Hormonal regulation of cyclic nucleotide phosphodiesterases.

0163-769X/91/1203-0218$03.00/0 Endocrine Reviews Copyright © 1991 by The Endocrine Society

Vol. 12, No. 3 Printed in U.S.A.

Hormonal Regulation of Cyclic Nucleotide Phosphodiesterases * MARCO CONTI, S.-L. CATHERINE JIN, LUCIA MONACO, DAVID R. REPASKE, AND JOHANNES V. SWINNEN The Laboratories for Reproductive Biology, Departments of Pediatrics and Physiology, University of North Carolina at Chapel Hill, and Institute of Histology and General Embryology, University of Rome, Italy

I. Introduction II. Multiple phosphodiesterase forms present in the mammalian cell A. Classification of phosphodiesterases B. Structure of phosphodiesterases C. Selective phosphodiesterase inhibitors III. Phosphodiesterase regulation by hormones that control intracellular cAMP A. Regulation of the cGI-PDE by cAMP-dependent phosphorylation B. Regulation of the CaM-PDE by cAMP-dependent phosphorylation C. Long term, cAMP-dependent induction of the cAMPPDEs D. Consequences of the short term and long term phosphodiesterase regulation IV. Phosphodiesterase activation by hormones that regulate intracellular calcium V. Phosphodiesterase regulation by insulin and other growth factors that activate tyrosine kinases A. Mechanism of insulin action B. Mechanism of insulin activation of the cGI-PDE VI. Phosphodiesterase regulation by hormones that control intracellular cGMP VII. Steroid hormone regulation of phosphodiesterases VIII. Conclusions and perspectives

protein kinases (6, 7). Although novel second messenger pathways have been discovered and dissected in some detail in the past 10 years (8, 9), cyclic nucleotides (cAMP and cGMP) remain the best characterized second messengers, and their role in the differentiation and control of metabolic processes of the endocrine cell is widely recognized. The components of the membrane-associated machinery that transduces external signals into changes in cyclic nucleotide levels have been isolated and characterized. These include many members of the family of membrane receptors (10-14), G proteins acting as transducers (15, 16), and adenylate cyclase effectors (15, 17). Furthermore, complementary DNAs that encode these components are becoming available (10-17), thus opening new avenues by which to study the structure and function of these membrane-associated, protein complexes. Many of the sequential steps that cause the hormone-receptor complexes to activate G proteins and adenylate cyclase are known (15). Receptors with intrinsic guanylate cyclase activity have also been purified and cloned, and an understanding of their structure/function relationship is advancing at a rapid pace (18, 19). Once cyclic nucleotides are synthesized by their respective cyclases, they either bind and activate specific protein kinases or they are degraded by cyclic nucleotide phosphodiesterases (PDEs). Although cGMP (20; see Ref. 21 for a review) and in one instance cAMP (22) can directly bind and activate other cyclic nucleotide binding proteins, activation of the cyclic nucleotide-dependent protein kinases is the basis of many effects of cyclic nucleotides on cell functions (23). There is also evidence that gene expression can be regulated by cAMP via activation of cAMPdependent protein kinases (reviewed in Ref. 24). The catalytic subunit of this kinase phosphorylates a transacting factor [cAMP responsive element binding protein (CREB)] which binds to specific regulatory elements present in many cAMP-regulated genes (24, 25). This activation of transcription, by changing the state of

I. Introduction

A

COMPLEX array of signals from the extracellular environment regulate many cell processes, including the entry and exit from the mitotic cycle and the induction and maintenance of differentiation (1-3). Cells elaborate these external stimuli via receptors, transducers, and second messengers (4, 5) which control intricate circuits that lead to protein phosphorylation by Address requests for reprints to: Dr. Marco Conti, The Laboratories for Reproductive Biology, Department of Pediatrics, CB 7500, MacNider 202H, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599. * The studies carried out in the authors' laboratory were supported by NIH Grants HD-20788 and P30-HD-18968.

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HORMONE REGULATION OF PHOSPHODIESTERASES

phosphorylation of trans-acting factors, is a crucial step in the cascade of events that serve to transduce the cAMP signal to the cell nucleus and may be a mechanism modulating the activity of other trans-acting factors. Together with a transport mechanism (26) for export of cyclic nucleotides outside the cell, cyclic nucleotide PDEs are responsible for the inactivation of the cyclic nucleotide messenger signals. In most systems, egression of cyclic nucleotides accounts only for a minor fraction of cyclic nucleotide disposal from the cell (26, 27). Therefore, intracellular cyclic nucleotide levels are mainly the result of a steady state of synthesis and degradation. Surprisingly, the possibility that hormones regulate cell functions by controlling the rate of cyclic nucleotide degradation has received little attention, even though enzymes that degrade cAMP were described at the same time this second messenger was isolated (28). One of the major problems encountered in studying cyclic nucleotide degradation is the complexity of the families of PDEs that catalyze this reaction (29). Multiple forms are present in any given cell, and this renders it difficult to single out a particular PDE to study its hormonal regulation. In addition, the hormone-dependent activation of PDE often does not survive cell homogenization. This, together with the fact that questions have been raised as to whether the PDE activity measured in the cell extract faithfully reflects the activity in the intact cells (30), has hampered the progress of the knowledge about the regulation of these enzymes. The data available on hormone control of PDEs are scarce when compared with the plentiful information available on adenylate cyclase regulation. In spite of the difficulties involved in studying these enzymes, considerable progress has been made in the last few years. As we will discuss, new tools, such as specific antibodies and cDNAs, have been developed to unequivocally identify the different forms present in a tissue. It is now clear that activation of PDEs can be the primary event that mediates the action of some hormones and external stimuli. Light activation of a cGMP-PDE coupled to a G protein (transducin) is an example of how a cell uses activation of cyclic nucleotide degradation for signal transduction (31). Therefore, PDEs can function as effectors of the hormone-dependent signal transduction. Second, by virtue of the fact that PDEs can be regulated by multiple stimuli (29), they provide a means of integration between the cyclic nucleotide and other signal transduction pathways. Thus, we shall review data showing that a PDE, whose activity is regulated by Ca++ and calmodulin (CaM), serves to integrate the cyclic nucleotide-dependent pathways with a Ca++- dependent signal transduction system. The knowledge that PDEs are regulated in a complex fashion is lending support to the concept that these enzymes might play an important

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role in many cellular processes. Short-term and longterm cellular adaptation to different stimuli might be one function of PDEs. This is, for instance, suggested by an observation made in Drosophila melanogaster. Memory formation in the fruit fly is impaired when mutations disrupt the expression of a PDE (32), suggesting that PDEs and cAMP are somehow involved in the adaptive processes causing memory formation. The picture that is emerging then is that cyclic nucleotide degradation is not a constitutive function of the cell. Conversely, signals are translated into changes in second messenger inactivation for fine tuning responses of the cell to the extracellular environment. Since it is becoming clear that switching off a signal may represent a positive stimulus that leads to expression of new functions in the cell, there has been a new surge in interest in these steps that lead to inactivation of second messenger-dependent stimuli. For instance, phosphatases, the enzymes that dephosphorylate protein substrates, have been involved in regulation of the cell cycle, and the possible carcinogenic effects of phosphatase inhibitors have given new impetus to the study of these enzymes (33). In this review, some background information will be provided on the structure of the different known PDEs. For a more comprehensive survey of the molecular structure of these enzymes we refer the reader to reviews that have recently appeared (29, 34). Here we will focus more on the established and potential functions of these enzymes, and we will review the mechanisms that control the different PDE forms present in mammalian cells.

