell signaling, and will conclude by highlighting possible applications of the pharmacology of PDEs for the treatment of endocrine disorders.


    MODULAR STRUCTURE OF PDEs
 TOP
 INTRODUCTION
 MODULAR STRUCTURE OF PDEs
 MECHANISMS OF REGULATION OF...
 SIGNALING CASCADES INVOLVING...
 HOW PDE PHARMACOLOGY MAY...
 REFERENCES
 
The PDE Superfamily
That more than one PDE protein degrades cyclic nucleotides in the cell was appreciated soon after PDEs were discovered. Different chromatographic PDE forms with clearly distinct kinetics, substrate specificity, and pharmacological properties were demonstrated in extracts from brain and other tissues (7). A more in-depth understanding of the PDE complexity in mammalian cells has come with the cloning and identification of the different PDE genes. The field is evolving rapidly, and new families and genes have been recently added to the PDE superfamily. The latest members have been identified taking advantage of the human genome project by using homologous searches of EST databases (8, 9, 10, 11, 12). At last count, 21 genes coding for cyclic nucleotide PDEs have been identified in mammals (Fig. 1Go). Using the most widely accepted nomenclature (13), the PDE family is indicated by an Arabic numeral, followed by a capital letter indicating the gene within a family, and a second Arabic numeral indicating the splicing variant derived from a single gene (for example PDE1C3: family 1, gene C, splicing variant 3).



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Figure 1. Modular Structure of the PDEs: Schematic Representation of the Domain Arrangement in Members of the 11 Families of PDEs

The number in parentheses next to the gene family indicates the number of known genes. In the PDE6 families, only the genes coding for catalytic subunits are reported. [Modified from Ref. 49.]

 
Structure/Function of PDEs
With the exception of a yeast and a Dictiostelium PDE, which may belong to a different family, all PDEs have a conserved region that corresponds to the catalytic domain (Fig. 1Go) (14). This domain is structurally related to other metal-dependent phosphohydrolases with conserved HD motif (15), pointing to the important role of divalent cations in cyclic nucleotide hydrolysis (16). In addition to the signature sequence AaxxHDxDHxG identified by sequence comparison, a number of conserved residues have been identified within this catalytic domain, and their mutagenesis often impacts catalysis (14). Spontaneous mutations in PDE6 within the catalytic domain and their association with major defects of the retina further support the importance of this domain in the enzyme function (17). The recent release of the crystal structure of the catalytic domain has reconciled the many site-directed mutagenesis studies done on this region of the PDEs (18). The PDE4 catalytic domain is a compact structure composed of 17 {alpha}-helices divided into three subdomains, with the most conserved residues involved in the formation of the catalytic pocket (18). More importantly, this structural analysis has confirmed the presence of metal ions in the catalytic pocket, and it will certainly provide conclusive answers regarding the exact interactions of this domain with cyclic nucleotide substrates and model inhibitors (19).

The arrangement of domains around this catalytic core, as well as the presence of a large number of splicing variants, points to a modular structure of PDEs (5, 14). Several domains with a variety of proven or putative functions have been identified at the amino terminus portion of most PDE forms (Fig. 1Go) and are distinctive characteristics of each family (14). These include protein-protein interaction domains as well as domains that bind small molecules such as cyclic nucleotides. In addition, phosphorylation domains that control the catalytic function have been mapped at the amino terminus of most PDEs (reviewed in Ref. 5). Domains present at the carboxyl terminus of PDEs may be involved in dimerization, as has been suggested for PDE4 (20) and PDE1 (21), or may function as a regulatory domain being a target for phosphorylation (22).

