A nonhydrolyzable reactive cAMP analogue, (S(p))-8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic S-(methyl)monophosphorothioate, irreversibly inactivates human platelet cGMP-inhibited cAMP phosphodiesterase at micromolar concentrations

We previously showed that 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate inactivates cAMP phosphodiesterase (PDE3A); however, millimolar concentrations were needed to inactivate PDE3A because of ongoing hydrolysis. We have now synthesized a nonhydrolyzable reactive cAMP analogue, (S(p))-8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic S-(methyl)monophosphorothioate (S(p)-8-BDB-TcAMPSMe). S(p)-8-BDB-TcAMPSMe inactivates PDE3A in a time-dependent, irreversible manner, exhibiting saturation kinetics with a k(max) of (19.5 +/- 0.3) x 10(-3) min(-1) and a K(I) of 3.5 +/- 0.3 muM. To ascertain whether S(p)-8-BDB-TcAMPSMe reacts in the active site, nonhydrolyzable analogues of the substrate cAMP, or the competitive inhibitor cGMP, were included to protect against the inactivation of PDE3A. The order of effectiveness of protectants in decreasing the rate of inactivation (with K(d) values in micromolar) is as follows: S(p)-cAMPS (18) > R(p)-cGMPS (560) and S(p)-cGMPS (1260) > 5'-AMP (17 660), R(p)-cAMPS (30 110), and 5'-GMP (42 170). We docked S(p)-8-BDB-TcAMPSMe into PDE3A, based on the structural model of PDE3A-cAMP and the kinetic data from site-directed mutants. The S(p)-8-BDB-TcAMPSMe fits into the active site in the model. These results suggest that inactivation of PDE3A by the affinity reagent is a consequence of reaction at the overlap between cAMP and cGMP binding regions in the active site. S(p)-8-BDB-TcAMPSMe has proven to be an effective active site-directed irreversible cAMP affinity label for platelet PDE3A and can be used to identify amino acids in the active site of PDE3A as well as in other cAMP phosphodiesterases.     

Cyclic nucleotide phosphodiesterases (PDEs) in human osteoblastic cells; the effect of PDE inhibition on cAMP accumulation

The regulation of the secondary messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), is crucial in the hormonal regulation of bone metabolism. Both cAMP and cGMP are inactivated by cyclic nucleotide phosphodiesterases (PDEs), a superfamily of enzymes divided into 11 families (PDE1-11). We compared the PDEs of cultured human osteoblasts (NHOst) and SaOS-2 osteosarcoma cells. The PDE activity of NHOst cells consisted of PDE1, PDE3 and PDE7, whereas PDE1, PDE7 and PDE4, but no PDE3 activity was detected in SaOS-2 cells. In line with the difference in the PDE profiles, rolipram, a PDE4 inhibitor, increased the accumulation of cAMP in SaOS-2, but not in NHOst cells. Expression of PDE subtypes PDE1C, PDE3A, PDE4A, PDE4B, PDE7A and PDE7B was detected in both cell types. NHOst cells additionally expressed PDE1A.     

Role of phosphodiesterase 2 in growth and invasion of human malignant melanoma cells

Cyclic nucleotide phosphodiesterases (PDEs) regulate the intracellular concentrations and effects of adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP). The role of PDEs in malignant tumor cells is still uncertain. The role of PDEs, especially PDE2, in human malignant melanoma PMP cell line was examined in this study. In PMP cells, 8-bromo-cAMP, a cAMP analog, inhibited cell growth and invasion. However, 8-bromo-cGMP, a cGMP analog, had little or no effect. PDE2 and PDE4, but not PDE3, were expressed in PMP cells. Growth and invasion of PMP cells were inhibited by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), a specific PDE2 inhibitor, but not by rolipram, a specific PDE4 inhibitor. Moreover, cell growth and invasion were inhibited by transfection of small interfering RNAs (siRNAs) specific for PDE2A and a catalytically-dead mutant of PDE2A. After treating cells with EHNA or rolipram, intracellular cAMP concentrations were increased. Growth and invasion were stimulated by PKA14-22, a PKA inhibitor, and inhibited by N⁶-benzoyl-c AMP, a PKA specific cAMP analog, whereas 8-(4-chlorophenylthio)-2′-O-methyl-cAMP, an Epac specific cAMP analog, did not. Invasion, but not growth, was stimulated by A-kinase anchor protein (AKAP) St-Ht31 inhibitory peptide. Based on these results, PDE2 appears to play an important role in growth and invasion of the human malignant melanoma PMP cell line. Selectively suppressing PDE2 might possibly inhibit growth and invasion of other malignant tumor cell lines.     