II. Multiple PDE Forms Present in the Mammalian Cell Early studies on the characterization of the PDE activity extracted from several tissues soon led to the discovery that multiple PDE forms are expressed in mammalian cells. Thompson and Appleman (35) reported that rat brain cortex extracts contain three PDE forms, which could be separated by ion exchange chromatography and distinguished on the basis of their kinetic properties. Also, fractionation of liver extracts by ion exchange chromatography resulted in multiple peaks of PDE activity (36). In the years following these early observations, considerable effort has been devoted to characterizing the different forms that are present in mammalian cells. A. Classification of PDEs

On the basis of their structural and kinetic characteristics, it is now accepted that the different forms present in mammalian cells can be classified into at least five major families of cyclic nucleotide PDEs (Table 1). The


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TABLE 1. PDE families of isoenzymes Family"

Abbreviation6

cAMP cGMP

I.

Ca++/calmodulin PDEs

CaM-PDE

10-50 1-3

II.

cGMP-stimulated PDEs

cGS-PDE

30 15

cGMP-inhibited PDEs

cGI-PDE

0.1-0.3 0.03-0.3

III.

IV.

cAMP-specific PDEs

cAMP-PDE

1-3 >300

V.

cGMP-specific PDEs

cGMP-PDE

>100 4-5/20

Hormones regulating activity

Specific inhibitors'*

Ca++ CaM phosphorylation

Muscarinic cholinergic agonists GnRH

8-MethoxymethylIBMX Phenothiazine

cGMP

ANF

None

105 kDa Soluble 105 kDa Particulate

Phosphorylation cGMP

Insulin Glucagon Dexametasone

Cilostamide Milrinone Fenoximone

110 kDa 135 kDa 63 kDa "dense vesicle"

cAMP

FSH, PGEj TSH /3-Adrenergic agonists

RO 20-1724 Rolipram

rat rat rat rat

Transducin cGMP?

Light

Dipyridamole Zaprinast

Intracellular modulator

Isoforms described

Ref."

59kDa 61kDa 63 kDa brain 67 kDa low Km 75 kDa lung

38 39-42 40-41 43 44

PDEl PDE2/RD1 PDE3 PDE4/DPD

99 kDa rod 99 kDa cone 93 kDa lung

45-46

47 48 49

50 50-51 52 53-54

55-57 55-58 59

a

The family denomination is based on the kinetic properties or the regulator of the different forms. It follows the nomenclature proposed in Ref. 37. 6 Abbreviation used throughout the text are based on common usage. c Km ranges derived from different forms are reported. Figures are given in micromolar units. d Representative inhibitors are reported. For more details see Ref. 73. e References describing the properties of the different isoforms are reported and are only representative. Additional references are cited in the text.

classification follows the one proposed by Beavo and Reifsnyder (37). The reader should be aware that there is still considerable confusion in the field about the nomenclature. Not all authors use the same name for a given isoenzyme, and the recent discovery of new forms has complicated an already arduous task. The different families of PDEs can be distinguished on the basis of their properties and activation. The CaM PDEs are homodimeric proteins which hydrolyze cGMP usually with higher affinity than cAMP and are activated by the Ca++/CaM complexes (38-42). The different forms isolated (Table 1) show subtle differences in subunit structure, in Km, and CaM activation. Two additional variant forms have been characterized, one from the mouse testis with high affinity for both cAMP and cGMP (43), and the other from lung tissue in which CaM is a subunit tightly bound to the enzyme (44). The other family that hydrolyzes both cAMP and cGMP with low affinity is the cGMP-stimulated PDE (Table 1). This family of enzymes is characterized by the presence of an allosteric cGMP binding site (45, 46). Occupancy of this site pro-

motes hydrolysis at the catalytic site. Only recently has it become clear that the PDEs hydrolyzing cAMP with low Km (0.1-3 JUM) can be subdivided into two distinct families: the cGMP-inhibited family of isoenzymes that are sensitive to the cardiotonic and platelet antiaggregant drugs (47-49) and a family of PDEs that hydrolyze cAMP with a high affinity but that are insensitive to cGMP inhibition (50-54). An additional distinctive characteristic of this latter family of PDEs is the inhibition by drugs such as rolipram and RO-201724. The cGMPspecific PDEs were originally described in the retina as the cGMP-hydrolyzing enzymes that are activated by the guanine nucleotide binding protein transducin (31, 5558). Enzymes specific for cGMP have been isolated from lung in virtue of their ability to bind cGMP and from platelets (59, 60). A common characteristic of this group of enzymes is the presence of a cGMP binding site distinct from the catalytic site. The function of this binding site is unknown. The primary sequence of a number of these enzymes is available from direct sequencing of the purified protein


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or from translation of the nucleotide sequence of cDNA clones (42, 46, 50-58, 61, 62). The comparison of these sequences has confirmed the presence of multiple genes that code for the different members of the families of PDEs. Another piece of information derived from the cloning of the PDE cDNAs is that even within a single PDE family, several genes encode similar but distinct forms, whose presence in some cases was not even suspected from the biochemical data available. For instance, evidence of the presence of multiple forms of cAMPPDEs with the same kinetic characteristics was not available (63) until clones corresponding to these PDEs were isolated (50-54). It is now clear that at least 4 genes that code for these cAMP PDEs (Table 1) are present in the rat (50-54). An additional level of complexity might also be present within a single family of PDEs. Charbonneau and co-workers (34, 64) have provided data indicating that the 61 kilodalton (kDa) and the 59 kDa CaM PDEs are identical except for a region at the amino terminal end, suggesting alternate splicing of a transcript from a single gene. The 5' regions of sequences of a cAMP PDE (rat PDE4) retrieved from the rat testis (65) are different from sequences derived from rat brain (53), again opening the possibility of alternate splicing also for the cAMP-PDE family. In addition, cAMP-PDE cDNAs with variable 5'-ends have been isolated independently by two laboratories for two additional forms (51, 52, 66). These cloning data need to be regarded with some caution because cloning artifacts might be a cause of these differences. Nevertheless, if these observations are confirmed, it will be proved that alternate splicing or multiple start sites of transcription increase the actual number of forms expressed in a cell. The presence of multiple messenger RNA transcripts in the different organs tested is also an argument in favor of multiple start sites or alternate splicing of the PDE mRNAs (5054). That multiple transcription start sites may have an important role is suggested by studies on Dictyostelium discoideum in which transcription of a PDE gene uses different promoters according to the developmental stage (67). The significance of this extreme heterogeneity is unknown. It can be speculated that enzymes with similar catalytic centers have evolved differences in regulatory domains to increase the possibility of control. We will discuss evidence in support of this concept later in this section. The possibility of a redundancy of the cyclic nucleotide degradative pathway also needs to be taken into consideration, as it may provide an evolutionary advantage. B. Structure of the PDEs There is general consensus that the PDEs present in mammalian cells have a common structure (Fig. 1). The