Although viewed by some as an oversimplification, it is probable that all these different domains regulate PDE catalysis by a common mechanism. The regulatory domains that flank the catalytic domains function as a sensor of intracellular signals. Reception of these signals produces a change in conformation of the PDE so that an inhibitory domain no longer exerts a negative constraint on the catalysis. The presence of an inhibitory domain is inferred by biochemical studies with controlled proteolysis of PDE1, PDE2, and PDE4 and deletion mutagenesis of PDE1, PDE3, PDE4, and possibly PDE7 (5). Moreover, the regulation of PDE6 by the inhibitory {gamma}-subunit again points to the important role of inhibitory constraint in PDE catalysis. Along the same line, PDE4s have two unique modules that are conserved in the four genes that compose this family. On the basis of their conservation, they have been named upstream conserved region 1 and 2 (UCR1 and UCR2) (23). Functionally, the UCR2 conserved domain corresponds to an autoinhibitory domain that negatively regulates the catalytic activity (24), while regulatory phosphorylation sites have been mapped in the UCR1 (25). A model for the interactions between these regions has been developed on the basis of domain binding and regulation of catalysis (26). While this model has been recently confirmed by others (27), it remains to be determined to what extent it may be applied to PDEs that belong to other families.

Significance of the PDE Complexity
Domain shuffling may explain why a large number of PDE variants with divergent amino and carboxyl termini have been identified. As an example, the PDE4D gene encodes five well characterized splicing variants, a property that is inherited from Drosophila (14). These variants are generated by alternate splicing and/or different promoter usage (14). More importantly, these variants are subjected to different regulations (see below) or are targeted to different subcellular compartments. Targeting domains have been identified in most PDEs. PDE3s have a domain that includes six transmembrane hydrophobic helices, which target them to the endoplasmic reticulum. This domain is absent in some soluble PDE3 splicing variants (28). Two variants with soluble and particulate distribution have been described for PDE2 and PDE7 (14). A domain that interacts with RACK1, a scaffold protein that binds activated protein kinase C (PKC) isoforms, was identified at the amino terminus of one of the five PDE4D variants (29), and putative SH3 interacting domains have been reported for one PDE4A and one PDE4D variant (30, 31). A scaffold protein that anchors PDE4D to the Golgi/centrosome structures in the vicinity of PKAs has also been reported (32, 33). Although the physiological impact of this differential targeting is largely unknown, these findings lend support to the idea that PDE subcellular targeting may have a role in signal compartmentalization (34).

The divergent properties and the presence of distinct regulatory domains in PDEs explain, at least in part, the heterogeneity of the PDE superfamily. A cell utilizes PDEs with different properties and regulations to adapt to the large variety of signals to which it is exposed, to control cyclic nucleotide accumulation in different subcellular compartments, and to integrate different signaling pathways. More difficult to understand is why multiple genes are present within a family because the corresponding proteins have largely overlapping properties and regulations. Recently, it has been shown that inactivation of only one of the four PDE4 genes present in the mouse produces profound and unexpected phenotypes, suggesting that the functions of different PDE genes do not overlap (35). In addition, in situ studies on PDE mRNA expression in brain have uncovered some specificity in the expression of genes belonging to the same family. PDE4B, for instance, is expressed in the granular layer of the cerebellum, while PDE4D is expressed in the Purkinje cells (36). Thus, it is also possible that gene duplication may have occurred to increase the control of PDE expression in a tissue- and developmental-specific fashion.


    MECHANISMS OF REGULATION OF PDEs
 TOP
 INTRODUCTION
 MODULAR STRUCTURE OF PDEs
 MECHANISMS OF REGULATION OF...
 SIGNALING CASCADES INVOLVING...
 HOW PDE PHARMACOLOGY MAY...
 REFERENCES
 
A wide range of regulations to control cyclic nucleotide hydrolysis are operating in the cell, completely refuting the initial idea that PDEs are housekeeping enzymes with a passive role in signaling. The nature of the stimuli that modulate PDE activity is diverse, ranging from posttranslational modification and binding of small ligands to protein-protein interaction.

Protein-Protein Interaction and PDE Function
PDE1s were among the first targets for calmodulin (CaM) to be identified, and activation of cGMP hydrolysis has been used as a CaM bioassay for more than 20 yr. CaM binding modules, which consist of a basic amphypatic helix, have been identified by protein homology and by deletion mutagenesis in all proteins derived from the three PDE1 genes (37). Several pieces of evidence indicate that the CaM binding domain affects the catalytic domain indirectly by controlling the interaction of an autoinhibitory domain with the catalytic domain (21). Using deletion mutagenesis, this autoinhibitory domain has been mapped to a region between the two CaM binding sites in PDE1A1 (37). Ca++ and CaM produce a major increase in PDE activity, suggesting that the enzyme may be completely inactive in the absence of Ca++. Of interest is the fact that the affinity for Ca++/CaM is different between the different PDE1 proteins. The PDE1A gene encodes two splicing variants, PDE1A1 and PDE1A2. CaM is 10 times more potent in activating A1 than A2, indicating that splicing is a means to regulate sensitivity to Ca++ and CaM (37). Additional data comparing isoenzymes from brain, heart, and lung have shown differences in the affinity of PDE1B and PDE1C for CaM (38). CaM binding is also regulated by PDE1 phosphorylation (see below).