A cell-based PDE4 assay in 1536-well plate format for high-throughput screening

The cyclic nucleotide phosphodiesterases (PDEs) are intracellular enzymes that catalyze the hydrolysis of 3,'5'-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5'nucleotide monophosphates. These enzymes play an important role in controlling cellular concentrations of cyclic nucleotides and thus regulate a variety of cellular signaling events. PDEs are emerging as drug targets for several diseases, including asthma, cardiovascular disease, attention-deficit hyperactivity disorder, Parkinson's disease, and Alzheimer's disease. Although biochemical assays with purified recombinant PDE enzymes and cAMP or cGMP substrate are commonly used for compound screening, cell-based assays would provide a better assessment of compound activity in a more physiological context. The authors report the development and validation of a new cell-based PDE4 assay using a constitutively active G-protein-coupled receptor as a driving force for cAMP production and a cyclic nucleotide-gated cation channel as a biosensor in 1536-well plates.     

Characterization of a novel cAMP-binding, cAMP-specific cyclic nucleotide phosphodiesterase (TcrPDEB1) from Trypanosoma cruzi

Trypanosoma cruzi, the causative agent of Chagas disease, encodes a number of different cAMP-specific PDE (phosphodiesterase) families. Here we report the identification and characterization of TcrPDEB1 and its comparison with the previously identified TcrPDEB2 (formerly known as TcPDE1). These are two different PDE enzymes of the TcrPDEB family, named in accordance with the recent recommendations of the Nomenclature Committee for Kinetoplast PDEs [Kunz, Beavo, D'Angelo, Flawia, Francis, Johner, Laxman, Oberholzer, Rascon, Shakur et al. (2006) Mol. Biochem. Parasitol. 145, 133-135]. Both enzymes show resistance to inhibition by many mammalian PDE inhibitors, and those that do inhibit do so with appreciable differences in their inhibitor profiles for the two enzymes. Both enzymes contain two GAF (cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coli FhlA) domains and a catalytic domain highly homologous with that of the T. brucei TbPDE2/TbrPDEB2 family. The N-terminus+GAF-A domains of both enzymes showed significant differences in their affinities for cyclic nucleotide binding. Using a calorimetric technique that allows accurate measurements of low-affinity binding sites, the TcrPDEB2 N-terminus+GAF-A domain was found to bind cAMP with an affinity of approximately 500 nM. The TcrPDEB1 N-terminus+GAF-A domain bound cAMP with a slightly lower affinity of approximately 1 muM. The N-terminus+GAF-A domain of TcrPDEB1 did not bind cGMP, whereas the N-terminus+GAF-A domain of TcrPDEB2 bound cGMP with a low affinity of approximately 3 muM. GAF domains homologous with those found in these proteins were also identified in related trypanosomatid parasites. Finally, a fluorescent cAMP analogue, MANT-cAMP [2'-O-(N-methylanthraniloyl)adenosine-3',5'-cyclic monophosphate], was found to be a substrate for the TcPDEB1 catalytic domain, opening the possibility of using this molecule as a substrate in non-radioactive, fluorescence-based PDE assays, including screening for trypanosome PDE inhibitors.     

Biochemical and pharmacological characterization of cyclic nucleotide phosphodiesterase in airway epithelium

According to their respective elution order, specificity for cAMP and cGMP, their sensitivity to calmodulin, and their modulation by cGMP and rolipram, four cyclic nucleotide phosphodiesterases (PDE) were separated from the cytosol: PDE I (calmodulin-sensitive), PDE II (stimulated by cGMP, PDE IV (cGMP specific-PDE and inhibited by rolipram) and PDE V (cGMP specific). PDE IV (K_m=1.4 M) was competitively inhibited rolipram (K_i=1.2 M) whereas PDE V (K_m=0.83 M) was competitively inhibited by zaprinast in the molar range (K_i=0.12 M). Moreover the microsomal fraction contained three PDE isoforms: PDE II, PDE III (inhibited by cGMP or indolidan) and PDE IV. These results show that cAMP degradation in cytosolic and membrane fractions is modulated by cGMP and selectively inhibited by rolipram and, in addition, by indolidan in membrane fractions. (Mol Cell Biochem140: 171–175, 1994)     

Molecular determinants of cGMP binding to chicken cone photoreceptor phosphodiesterase