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STRUCTURE OF THE CYCLIC NUCLEOTIDE PHOSPHODIESTERASES

catalytic domain

COOH

Calmodulin binding site

Highly conserved domain Putative hinge region

i

I V

FIG. 1. Structure of the cyclic nucleotide PDEs. The filled box corresponds to the domain conserved in most PDEs (see text). The length of boxes corresponding to the amino- and carboxy terminus domains are not in scale, as they vary widely in length in the different PDEs. Arrows point to the approximate location of the binding site for calmodulin and for cGMP.

comparison of the different PDE sequences available (with the exception of a Dictyostelium discoideum PDE and a Saccharomycies cereuisiae PDE) shows that a region of 270 amino acids is remarkably conserved among families (34, 42, 46, 50-58, 61, 62, 66). In this region several histidine, serine, and threonine residues are invariant in 10 different PDE sequences, suggesting that these residues might have an important function in the PDE protein. This domain is surrounded by regions with no obvious similarities (Fig. 1). Several experimental pieces of evidence indicate that the domain conserved in all these PDEs corresponds to the catalytic domain. Controlled proteolysis of a CaM PDE purified from bovine brain results in a proteolytic fragment that can still hydrolyze cyclic nucleotides, but cannot be regulated by CaM (34, 64, 68, 69), indicating that a catalytic domain can be separated from regulatory domains. An antipeptide antibody against the conserved domain of this CaM PDE recognizes this proteolytic fragment (34, 64). Similarly, controlled proteolysis can separate an activated catalytic domain from a cGMP binding site of a cGMP-stimulated PDE (29, 70). This latter fragment roughly corresponds again to the conserved domain. Fur-


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thermore, deletion mutation studies on a cAMP-PDE (rat PDE3) suggest the same conclusion (Jin, S-L. C, J. V. Swinnen, and M. Conti, unpublished observation). Removal of 100 amino acids at the amino or carboxy terminus of this protein yields an enzyme that is still catalytically active. Conversely, deletions removing larger segments at the amino terminus affecting the conserved domain produce an inactive protein. In addition, point mutations targeting the invariant residues in the conserved domain abolish catalysis. Therefore, this region is most likely directly involved in the structure of the catalytic center of the PDEs. The lack of structural similarity at the amino and the carboxy termini suggests that these domains serve different functions in the various PDEs. Since the PDE families described above are regulated by different mechanisms, it is reasonable to believe that these unrelated regions of the protein serve as regulatory regions (Fig. 1). Evidence in support of this view is available for the CaM-PDE and the cGMP-stimulated PDE (cGS-PDE). It has been shown that the amino terminus of the 59 kDa and the 61 kDa CaM-PDE from bovine tissues contains a sequence with structural features (a-helical and charge distribution) that resemble other CaM binding domains (34). Peptides synthesized on the basis of this sequence are able to interact with CaM in a Ca++dependent manner, confirming that this is the domain interacting with CaM (34, 64). Along the same line, photoaffinity labeling of the allosteric cGMP binding site in the cGMP-stimulated PDE maps this binding site to the amino-terminal region of this protein (70). Therefore, it is clear for at least two PDE families that the amino-terminal domain functions as a modulator of the activity of the catalytic domain. Additional functions for this and the carboxy domains that are not conserved in the different PDEs are to be expected. For instance, it is not known where sites of phosphorylation are located in those PDEs that are substrates for protein kinases. Another possibility that has not been explored is that the amino- and the carboxy-terminal domains may contain signals for targeting the PDEs to different intracellular compartments. In summary, PDEs appear to be composed of a catalytic domain structurally homologous in all PDEs, indicating that diverse enzymes have developed from a common ancestor. This domain is connected via hinge regions to the amino- and carboxy-terminal domains which serve as modulators of the catalytic center. C. Selective PDE inhibitors While in the near future the molecular cloning and sequencing of the different PDE forms should provide a complete picture of the structure of these enzymes, other

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approaches can be used to test the role of different PDEs in cell functions. Early studies on the role of PDEs, in intact tissues or cells in culture, used nonselective PDE inhibitors such as theophilline, caffeine, or 3-isobutyl-lmethylxanthine (IBMX) (71). Considerable progress has been made in the synthesis and characterization of more selective PDE inhibitors. New generations of derivatives are now available that allow the selective inhibition of CaM PDEs, cGI-PDE, and cAMP-PDE (Table 1) (72, 73). These inhibitors can be used to test the role of a certain PDE isoenzyme in the overall cyclic nucleotide degradative pathway of the intact cell. Here are some brief examples of the use of these inhibitors, and for more details, the reader is referred to comprehensive reviews of this field (72, 73). A striking example of selectivity is the difference in inotropic effects on cardiac ventricular musculature of different PDE inhibitors. In the guinea pig, dog, rabbit, and probably human heart (74-77), there are two PDEs that hydrolyze cAMP with high affinity. These have been characterized as a cGI-PDE and a cAMP-PDE (74-77). The cGI-PDE is inhibited by cilostamide and milrinone in the submicromolar range (Table 1), while 100-fold higher concentrations of these compounds are required to inhibit the activity of the cAMP-PDE (74-77). Conversely, the cAMP-PDE is effectively inhibited by rolipram and RO-201724 (Table 1), which have minimal effects on the cGI-PDE activity (74-77). This selectivity has been used to test the role of the two PDEs in the cAMP-dependent inotropic effects in the heart. In perfused heart preparations, milrinone and cilostamide have marked inotropic effects while rolipram or RO 201724 do not significantly alter the strength of contraction (74, 77), suggesting that different PDE isoenzymes have different functions in cardiomiocytes. Another example of selectivity comes from the studies of Lorenz and Wells (78). Using vascular smooth musculature preparations, they showed that several substituted xanthines potentiate the relaxant effect of isoproterenol (which acts through the cAMP pathway) and of sodium nitroprusside (which acts through the cGMP-dependent pathway). This potentiation correlated with their ability to inhibit cAMP-PDEs or cGMP-PDEs (78). Similarly, cilostamide, which is a potent inhibitor of the 3T3 adipocyte particulate cGI-PDE, the enzyme mediating insulin's antilipolytic effects, is a more potent stimulator of lipolysis than RO 20-1724, which instead inhibits a soluble cAMP-PDE (79). In human basophils, antigen-stimulated histamine release can be inhibited by an increase in intracellular cAMP (80). It has been shown that a selective inhibitor of the cAMP-PDE, rolipram, blocks histamine release by these cells, while the inhibitor of the cGMP-PDE, zaprinast (M&B 22,948) is without effect (80).


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An increasing number of pharmacological agents are therefore available to test which role different PDE forms play in a cellular response. Surprisingly, .while a large body of work has been done on cardiac muscle or smooth muscle contractility, few reports have used these selective inhibitors to study the impact of different PDE forms on the response of endocrine cells.

III. PDE Regulation by Hormones that Control Intracellular cAMP It is now clear that an increase in intracellular cAMP caused by those hormones that regulate adenylate cyclase also produces changes in PDE activity with consequent changes in rates of cyclic nucleotide degradation. Early studies showed that an increase in intracellular cAMP would bring about an increase in PDE activity in fibroblasts (81, 82), lymphoma cells (83), and in liver (84). Only after methods were developed to distinguish between the different PDEs has it become clear that multiple regulations affecting different PDE forms are operating in a cell. The biochemical mechanisms that mediate these activations will be reviewed here for each of the different PDE families. The current opinions on the physiological significance of these regulations will also be discussed.