The role of PDEs in light perception underscores the importance of PDEs in signaling. We are able to sense visual cues because light causes a dramatic decrease in cGMP in the retina via activation of a PDE. In this pathway, light-activated rhodopsin interacts with the G protein transducin that, in turn, activates PDE6, which hydrolyzes cone and rod cGMP (39, 40). The decrease in cGMP results in closure of cGMP-gated channels in the membrane, thus causing hyperpolarization. The PDE6 expressed in the retina is a tetramer composed of two distinct {alpha}- and ß-subunits and two {gamma}-subunits in rods, with a slightly different {alpha}2-dimer expressed in cones (41, 42). Two {gamma}-subunits bind the function as inhibitors of the cGMP hydrolytic activity of the {alpha}- and ß-subunits. In addition, a {delta}-subunit copurifies with PDE6 and may play a role in the membrane association of this PDE (43). The site of interaction of the {gamma}-subunit on the {alpha}ß-subunits has been identified by several laboratories and mapped to regions surrounding the catalytic domain (44). This interaction completely suppresses cGMP hydrolysis. Transducin controls the interaction between {gamma}- and {alpha}ß-subunits, but it is unclear whether it interacts directly with the {gamma}-subunits or with the {alpha}ß-catalytic subunits.

PDE5, which is widely expressed in tissues outside the retina, has considerable homology with PDE6. Hence, the idea has been put forward that the activity of PDE5 may be regulated by a homolog of the {gamma}-subunit. Indeed, there are reports suggesting that proteins immunologically related to the retina {gamma}-subunit are expressed outside the retina (45). The exact function of these novel proteins remains to be determined. Other sensory cues may use membrane signal transduction machinery involving G protein interaction with a PDE, as suggested for the taste buds (46).

Cyclic Nucleotide and Other Allosteric Regulations of PDEs
The regulation of PDEs through allosteric binding of cyclic nucleotides was discovered in the 1970s (47). PDE2 binds cGMP with an affinity of approximately 100 nM and produces an allosteric change in the catalytic domain. Because of this allosteric regulation, the enzyme hydrolyzes both cAMP and cGMP with positive cooperative kinetics. However, in the intact cell this enzyme probably functions as a cGMP-stimulated cAMP PDE. This property allows integration of the cGMP- and cAMP-regulated pathways, as suggested for atrial natriuretic factor (ANF) signaling (48).

Structurally related cGMP binding domains have been identified in PDE5, PDE6, PDE9, and PDE10 (49). In PDE5, occupancy of this site may modulate the ability of the enzyme to be phosphorylated by protein kinase G (50, 51). In PDE6, cGMP binding regulates the affinity of {alpha}ß-dimers for the inhibitory {gamma}-subunit (52), while little is known about the role of cGMP binding in PDE9 and PDE10. Similar domains have been found in proteins other than PDEs and have been termed GAF domains (cGMP-specific and cGMP-stimulated PDEs, Anabaena adenylyl cyclase and Escherichia coli Fh1A) (53). The presence of these domains in species where cGMP is not produced has led to the recent proposal that the GAF domain in PDEs may not serve to bind cGMP but is involved in interactions with other unknown small ligands (49).

On the basis of sequence homology with a domain found in proteins from bacteria to eukariots, a PAS (Period, Arnt, Sim) domain was identified in PDE8 (54). This domain functions as a signal detector and is usually associated with a heme or a chromophore cofactor (55). In archaea, the PAS domain of FixL is a sensor for oxygen or possibly nitric oxide (56). Although the function of the PAS domain in PDE8 is not known, it may be important for protein-protein interaction or for sensing concentrations of a small ligand (49), suggesting a novel mode of regulation for PDEs.