Structural studies on photoreceptor phosphodiesterases type 6 (PDE6s) have been hampered by an inability to express and purify substantial amounts of enzyme. Here we describe bacterial expression and characterization of the chicken cone PDE6 regulatory GAF-A and GAF-B domains. High affinity cGMP binding was found only for GAF-A as predicted from sequence alignments with the GAF domains of PDE2 and PDE5. A homology model of the GAF-A domain of chicken cone PDE6 based on the crystal structure of mouse PDE2A GAF-B was used to identify residues likely to make contact with cGMP. Alanine mutagenesis of 4 of these residues (F123A, D169A, T172A, and T176A) showed that each was absolutely required for cGMP binding. Three of these residues map to the H4 helical structure of the GAF-A domain indicating this region as a key structural component for cGMP binding. Mutagenesis of another residue, S97A, decreased cGMP binding affinity 5-fold. Finally mutagenesis of Glu-124 indicated that it is responsible for part but not all of the high specificity for cGMP binding to PDE6 GAF-A. Since little data is available on the properties of the chicken cone PDE6 holoenzyme, we also characterized the native PDEs of chicken retina. Two histone-activated PDE6 peaks were separated by ion exchange chromatography and identified by mass spectrometry as cone and rod photoreceptor PDE6s, respectively. Both of these PDEs had cGMP binding and kinetic properties similar to their corresponding bovine photoreceptor PDEs. Moreover the cGMP binding properties of chicken cone PDE6 holoenzyme were very similar to those of the bacterially expressed individual GAF-A or GAF-A/B domains.     

Cross talk between NO and cyclic nucleotide phosphodiesterases in the modulation of signal transduction in blood vessel.

An increase in cAMP and/or cGMP induces vasodilation which could be potentiated by endothelium or NO-donors. Cyclic nucleotide phosphodiesterases (PDE) are differently distributed in vascular tissues. cAMP hydrolyzing PDE isozymes in endothelial cells are represented by PDE2 (cGMP stimulated-PDE) and PDE4 (cGMP insensitive-PDE), whereas in smooth muscle cells PDE3 (cGMP inhibited-PDE) and PDE4 are present. To investigate the role of NO in vasodilation induced by PDE inhibitors, we studied the effects of PDE3- or PDE4-inhibitor alone and their combination on cyclic nucleotide levels, on relaxation of precontracted aorta and on protein kinase implication. Furthermore, the direct effect of dinitrosyl iron complex (DNIC) was studied on purified recombinant PDE4B. The results show that: 1) in endothelial cells PDE4 inhibition may up-regulate basal production of NO, this effect being potentiated by PDE2 inhibition; 2) in smooth muscle cGMP produced by NO inhibits PDE3 and increases cAMP level allowing PDE4 to participate in vascular contraction; 3) protein kinase G mediates the relaxing effects of PDE3 or PDE4 inhibition. 4) DNIC inhibits non competitively PDE4B indicating a direct effect of NO on PDE4 which could explain an additive vasodilatory effect of NO. A direct and a cGMP related cross-talk between NO and cAMP-PDEs, may participate into the vasomodulation mediated by cAMP activation of protein kinase G.     

Hydrolysis of cyclic GMP in rat peritoneal macrophages

Intact rat peritoneal macrophages (rPM) treated with 3-isobutyl-1-methylxanthine (IBMX), an inhibitor of phosphodiesterases (PDEs), accumulated more cGMP than untreated cells. A PDE activity toward [(3)H]cGMP was detected in the soluble and particulate fractions of rPM. The hydrolysis of cGMP was Ca(2+)/calmodulin-independent but increased in the presence of cGMP excess. Similar results were obtained when [(3)H]cAMP was used as a substrate. The hydrolytic activity towards both nucleotides was inhibited in the presence of IBMX. Therefore, the PDEs of families 2, 5, 10 and 11 are potential candidates for cGMP hydrolysis in the rPM. They may not only regulate the cGMP level in a feedback-controlled way but also link cGMP-dependent pathways with those regulated by cAMP.     

Phosphodiesterase inhibition by a gastroprotective agent irsogladine: preferential blockade of cAMP hydrolysis