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cAMP (91). Degradation of this compound was enhanced when intact hepatocytes were challenged with glucagon (91). The high affinity PDE, which is activated in a cAMPdependent manner, has been further characterized and purified, and there is now general consensus that this enzyme belongs to the cGI-PDE family of enzymes (48, 49). This enzyme is, in most instances, particulate and can be solubilized by nonionic detergents (48), or by limited proteolysis (92). There is also agreement that a phosphorylation catalyzed by a cAMP-dependent protein kinase is responsible for its activation. Several lines of evidence support this conclusion. Purified cGI-PDE derived from rat adipocytes (93) and human platelets (95, 96) can be efficiently phosphorylated in vitro by the purified catalytic subunit of a cAMP-dependent protein kinase. This phosphorylation brings about an increase in hydrolytic activity (Fig. 2). In addition, isoproterenol produces an increase in 32P-labeling of a 135 kDa polypeptide that has the same molecular mass and is immunologically indistinguishable from the cGI-PDE (94). It should be pointed out that the same enzyme is under the control of insulin, again via phosphorylation at a site that might not coincide with the protein kinase A (PKA)catalyzed phosphorylated site (94).

A. Regulation of the cGI-PDE by cAMP-dependent phosphorylation It has been shown that glucagon produces a rapid increase in the PDE activity present in the rat liver (84). The PDE form activated by glucagon is mostly particulate, and its activity is inhibited by micromolar concentrations of cGMP (84). Houslay and collaborators (85) have been able to separate several different PDE forms from the particulate fraction of rat hepatocytes. They have shown that glucagon, as well as cholera-toxin and (Bu^cAMP, produces an increase in the activity of a "dense vesicle" form which can be separated from another membrane-bound PDE (peripheral PDE) (49, 85). The "dense vesicle" PDE form has kinetic properties similar to those described in an earlier report (84). A similar PDE activation has been observed in rat adipocytes, in which the adrenergic agonist isoproterenol produces a rapid, transient activation of a membrane-bound PDE with ti/2 of about 1 min (86-88). Studies carried out using 18O labeling of adenine nucleotides of platelets have shown that agents that increase intracellular cAMP produce an increase in both synthesis and degradation of the cyclic nucleotide, suggesting that a rapid activation of PDE indeed occurs in an intact cell (89, 90). Another piece of information indicating that a cAMP-dependent activation of PDE occurs in the intact cell comes from studies using the cAMP analog 8-p-chlorophenyl-thio-

Nucleus FIG. 2. Summary of the mechanisms of regulation of cGI-PDE and cAMP-PDE by hormones that regulate intracellular cAMP. The scheme is based on data reported in references. Rs, Stimulatory receptor; Ri, inhibitory receptor; Gs, stimulatory guanine nucleotide binding protein; Gi, inhibitory guanine nucleotide binding protein; C, catalytic subunit of the adenylate cyclase complex; PKA, cAMP-dependent protein kinase; ER, endoplasmic reticulum/microsomal fraction.


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B. Regulation of CaM-PDE by cAMP-dependent phosphorylation Sharma and co-workers (97, 98) have studied the possibility that CaM-PDE activity is regulated by a cAMPdependent phosphorylation. Incubation of purified bovine brain or bovine heart 60 kDa CaM-PDE and the catalytic subunit of cAMP-dependent protein kinase leads to phosphorylation of the PDE (97). This reaction appears to be specific because other kinases (98), with the exception of the CaM-kinase II which phosphorylates the 63 kDa CaM-PDE (99), are unable to phosphorylate these PDEs. Interestingly, this phosphorylation produces a decrease in CaM affinity of the enzymes and a decrease in PDE activation by Ca++/CaM (97,98). Thus, the dosedependent activation of these PDEs is shifted to the right when these proteins are in the phosphorylated state. This has led the authors to propose that an increase in intracellular cAMP brings about an increase in phosphorylation of these CaM-PDE isoenzymes (97, 98) in the intact cell. This phosphorylation in turn might affect their activity under basal Ca++ concentrations, or partially suppress the CaM activation, if an increase in intracellular Ca++ follows the activation of the cAMPdependent pathway. According to the authors, this transient decrease in activity of the 60 kDa CaM-PDE might serve to increase the amplitude on one hand, and shorten the duration on the other, of the spike in cAMP concentration (98). It should be pointed out that data showing that these CaM-PDEs are phosphorylated in the intact cell in a cAMP-dependent manner are not yet available. In the Sertoli cell of the rat testis, it has been shown that FSH produces a short-term inactivation of the PDE present in the cell (100, 101). This inhibition reaches a nadir in about 15 min. Inhibition of the activity is observed whether cAMP or cGMP is used in the PDE assay, suggesting that a PDE form which hydrolyzes both nucleotides is involved in this regulation. Since a CaM-PDE is the major form of PDE present in these cells before hormone stimulation (102) and since FSH activates the cAMP-dependent protein kinase (100), it is possible that this short-term inhibition is due to a cAMP-dependent phosphorylation of this enzyme. The reason why this is one of the few systems where this short-term inhibition can be readily detected is that in the immature cells, CaM-PDE is essentially the only cyclic nucleotide hydrolytic activity present in the cytosol. If this hypothesis is confirmed by further data, there would be a demonstration that the regulatory mechanism proposed by Sharma and co-workers is indeed operating in the intact cell. C. Long-term, cAMP-dependent induction of the cAMPPDEs The above described regulations occur within seconds or minutes of hormone activation of the target cell. It is

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now clear that stimulation of a distinct group of PDEs occurs in the cell after hormone activation of the cyclic nucleotide-dependent pathway and that this activation follows a much longer time course. For more than 15 years it has been known that in fibroblasts (81, 82, 103), lymphoma cells (83), or glioma cells (104-107) exposed to epinephrine or prostaglandins, there is a sustained activation of a soluble high affinity PDE. This activation follows a slow time course and requires ongoing protein synthesis to occur. Similarly, it has been shown that FSH, which controls cAMP levels in Sertoli and granulosa cells of the male and female gonads, produces a long-term increase in PDE activity (102, 108, 109). Again, both protein and RNA synthesis were required for the FSH stimulation to occur (102,108, 109). This gonadotropin-dependent activation of a PDE activity has been observed not only in vitro but also in vivo, indicating that this regulation is also operating under physiological conditions (110). A similar in vivo effect on a PDE has been observed in the pineal gland after injection of epinephrine (111). A common finding in all the above described systems is that a comparable increase in PDE activity is caused not only by those hormones that regulate cAMP in that particular cell, but also by cholera toxin and forskolin or by cAMP analogs. The reverse is true for the Sertoli cell, in which a decrease in intracellular cAMP brought about by inhibitory adenosine receptors also blocks the FSH-dependent PDE induction (112). It is clear that the activation of a cAMPdependent protein kinase is required for this long-term increase in PDE activity. This is demonstrated by comparison of the PDE activation in wild type S49 lymphoma cells and kinase mutants. While epinephrine stimulates the PDE activity in the wild type cells (83), such a stimulation is absent in S49 cells bearing a mutation in a cAMP-dependent protein kinase (Kin-). Furthermore, additional mutants have been described in which a decrease in PDE activity is associated with kinase mutations even under basal conditions (113). The above data from a wide variety of in vitro and in vivo experimental systems strongly support the idea that the long-term regulation of PDEs is a widespread phenomenon. In addition, they provide compelling evidence that this activation is mediated by the cAMP-dependent pathway. As for the properties of the PDE involved in this regulation, the early studies performed did not have the tools available to identify this PDE isoenzyme. Nevertheless, a survey of all the data published permits the conclusion that the long-term, cAMP-dependent activation targets a high affinity cAMP-PDE (Table 1). These forms are not inhibited by cGMP and are sensitive to compounds such as rolipram and RO 20-1724 (Table 1) more than to cilostamide and milrinone (52, 72, 73,114).