Posttranslational Modification
There are now reports demonstrating phosphorylation of PDE1, PDE3, PDE4, PDE5, PDE6, and possibly PDE7 (reviewed in Ref. 5). With some rare exceptions, phosphorylation occurs on regulatory domains present at the amino terminus of the PDE protein. Several kinases including PKA, protein kinase B (PKB), mitogen-activated protein kinase (MAPK), and calmodulin kinase (CaMK) catalyze these regulatory phosphorylations (see below).


    SIGNALING CASCADES INVOLVING PDEs IN ENDOCRINE CELLS
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 INTRODUCTION
 MODULAR STRUCTURE OF PDEs
 MECHANISMS OF REGULATION OF...
 SIGNALING CASCADES INVOLVING...
 HOW PDE PHARMACOLOGY MAY...
 REFERENCES
 
Here we will focus on PDE regulations that have the greatest impact on endocrine systems. In several instances a PDE serves as a connection between two different pathways allowing signal integration. Some of these regulations have been extensively reviewed elsewhere in the context of cardiovascular or central nervous system functions (41), PDE1 being an example of a Ca++ signal regulating cAMP and cGMP concentration.

The PI-3 Kinase Pathway and Activation of PDE3
Insulin and IGF-I binding activates the receptor tyrosine kinase with phosphorylation and recruitment of adapter proteins, including insulin receptor substrate 1–4 (IRS1–4) (57). Once phosphorylated, these adapters recruit several effectors including the lipid phosphatidylinositol 3 kinase (PI-3K). The phosphatidyl triphosphate lipid formed serves as an anchor and recruits to the membrane the kinase PDK1/2 and downstream kinase PKB/AKT (Fig. 2Go) (58). There is now ample evidence that PDE3s integrate this PI-3K signaling cascade with the cyclic nucleotide-regulated pathway. A large number of observations are consistent with the presence of this signaling cascade in the cell. Activation of PI-3 kinase by insulin is associated with an increase in PDE3 activity in adipocytes, and the PDE3 activation is blocked by wortmannin and LY 294002, both of which are inhibitors of PI-3 kinases (59). In addition, insulin treatment causes the incorporation of 32P-phosphate in PDE3B (60, 61). That PDE3B is directly phosphorylated by PKB is demonstrated by cell-free experiments with recombinant proteins (62). Two possible phosphorylation sites have been identified in PDE3B. Ser302 of rat PDE3B was identified by phosphopeptide mapping of PDE3B from insulin-stimulated cells (63). Conversely, site-directed mutagenesis has indicated Ser273 as the major site of PKB phosphorylation (64). While the sequence surrounding Ser273 conforms with the consensus for PKB phosphorylation, Ser302 is an anomalous site because it is also phosphorylated by PKA. Whether both sites are used in a cell-specific fashion is unclear and requires further experimentation. Recently, Rondinone et al. (65) have proposed that an additional mechanism of PDE3 activation by insulin may directly involve phosphorylation of PDE3B by the PI-3K associated with the insulin receptor.



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Figure 2. Signaling Pathways That Control PDE3 and Lipolysis in the Cell

See text for details.

 
The PI-3K-PKB-PDE3B signaling module has important physiological implications because it is used in several endocrine regulations and in growth factor control of the entry and exit from the cell cycle. For example, hormone-sensitive lipase (HSL) is the enzyme that catalyzes the hydrolysis of triglycerides stored in adipose tissue and is thought to be the rate-limiting enzyme for the mobilization of FFA (66). The activity of this enzyme is under the control of catecholamines and other lipolytic hormones that stimulate cAMP accumulation (Fig. 2Go). The PKA activation that follows an increase in cAMP causes activation of HSL by phosphorylation on one or more sites (66). In adipocytes, insulin inhibition of lipolysis is mediated by a decrease in cAMP levels and is associated with a decreased phosphorylation of HSL. Both in vitro and in vivo insulin effects are blocked by specific PDE3 inhibitors (28, 67) pointing to an important role of this PDE. Moreover, the phosphorylation and activation of PDE3B correlates with the inactivation of PKA and the dephosphorylation of HSL. Thus, PDE3 phosphorylation appears to be a crucial step in mediating the effect of insulin on lipid metabolism. The antiglycogenolytic effects of insulin may also be mediated, at least in part, by the same pathway involving a PDE3 (68).