4

"

(

j

August, 1991


These forms are, therefore, different from the cGI-PDE, which is transiently activated by a cAMP-dependent phosphorylation (Fig. 2). Since the cloning of cDNAs that encode cAMP-PDEs (50-54), it has become possible to confirm that these isoenzymes are the target of this regulation. The sequence of events leading to the increase in PDE activity has also been elucidated. Studies conducted in our laboratory have shown that cAMP regulates the level of mRNAs that encode such cAMP-PDEs both in the Sertoli cell and in C6 glioma cell line (52). In the Sertoli cell, in which transcripts corresponding to two cAMPPDE isoforms (rat PDE3 and rat PDE4; see Table 1) are present, both messages are increased, although to a different degree, after stimulation of the cell with FSH, (Bu)2cAMP, cholera toxin, or forskolin (52, 65, 66). There is also evidence that a cAMP-dependent increase in transcription of one of the cAMP-PDE genes (rat PDE3) precedes and is one probable cause of the observed increase in mRNA levels (65). Preliminary evidence from our laboratory also suggests that cAMPmediated stabilization of PDE mRNAs might contribute to the increase in steady-state levels of the PDE mRNA. Finally, the increase in cAMP-PDE mRNA is followed in the Sertoli cell by an increase in the PDE protein as quantified by immunoblot analysis. The increase in PDE protein per se is probably sufficient to produce the 10fold increase in PDE activity measured in the Sertoli cell homogenate. However, it is also possible that posttranslational modifications of the PDE protein are necessary to bring about the full catalytic potential of the enzyme. Thus, these data provide evidence that a regulation of a cAMP-PDE gene expression is present in the hormone target cell (Fig. 2). This can be viewed as an intracellular feedback mechanism by which cAMP regulates the expression of its own degrading enzymes. It should be mentioned that the presence of a cAMPPDE in a cell does not necessarily imply that this enzyme is regulated by cAMP. As previously described, there are at least four genes that code for similar cAMP-PDEs in the rat. Preliminary data available from our laboratory (De Negri, S., J. V. Swinnen, and M. Conti, unpublished, and Ref. 115) indicate that these four genes are not equally sensitive to hormonal and cAMP stimulation. In the MA-10 Leydig tumor cell line, the major cAMP-PDE present corresponds to rat PDE2 (115). Neither the activity nor the protein levels of this PDE are affected by increasing cAMP by either human CG, forskolin, or (Bu)2cAMP. That this enzyme is not regulated by cAMP is further supported by Northern blot analysis, by which no significant changes in rat PDE2 mRNA levels are detectable after manipulation of intracellular cAMP. Similar conclusions are reached from studies in FRTL-5

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thyroid cells (116), in which rat PDE3 and rat PDE4 but not rat PDE2 are regulated by TSH or by (Bu)2cAMP. A long-term regulation of PDE expression mediated by cAMP might not be restricted to the family of the cAMP-specific PDEs. Differentiation of 3T3-fibroblasts to adipocytes is induced by treatment of the cells with (Bu)2cAMP (117, 118). This differentiation is associated with a several fold increase in cGI-PDE in the particulate fraction. The development of cDNA probes for the cGIPDE should answer questions concerning the exact mechanism underlying this regulation. D. Consequences of short-term and long-term PDE regulation From the above described data it is clear that an elaborate set of changes in the activity of several PDE forms follows hormone activation of the cAMP-dependent pathway. By virtue of the multiple forms involved in this regulation, the changes in rate of cyclic nucleotide hydrolysis may span over a period of minutes to days. The following cascade of events is anticipated when a hormone binds to receptors on a hypothetical cell that expresses a membrane-bound cGI-PDE and soluble CaM-PDEs and cAMP-PDEs (see Fig. 2). Within seconds after the hormone binds to the receptor, adenylate cyclase is activated and cAMP accumulates in the cytosol causing an activation of a cAMP-dependent protein kinase. The activated kinase phosphorylates a membranebound cGI-PDE, thus increasing its activity. At the same time, the soluble CaM-PDE becomes phosphorylated. This causes a change in its activity depending on the degree of activation by Ca++/CaM. At the time when activation of the cGI-PDE is dissipating, a new, probably larger increase in PDE activity follows the accumulation of newly synthesized cAMP-PDE. If the stimulus that activates the cAMP cascade is not removed, the cAMPPDE induction causes an increase in PDE activity that may- persist longer than 24 h. However, some caution should be exercised in evaluating these regulations, since no experimental data have addressed the possibility that all these PDEs and their regulatory mechanisms are operating within the same cell. One can offer several plausible explanations for these fluctuations in the rate of cyclic nucleotide hydrolysis, even though the exact significance of these events is still not entirely clear. The reason for a simultaneous increase in cAMP synthesis and cAMP degradation in a cell is elusive. As has been shown for platelets in which prostaglandin I (PGI) stimulates both adenylate cyclase and PDE (89, 90), this simultaneous increase in synthesis and degradation produces an accelerated cAMP turnover. This accelerated cAMP turnover may serve to increase the sensitivity of the c AMP-dependent system


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by producing more rapid changes in PKA activation/ deactivation. This hypothesis is supported by studies on a cell-free reconstituted system, in which PKA activation was more effectively produced in the presence of an active PDE (a CaM-PDE in this case) (119). Goldberg and colleagues (120) have proposed that cyclic nucleotide hydrolysis generates free energy that is utilized by the cell with an unknown mechanism. There is also the possibility that the short-term activation is the expression of a mechanism that serves to terminate the hormonal stimulation or to limit cell response to hormones (93-96, 121, 122). This early activation of the cGI-PDE has the same time course of other desensitizing mechanisms mediated by phosphorylation, such as receptor phosphorylation and uncoupling from the adenylate cyclase (123, 124). There might then be a cooperation of different desensitizing mechanisms that control both synthesis and degradation of cyclic nucleotides and serve to dissipate an external stimulus (93-96,121,122). Also, the long-term cAMP-PDE induction has a major impact on cell responsiveness. Data available for the Sertoli cell indicate that this PDE activation contributes to approximately 50% of the reduction of hormone responsiveness of the Sertoli cell (101,125), the remaining reduction being accounted for by adenylate cyclase desensitization and receptor down-regulation (101). Experiments using PDE inhibitors that are selective for the PDE induced by FSH show that upon inhibition of the PDE, responsiveness of the cell is restored (125). That an increase in cAMP-PDE activity by itself produces a reduction in hormone responsiveness is also supported by studies on stable transfected MA-10 cells (115). These cells respond to LH/human CG with an increase in intacellular cAMP and increased steroidogenesis (126). A 2- to 5-fold increase in overall PDE activity can be produced in these cells by stable transfection with an expression vector carrying a cAMP-PDE cDNA (115), the same PDE that is activated by FSH in the Sertoli cell. This increase is accompanied by a commensurate reduction in human CG-dependent cAMP and steroidogenic response. Thus, it is clear that a sustained increase in cytosolic cAMP-PDE activity can lead to a reduction of the sensitivity of the cell to hormone. There are some noteworthy differences between the short-term activation of the cGI-PDE and the delayed induction of the cAMP-PDE. Activation of the cGI-PDE occurs at a time in which cAMP has reached its peak concentration (88). The induced cAMP-PDE, instead, is activated when cAMP levels are returning toward the basal levels (101). These intracellular cAMP concentrations are below the Michaelis-Menten constant (Km) of the enzyme for cAMP so that the enzyme will express its full catalytic potential only when cells are rechallenged with hormone.

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Studies on a genetic disorder affecting the hormonal control of water permeability in the kidney may provide some important clues about the role of cAMP-PDE regulation in cellular responsiveness. A strain of mice (DI+/+ severe) with hereditary nephrogenic diabetes insipidus (NDI mice) has been characterized (127, 128). The molecular basis of this disorder appears to be an impaired response of the distal portion of the nephron to arginine vasopressin (AVP). In normal animals, AVP stimulates adenylate cyclase and activates a cAMP-dependent protein kinase, thus regulating hydraulic water permeability in the collecting duct (129). On the contrary, the entire medullary collecting duct of the NDI mice does not respond to AVP with an increase in intracellular cAMP (129,130), even though AVP apparently stimulates adenylate cyclase in these cells to the same extent as in normal cells (130). Dousa and collaborators (131) have studied the PDE activity expressed in the inner medullary collecting duct of these NDI mice and have found an increase in a cAMP-PDE activity, suggesting that an altered cAMP hydrolysis might be one of the causes of the observed phenotype (131). Finally, the selective cAMP-PDE inhibitor rolipram can restore the AVP-stimulated cAMP accumulation in the inner medullary collecting duct of the NDI mice (132). Rolipram administered to NDI mice also increases urine osmolarity and ameliorates the diabetic condition. These studies then support the hypothesis that the nephrogenic diabetes insipidus of the NDI mice is caused by an abnormal cAMP catabolism. An altered expression or regulation of a cAMP-PDE or a mutation affecting the activity of this enzyme might be at the basis of this genetic disorder. These data then suggest that a cAMPPDE plays a crucial role in the control of cell responsiveness to hormones. It seems unlikely that the above described regulations are merely a safety mechanism that rescues the cell from extreme, nonphysiological stimulations. Both short-term and long-term activations occur at hormone concentrations well within the physiological range. Thus, these PDE activations are more likely part of the mechanisms that control normal cell homeostasis. Since FSH also induces the differentiation of the Sertoli cell (133), it is possible that induction of a cAMP-PDE and the consequent changes in the pattern of cAMP degradation are simply an expression of the differentiated phenotype. That changes in PDE activity accompany differentiation has been observed in several instances. Adipocyte differentiation is accompanied by an increase in cGI-PDE expressed in these cells (117, 118). In a similar fashion, an increase in cAMP-PDE is observed during in vivo maturation of the Sertoli cell. It is also interesting to notice that the responsiveness of the Sertoli cell to FSH decreases during maturation (134), indicating that re-


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sponse to gonadotropin is inversely proportional to the intracellular concentration of PDE. With a time course similar to the cGI-PDE activation, a CaM-PDE is phosphorylated by the cAMP-dependent protein kinase, thus becoming less active. The overall activity of this latter enzyme is dependent on the intracellular Ca++ concentrations at the time at which the hormone is stimulating cAMP synthesis. The reason for activating one PDE and inhibiting another PDE at the same time is not clear. The cGI-PDE is mostly particulate while the CaM-PDE is usually soluble. It is then possible that these opposing regulations occur in two different cellular compartments. The inactivation of the CaM-PDE could also be a mechanism by which Ca++ regulation of cAMP levels is turned off during the activation of the cAMP-dependent pathway.

IV. PDE Activation by Hormones that Regulate Intracellular Calcium The CaM-PDE forms that have been isolated and characterized (38-44, 135, 136) share the property of being activated by Ca++-CaM complexes in a cell-free system. The properties of the binding of Ca++ CaM to PDE have been studied extensively (97, 98), as one of the established biological assays for CaM is the activation of the hydrolytic activity of this PDE. On the basis of this cell-free regulation, it has been logical to hypothesize that also in the intact cell in vivo, fluctuations in intracellular Ca++ levels can produce changes in CaMPDE activity and consequently fluctuation in intracellular cAMP levels (Fig. 3). That this indeed occurs in the intact cell has been shown in several systems. In the 132INI human astrocytoma cell line, accumulation of cAMP in the presence of isoproterenol or prostaglandin Ei is attenuated by muscarinic cholinergic agonists (137, 139, 140). Similarly, in dog thyroid slices the TSHdependent increase in intracellular cAMP is inhibited by carbachol (138, 141, 142). That PDE activation is responsible for the muscarinic agonist inhibition was initially suggested by studies employing PDE inhibitors (137-145). In the presence of the nonselective inhibitor IBMX, oxotremorine no longer prevents /3-adrenergic agonist stimulation of cAMP accumulation in astrocytoma cells. Equally intriguing is the observation made in dog thyroid cells, where two types of negative control of cAMP levels are operating (138). It was found that norepinephrine, through an a2-receptor, decreases cAMP accumulation, but this decrease is not blocked by the PDE inhibitor IBMX. This effect is a direct negative regulation of adenylate cyclase, via activation of a Gi protein, since pertussis toxin blocks this inhibition (138, 143). Conversely, acetylcholine and carbachol, through a muscarinic receptor, inhibit cAMP accumulation, but

227 Muscarinic receptor

ER cisternae

FIG. 3. Mechanism of regulation of the Cam-PDE by those hormones that regulate phosploinositide turnover and intracellular calcium. The scheme is based on the mechanism proposed in Refs. 137 and 138. PLC, Phospholipase C; Gx, putative guanine nucleotide binding protein; ER, endoplasmic reticulum cisternae sequestering calcium.

this inhibition is sensitive to IBMX, indicating that an alternative mechanism of reduction of cAMP accumulation, probably involving a PDE, is activated in these cells. This hypothesis is supported by the measurement of the rate of cAMP degradation in intact astrocytoma cells, which shows that oxotremorine increases cAMP degradation approximately 3-fold (139). A similar increase in the rate of cAMP degradation was obtained by the ionophore A23187, indicating that an increase in intracellular Ca++ plays a role in this regulation (139, 142). The finding that pertussis toxin does not block the muscarinic agonist inhibition points to the conclusion that muscarinic receptors are coupled to inhibition of the cAMP-dependent pathway via a unique mechanism (137, 138) (Fig. 3). This involves muscarinic receptor-dependent increase in phosphoinositide turnover, increase in intracellular Ca++, and activation of a CaM-PDE. That a Ca++ CaM PDE is target for this regulation is also suggested by studies with selective PDE inhibitors conducted both on the thyroid and astrocytoma cells (144, 145). It should be remembered that the 63 kDa PDE present in the brain is phosphorylated by a CaM-dependent kinase, and the phosphorylated PDE becomes less sensitive to CaM (97, 99). It is not clear how this regulation fits in the above charted pathway. Although not as well characterized as the above described systems, an activation of CaM-PDE might also be responsible for GnRH inhibition of LH-stimulated cAMP accumulation in granulosa cells (146). In fact, GnRH inhibition of cAMP accumulation is dependent


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on Ca++ and can be obliterated by incubation of the cells with the PDE inhibitor IBMX. Similarly, in the Sertoli cell, serum inhibition of the FSH-dependent cAMP accumulation might be mediated by an increase in activity of a CaM-PDE (147). Serum effects are again blocked by the PDE inhibitor IBMX (147). On the basis of the above findings, one can conclude that Ca++ and cAMP intracellular levels are linked to each other, and that a CaM-PDE is one of the chips integrating these two circuitries. It is also predicted that those hormones that increase intracellular Ca++ should also attenuate cAMP-dependent responses in the target cell (Fig. 3).