A similar PI-3K, PKB, and PDE3B cascade is activated by leptin (OB), a recently discovered hormone involved in the control of fat metabolism and food intake (69). The peripheral effects of leptin are mediated by the activation of receptors that are structurally related to the cytokine receptors which signal through the janus kinases. This kinase, in turn, phosphorylates IRS-1 and IRS-2 promoting the recruitment and activation of PI-3 kinase (Fig. 2Go). Similar to what has been shown for insulin, PI-3 kinase activation causes PKB and PDE3B activation (70). The resulting decrease in cAMP mediates the antiglycogenolytic effects of leptin in hepatocytes.

Because IGF-I shares the same signaling pathway with insulin, IGF-I regulation of PDE3 may be important in the regulation of cell entry and exit from the cell cycle. In Xenopus eggs, a PDE with the properties of PDE3 is activated by AKT and is an important step in insulin-like growth factor (IGF)-induced resumption of meiosis (71, 72). A PDE3A is the predominant form that is also expressed in mammalian oocytes, and inhibition of this enzyme blocks the resumption of meiosis that follows the gonadotropin stimulation in vitro and in vivo (73, 74). With the same signaling cascade, IGF-I regulates insulin secretion in islet ß-cells by regulating a PDEB (75, 76). In general, it is expected that all the growth factor pathways that use PI-3K may use the PDE3 activation to regulate cAMP levels. This could provide a means to modulate the gating effects of cAMP (77) on the mitogen-activated protein (MAP) kinases signaling pathway, and to control exit and entry from the cell cycle.

PDEs as Homeostatic Regulators
Manipulation of hepatocytes with nonhydrolyzable cAMP analogs demonstrated that a rapid feedback controlling cAMP is operating in these cells (78). Accumulation of the cAMP analog in the cells activates PKA, which in turn activates a PDE. The ultimate result is a decrease in endogenous cAMP levels. With the discovery that a PDE3 is a substrate for PKA, it was proposed that this PDE is involved in these feedback mechanisms (79, 80). More recently, data in thyroid cells also have shown that PDE4 is activated by hormones that increase cAMP via a PKA-dependent mechanism (81). PKA phosphorylates PDE4D3, one of the variants derived from the PDE4D genes, and phosphorylation is associated with an increase in PDE activity (25, 81). The residues phosphorylated by PKA have been mapped by site-directed mutagenesis to the amino terminus of PDE4D3 (25). These observations have been confirmed and extended by demonstrating that introduction of a negatively charged amino acid in position 54 produces an activated enzyme (82). Thus, PDE3 and PDE4 are both rapidly activated by PKA, depending on the cell in which they are expressed (Fig. 3Go).



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Figure 3. Feedback Regulation of PDE3 and PDE4 and Hormone Responsiveness

See text for details.

 
The presence of the PKA-PDE4D feedback loop in intact cells has been recently confirmed by demonstrating that PKA inhibitors block the PDE4D3 phosphorylation/activation and cause a potentiation of TSH-dependent cAMP signaling (83). In a similar fashion, a stable cell line expressing PDE4D3 produces a major change in cAMP accumulation stimulated by hormones, whereas cell lines expressing a phosphorylation-deficient PDE4D3 have normal responses (83). Interestingly, it was observed that inactivating this feedback regulation has a major effect on the intensity of the cAMP spike without any major change in the duration of the signal (83).

During the characterization of the mechanisms causing desensitization, it was observed that an increase in cyclic nucleotides produces an increase in PDE activity (84). This activation was thought to be a mechanism of desensitization that cooperated with receptor/G protein uncoupling. With the limited knowledge of PDE heterogeneity available at that time, little was known about the PDE involved except that the enzyme was a cAMP-specific PDE and that the regulation required protein synthesis and PKA activation (84). A better understanding of this second feedback mechanism has come with the cloning of the PDE4 genes. Stimulation with hormones that increase cAMP invariably produces an increase in PDE4 mRNA and de novo synthesis of PDE4 proteins (85). This is most evident for the PDE4D gene. Long-term FSH stimulation of Sertoli cells causes more than a 100-fold increase in PDE4D mRNA, the accumulation of PDE4D1/D2 variants, and more than a 10-fold increase in PDE activity (86). Identical induction has been observed in most cells (14), suggesting that this is a ubiquitous feedback regulation of cAMP, thus providing a mechanistic explanation of the early findings on long-term, cycloheximide-sensitive PDE activation (Fig. 3Go).