V. PDE Regulation by Insulin and Other Growth Factors that Activate Tyrosine Kinases A. Mechanism of insulin action The mechanism by which insulin exerts its metabolic and pleiotropic effects in liver and adipose tissue is still not fully understood. The discovery that the receptor is a tyrosine kinase (reviewed in Ref. 148) has substantiated the possibility that a cascade of phosphorylations involving both tyrosine and serine/threonine kinases is the major signaling pathway used by this hormone (149). Some of the steps of this cascade have been elucidated (reviewed in Ref. 149). The release of "insulin mediators" might be an alternative or concomitant pathway of cellular activation (150, 151). Insulin opposes the effects of several catabolic hormones. Glucagon, catecholamines, and ACTH, which stimulate adenylate cyclase in adipocytes, are potent stimulators of fatty acid transport and triacylglycerol hydrolysis. The cascade of events triggered by these hormones includes activation of adenylate cyclase, increase in intracellular cAMP levels, and activation of a cAMP-dependent protein kinase (reviewed in Ref. 152). In turn, this protein kinase phosphorylates a hormonedependent lipase causing its activation (153, 154). The activated lipase hydrolyzes triacylglycerol, thus producing the release of glycerol and free fatty acids. These lipolytic hormones also stimulate long chain fatty acid transport across the membrane of adipocytes with a mechanism that involves cAMP (155). In several systems insulin blocks both lipolysis and fatty acid transport. It is believed that some of the insulin effects are dependent on a decrease in intracellular cAMP. Insulin inhibition of lipolysis is associated with an inhibition of epinephrine-dependent cAMP accumulation (156). Insulin also decreases the activity of cAMP-dependent protein kinase, a finding consistent with the view that a decrease in intracellular cAMP follows insulin treatment (157). While there have been conflicting reports as to whether insulin inhibits adenylate cyclase activity (158,159,160),

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several lines of evidence indicate that insulin exerts its effects by activation of a PDE. Beebe and co-workers have screened a large number of cAMP analogs for their susceptibility to PDE hydrolysis and activation of cAMP-dependent protein kinase (161). They found that the stimulation of lipolysis by those compounds that were more readily hydrolyzed by PDEs was efficiently antagonized by insulin, while the stimulation by those compounds resistant to the PDE was not competed for by insulin. In addition, PDE inhibitors with a different structure were able to block the antilipolytic effects of insulin, suggesting an involvement of a PDE in the insulin-inhibitory response (79, 161). Also, the insulin-inhibitory effects on fatty acid transport could be blocked by the PDE inhibitor IBMX (162). Experiments in which cAMP levels were decreased with adenosine-receptor agonists have provided results consistent with the conclusions drawn in the PDE inhibitor studies (88,153). Manipulation of intracellular cAMP in adipocytes with adenosine deaminase shows that an increase in cAMP counteracts insulin effects, again suggesting that this hormone acts by decreasing intracellular cAMP (153). All these data in intact cells are consistent with the view that insulin activation of a PDE plays a crucial role in the inhibition of lipolysis. Indeed, measurement of the PDE activity in adipocytes or liver cells shows that insulin increases the hydrolytic activity present in the homogenate (84, 85,88, 92, 94,163-167). Several reports have agreed on the fact that the insulin-stimulated PDE is a high affinity cAMP PDE, which is sensitive to cGMP inhibition (cGI-PDE). The enzyme is membrane bound and can be solubilized by different means (48, 49,85, 92). The insulin-regulated enzyme has been purified from adipocytes (48) and liver (49) and shown to be similar to forms purified from platelets and heart (168, 169). As the methods to characterize the PDEs have improved, it has become clear that the insulin-regulated enzyme is similar or identical to the enzyme that is activated in a cAMP-dependent manner in the same tissues (88, 94, 170, 171). For sake of completeness, it should be mentioned that Houslay and collaborators (172) have reported that a different, membrane bound, "peripheral" PDE is also activated by insulin. The properties of this enzyme resemble those of a type IV, cAMP-PDE which is inhibited by rolipram. Similarly to what has been shown for insulin in adipocytes and liver, both insulin and IGF-I, which binds to a similar receptor/tyrosine kinase, stimulate cAMP hydrolysis (173) in Xenopus oocytes and promote resumption of meiosis. Therefore, other biological effects of these hormones are mediated by an activation of a PDE. Interestingly, injection of activated Ha-ras protein in these oocytes also activates cAMP hydrolysis (173).


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B. Mechanism of insulin activation of the cGI-PDE It has been shown that the increase in PDE activity that follows insulin stimulation of the intact cell is still present after isolation of the particulate fraction, solubilization by detergent, and in the initial steps of purification (48). This suggests that a covalent modification of the enzyme is probably responsible for activation. Houslay and collaborators (174) showed that insulin action in the liver is associated with the rapid phosphorylation of a membrane protein substrate. With the development of specific antibodies that made possible the rapid isolation of this PDE, it has been shown that this enzyme can be phosphorylated in an insulin-dependent manner on a serine residue (94) and that this phosphorylation is probably responsible for the activation (Fig. 4). On the basis of several pieces of evidence, Manganiello and coauthors (170) have suggested that this residue is most likely different from the serine which is phosphorylated by the cAMP-dependent protein kinase. The nature of the kinase that is activated by insulin and that phosphorylates the PDE is unknown. The presence of such an enzyme has been also inferred from recent observations that particulate PDE can be activated by addition of soluble extracts of liver provided that ATP is present in the reaction mixture (175, 176). Thus, it is believed that insulin, via the activation of a cascade of kinases, phosphorylates the cGI-PDE present in the particulate fraction of adipocytes and liver (Fig. 4). This activation is responsible for the decrease in intracellular cAMP that had been observed. Whether insulin activation of the cGI-PDE and other insulin-dependent effects, Insulin receptor

FIG. 4. Mechanism of insulin regulation of the cGI-PDE. The scheme is based on the model described in Ref. 170. PK, putative protein kinase which is distal to insulin activation of the receptor tyrosine kinase and that may phosphorylate the cGI-PDE. The possibility exists that more than one kinase is present at this point.