The impact of the PDE feedback on cAMP signaling is emerging from studies on PDE4D knockout mice (35). PDE4D-null mice display a 30–40% decrease in growth rate during puberty, and the adults are usually smaller than their littermates. The decreased growth is associated with a decrease in circulating IGF-I levels, suggesting a disruption of the GH-IGF-I axis. In addition, the homozygous PDE4D-null females display reduced fertility with litter size approximately one-third of normal. This reduction in fertility is associated with a 70–80% decrease in ovulation rate compared with wild-type littermates (35). Surprisingly, when the sensitivity to gonadotropin is measured in granulosa cells from the PDE4D-null mice, a decrease in responsiveness to hCG was observed (35). This decreased response is difficult to reconcile with the common tenet that PDE inhibition leads to an increased cAMP accumulation and cAMP signaling. Pending additional experiments to clarify the exact cause of this decreased response, we propose that inactivation of the PDE4D-PKA feedback loop causes a desensitization of the cAMP signaling pathway at the level of receptor/G protein. Thus, PDE4 regulation allows appropriate cAMP signaling by protecting from desensitization. If confirmed, this concept may have important implications in human diseases as end-organ resistance may be associated with inherited PDE4 inactivating mutations.

Disruption of PDE4D expression also affects muscarinic cholinergic responses in the airway (87). Mice deficient in PDE4D do not respond to methacholine with an increase in airway resistance, in spite of a normal complement of muscarinic cholinergic receptors. This phenotype may be caused by an increase in sensitivity to noradrenaline, which causes relaxation of smooth muscle cells. Alternatively, PDEs may directly play a role in M3 muscarinic receptor signaling that mediates the contractile response of acetylcholine (87), again supporting a role for PDE4D as a homeostatic regulator of signaling.

The PDE4 feedback loop may have an important impact under those pathological conditions in which cAMP accumulation is deregulated. Mutations in Gs{alpha} produce a constitutively active protein that maintains adenylyl cyclase in a chronically activated state. These mutations are responsible for the phenotype of patients with McCune-Albright syndrome (88) and are probable causes of a number of adenomas of the pituitary and thyroid (89). There is also abundant literature for constitutive activation of pituitary hormone receptors that cause chronic cAMP elevation (90). All these spontaneous mutations cause a marked induction of PDE4 and possibly other PDEs both in vitro (91) and in vivo (92). The PDE4 activation must have an impact on growth as the proliferative effects of the Gs{alpha} mutations are seen in vitro only after inhibition of PDE4 (93). Therefore, it is possible that the abnormal growth induced by Gs{alpha} or receptor mutations is modified by the presence of different PDE alleles. Ongoing experiments will determine whether polymorphisms or mutations of a PDE4 exist in humans.


    HOW PDE PHARMACOLOGY MAY IMPACT ENDOCRINOLOGY
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 INTRODUCTION
 MODULAR STRUCTURE OF PDEs
 MECHANISMS OF REGULATION OF...
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 REFERENCES
 
The profound cardiovascular and central nervous system effects of PDE inhibition have been known and exploited for more than 2000 yr. Xanthines, including caffeine and theophylline present in coffee and tea, are the best known PDE inhibitors and have been used for treatment of cardiovascular and pulmonary disorders (41). Their multiple effects are due to their lack of selectivity because they inhibit all PDEs expressed in mammals except PDE8 and PDE9. With the discovery of new genes and the realization that more than 50 PDE proteins are present in mammals, new windows of opportunity are opening for pharmacological intervention. That the PDE catalytic domains are divergent enough to discriminate between different compounds was demonstrated with the synthesis of second-generation inhibitors (41). Drugs that display a 1000-fold selectivity toward PDE3, PDE4, PDE5/6, and possibly PDE1 have been synthesized and are being developed for numerous indications (94). The recent fortunes of the selective PDE5 inhibitors [sildenafil or Viagra (Pfizer, Inc., New York, NY)] underscore the efficacy of this approach and have added momentum to the field (95). Furthermore, drugs that distinguish between different members of one family are being actively developed, and some progress has been made toward this goal. When these drugs become available, selectivity should be greatly improved, thus opening a new host of applications including the manipulation of endocrine functions.

In spite of well established effects in vitro and advances in PDE pharmacology, the use of PDE inhibitors to manipulate endocrine cell responses is an unexplored field. For example, little is known about the functions of PDE8B expressed in the thyroid gland (10). Because of this restricted pattern of expression, this enzyme may be a target to specifically manipulate cAMP levels in thyrocytes and to enhance thyroid hormone production and/or to increase sensitivity of the gland to TSH stimulation.

There are two major areas where PDE inhibitors may be used to manipulate endocrine systems. They may be used for the control of hormone secretion and in the manipulation of end-organ sensitivity to hormones. Several studies have indicated that PDE inhibition may be used as an insulin secretagogue and as hypoglycemic agents. Glucose is the major regulator of insulin secretion by ß-cells of the islets of Langerhans, and its actions are potentiated by an increase in intracellular cAMP (96). In clonal insulin secreting cell lines as well as human and rat islet cell preparations, PDE3 inhibition augmented glucose-induced insulin secretion (97). In support of this concept, insulin secretagogues that were effective in vivo were found to be PDE3 and PDE4 inhibitors (98). Thus, PDE inhibition may be a viable strategy to manipulate insulin secretion and may be used in conjunction with other agonists that increase cAMP in ß-cells. Because PDE4 inhibitors have antiinflammatory effects, the inflammatory component of type 1 diabetes may be ameliorated by PDE inhibition, as has been shown in the NOD mouse model (99).

Pituitary hormone secretion may be another possible site of PDE manipulation. The hypothalamic pituitary adrenal axis is very sensitive to the PDE4 inhibitor dembuphyllin (100, 101). Given orally or intraperitoneally, a marked rise in serum corticosterone follows administration of this inhibitor. LH release is also stimulated by these inhibitors, suggesting that this may be a strategy to manipulate pituitary secretion in vivo.

In view of their marked synergism with hormones that signal through cAMP, PDE inhibitors may be used to augment hormonal stimulation. The power of this approach is underscored by data obtained in the ovarian follicle. Treatment of these follicles with a PDE4 inhibitor causes oocyte maturation and ovulation, mimicking the LH effects (72). It should then be possible to use these inhibitors to potentiate the effect of gonadotropins in the ovary. Conversely, PDE3 inhibitors have been shown to block oocyte maturation without affecting granulosa cells, providing a paradigm for novel contraceptive strategies (72, 73).

In conclusion, we believe that manipulation of PDE activity in endocrine cells may have considerable therapeutic potential. Once the repertoire of PDE expressed in endocrine cells is established, it should not be long before new generations of selective PDE inhibitors are used to manipulate endocrine cell responses and hormone secretion.


    ACKNOWLEDGMENTS
 
The author is indebted to the members of his laboratory for their contribution to the development of the ideas described in this review and to his colleagues at Stanford University for the many stimulating discussions. The space limitations of the minireview format preclude inclusion of all citations relevant to this topic. We have therefore elected to provide representative citations, referring to many reviews of the field where the original papers have been extensively discussed.


    FOOTNOTES
 
Address requests for reprints to: Marco Conti, M.D., Department of Gynecology and Obstetrics, Stanford University School of Medicine, 300 Pasteur Drive, Room A344, Stanford, California 94305-5317.

Work in the author’s laboratory is supported by NICHD Grants HD-20788, HD-31544, and the U54 Center grant HD-31398

Received for publication May 2, 2000. Accepted for publication June 29, 2000.


    REFERENCES
 TOP
 INTRODUCTION
 MODULAR STRUCTURE OF PDEs
 MECHANISMS OF REGULATION OF...
 SIGNALING CASCADES INVOLVING...
 HOW PDE PHARMACOLOGY MAY...
 REFERENCES
 

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