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i.e. activation of glucose transporters (177), are mediated by the same pathway is not known. It should also be mentioned that an inositol glycan purified from cultured myocytes can activate partially purified PDE preparations (151). It is not known how this piece of information fits in the above described regulation by phosphorylation. It has been proposed (151) that an insulin mediator can bind and directly activate a unique PDE form. Alternatively, the PDE preparations employed in these studies may contain a kinase that is activated by the mediator and that phosphorylates the PDE, thus producing the observed activation. VI. PDE Regulation by Hormones that Control Intracellular cGMP From the properties of the cGS-PDE determined in vitro, it is presumed that hormones that activate guanylate cyclase and that regulate intracellular cGMP should affect the cGS-PDE activity either by phosphorylation catalyzed by a cGMP protein kinase and/or by cGMP binding to the PDE itself. In some instances, even though a cGMP binding site has been extensively characterized, such as in the lung cGMP-specific PDE (59, 178) or in the 7-subunit of the retina PDE (55-58), the biological effect of occupancy of the cGMP binding site is still unknown. It is instead clear that in the cGS-PDE, cGMP binding to an allosteric binding site produces an activation of the catalytic site and an increase in hydrolytic activity (45, 46, 179). Direct evidence of intact cell activation of a cGS-PDE is still not available. The observation made by Hartzell and Fichmeister (180) points to a potentially important role of this form in mediating hormone action. These authors have found that cGMP inhibits the slow inward Ca++ current induced by agents that stimulate cAMP in frog cardiac myocytes (180). Studies with inhibitors and cGMP analogs have provided evidence that this cGMP effect is probably mediated by activation of a cGS-PDE (180, 181). High concentrations of cGS-PDE have been detected in the adrenal cortex, particularly in the zona glomerulosa (182). On the basis of this remarkable distribution the hypothesis has been put forward that hormones that activate guanylate cyclase in the adrenal cells such as atrial natriuretic peptide (ANP), inhibit cAMP-mediated events via activation of the cGS-PDE (182). The cascade of reaction that has been proposed is the following: ANP stimulates guanylate cyclase thus increasing intracellular cGMP, and in turn cGMP stimulates the cGS-PDE to hydrolyze cAMP producing a decrease in intracellular cAMP (182). This could be one of the mechanisms by which ANP inhibits the cAMP-dependent steroidogenesis in these cells.


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VII. Steroid Hormone Regulation of PDE

Acknowledgments

Several studies have dealt with the possible effects of steroid hormones on PDEs. It has been shown that in vivo estrogen administration causes an increase in the PDE expressed in the oviduct of the quail (183). Although the enzyme up-regulated by estrogen has been only partially characterized, it is similar to a cAMP-PDE (183). Since estrogen treatment indirectly causes an increase in cAMP in the quail oviduct (184), it is possible that a cAMP-dependent mechanism of the cAMP-PDE gene expression described earlier {Section IIIc) may play a role in this steroid effect. Also, the PDE activity present in the prostate is affected by androgen treatment via increased production of a heat-stable, dialyzable modulator (185). Incubation of 3T3 adipocytes with the glucocorticoids, such as dexamethasone, produces a decrease in insulin- and epinephrine-dependent activation of the cGI-PDE (118). The physiological significance of these steroid effects is not clear, but they might be part of the permissive or suppressive effects of these hormones on the cyclic nucleotide system.

The authors are indebted to to Dr. Joe Beavo and Jackie Corbin for the helpful discussion, to Dr. Tom Dousa for the unpublished material that he has shared, to Kathleen Homer for reviewing the manuscript, and to Frank S. French for his continuous support during the completion of our studies reviewed herein.

VIII. Conclusions and Perspectives The data reviewed indicate that cyclic nucleotide degradation by PDE is regulated by hormones in a complex fashion. In many instances the biochemical steps that lead to these regulations have been worked out. PDEs can be regulated by hormones that regulate intracellular cAMP and cGMP, intracellular Ca++, and by growth factors which act through tyrosine kinases. In practice, any external signal will be translated into a change in PDE activity. Less clear, however, is the physiological significance of the regulatory mechanisms that have been charted. Certainly, the astounding complexity of the circuits that control PDEs indicates an important role in cell homeostasis. The development of powerful tools to study PDEs should help to answer some major questions on the role of the mechanism regulating cyclic nucleotide degradation in different cellular processes. For instance, although it has been shown that a PDE is interacting with a G protein in the retina (31), it is not known whether G proteins, and therefore receptors, are directly coupled to PDEs in other cells. Some reports have addressed this question (173, 186-188), but a conclusive answer will hopefully come in the near future. Furthermore, very little is known about compartmentalization of PDEs. Cloning and site-directed mutagenesis should clarify the signals that target a PDE to a particular domain of the cell. It should also be possible to understand why different enzymes are directed to different cellular compartments. This knowledge could help to clarify if and why cyclic nucleotides themselves are compartmentalized.

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Cyclic nucleotide phosphodiesterases, insulin and thyroid hormones.

Cyclic nucleotide phosphodiesterases: pharmacology, biochemistry and function.

Advances in targeting cyclic nucleotide phosphodiesterases.

Roles of phosphodiesterases in the regulation of the cardiac cyclic nucleotide cross-talk signaling network.

Cyclic nucleotide phosphodiesterases (PDEs): coincidence detectors acting to spatially and temporally integrate cyclic nucleotide and non-cyclic nucleotide signals.

Higher-plant cyclic nucleotide phosphodiesterases. Resolution, partial purification and properties of three phosphodiesterases from potato tuber.

Temporal and spatial regulation of cAMP signaling in disease: role of cyclic nucleotide phosphodiesterases.

Cyclic nucleotide phosphodiesterases of Hyalophora cecropia silkmoth fat body.

Cyclic AMP compartments and signaling specificity: role of cyclic nucleotide phosphodiesterases.

Thyroid hormone control of cyclic nucleotide phosphodiesterases and the regulation of the sensitivity of the liver to hormones.

Isozymes of cyclic-3',5'-nucleotide phosphodiesterases in renal epithelial LLC-PK1 cells.

Characterization of cyclic nucleotide phosphodiesterases from cultured bovine aortic endothelial cells.

Characteristics of the cyclic nucleotide phosphodiesterases of normal and leukemic lymphocytes.

Selective inhibition of cyclic nucleotide phosphodiesterases by analogues of 1-methyl-3-isobutylxanthine.

Dihydro- and tetrahydroisoquinolines as inhibitors of cyclic nucleotide phosphodiesterases from dog heart. Structure-activity relationships.

Adenylate cyclase, guanylate cyclase and cyclic nucleotide phosphodiesterases of guinea-pig cardiac sarcolemma.

The cyclic nucleotide phosphodiesterases of Dictyostelium discoideum: molecular genetics and biochemistry.

Characterization of membrane-bound cyclic nucleotide phosphodiesterases from bovine aortic smooth muscle.

2',3'-cyclic nucleotide 3'-phosphodiesterases inhibit hepatitis B virus replication.

Cyclic nucleotide phosphodiesterases of rabbit renal cortex. Characterization of brush border membrane activities.

Inhibition of acyl coenzyme A:lysolecithin acyltransferases by local anesthetics, detergents and inhibitors of cyclic nucleotide phosphodiesterases.

Selective inhibition of cGMP-inhibited and cGMP-noninhibited cyclic nucleotide phosphodiesterases and relaxation of rat aorta.

Differential hemodynamic responses to selective inhibitors of cyclic nucleotide phosphodiesterases in conscious rats.

Select 3',5'-cyclic nucleotide phosphodiesterases exhibit altered expression in the aged rodent brain.