The effect of irsogladine [2,4-diamino-6-(2,5-dichlorophenyl)-s-triazine maleate], an antiulcer drug, on contents of cyclic nucleotides including cAMP and cGMP was investigated in rat stomachs. Irsogladine concentration-dependently increased cAMP content in rat glandula stomach. However, irsogladine at higher concentration (10(-5) M) was unable to further increase cAMP level in the presence of non-selective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine, although 3-isobutyl-1-methylxanthine by itself increased cAMP level. On the other hand, irsogladine had no effect on the glandula cGMP content. Subsequently, the effect of irsogladine on the cyclic nucleotide degradation by purified bovine brain and heart PDEs was investigated. The cAMP degradation by purified bovine brain PDE was partially suppressed by PDE1 inhibitor vinpocetin, PDE2 inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride and PDE4 inhibitor rolipram but not by PDE3 inhibitor cilostamide, and completely inhibited by 3-isobutyl-1-methylxanthine, suggesting that is attributed almost exclusively to PDE1, PDE2 and PDE4. Meanwhile, cGMP degradation by purified bovine brain PDE was partially suppressed by erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride. Irsogladine preferentially inhibited the response to cAMP degradation compared with cGMP degradation by this brain PDE. The cAMP degradation by bovine heart PDE was almost completely inhibited by the combination with vinpocetine and cilostamide, indicating that is mediated almost exclusively by PDE1 and PDE3. Irsogladine suppressed this cAMP degradation measured in the presence of vinpocetine to almost the same extent as that determined in the presence of cilostamide. These results indicate that irsogladine produces the increase of intracellular cAMP content via non-selective inhibition of PDE isozymes, which may be a key mechanism involved in its gastroprotective actions.     

Molecular Cloning and Expression of a Human Phosphodiesterase 4C

A cDNA coding for a human phosphodiesterase 4C (PDE4C2) was isolated from the mRNA prepared from the glioblastoma cell line, U87. The cDNA contained an ORF of 1818 bp corresponding to a 605 amino acid polypeptide. The sequence differed at the 5′ end from the human PDE4C previously reported (Engels, P. et al, 1995 FEBs Letters 358, 305-310) indicating that it represents a novel splice variant of the human PDE4C gene. Evidence was also obtained for a third 5′ splice variant. The PDE4C2 cDNA was transfected into both COS 1 cells and yeast cells, and shown to direct the expression of an 80 kD polypeptide by Western blotting using a PDE4C specific antiserum. The activity of cell lysates was typical of PDE4 being specific for cAMP and inhibitable by the selective inhibitor, rolipram. However, the K_m for cAMP of the enzyme produced in COS cells was 0.6 μM compared to 2.6 μM for the yeast 4C activity. In addition the COS cell PDE4 activity was much more sensitive to R rolipram than the yeast PDE4 enzyme (IC₅₀ of 23 nM compared to 1648 nM). This difference in rolipram sensitivity was associated with the detection of a high affinity [³H] R rolipram binding site on the COS cell 4C enzyme but not on the yeast expressed enzyme. The results indicate that the enzyme can adopt more than one active conformation, which are distinguished by their interaction with rolipram.     

Identification of overlapping but distinct cAMP and cGMP interaction sites with cyclic nucleotide phosphodiesterase 3A by site-directed mutagenesis and molecular modeling based on crystalline PDE4B

Cyclic nucleotide phosphodiesterase 3A (PDE3A) hydrolyzes cAMP to AMP, but is competitively inhibited by cGMP due to a low k(cat) despite a tight K(m). Cyclic AMP elevation is known to inhibit all pathways of platelet activation, and thus regulation of PDE3 activity is significant. Although cGMP elevation will inhibit platelet function, the major action of cGMP in platelets is to elevate cAMP by inhibiting PDE3A. To investigate the molecular details of how cGMP, a similar but not identical molecule to cAMP, behaves as an inhibitor of PDE3A, we constructed a molecular model of the catalytic domain of PDE3A based on homology to the recently determined X-ray crystal structure of PDE4B. Based on the excellent fit of this model structure, we mutated nine amino acids in the putative catalytic cleft of PDE3A to alanine using site-directed mutagenesis. Six of the nine mutants (Y751A, H840A, D950A, F972A, Q975A, and F1004A) significantly decreased catalytic efficiency, and had k(cat)/K(m) less than 10% of the wild-type PDE3A using cAMP as substrate. Mutants N845A, F972A, and F1004A showed a 3- to 12-fold increase of K(m) for cAMP. Four mutants (Y751A, H840A, D950A, and F1004A) had a 9- to 200-fold increase of K(i) for cGMP in comparison to the wild-type PDE3A. Studies of these mutants and our previous study identified two groups of amino acids: E866 and F1004 contribute commonly to both cAMP and cGMP interactions while N845, E971, and F972 residues are unique for cAMP and the residues Y751, H836, H840, and D950 interact with cGMP. Therefore, our results provide biochemical evidence that cGMP interacts with the active site residues differently from cAMP.     

A new nonhydrolyzable reactive cAMP analog, (Sp)-adenosine-3',5'-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate irreversibly inactivates human platelet cGMP-inhibited cAMP phosphodiesterase

Levels of cAMP that control critical platelet functions are regulated by cGMP-inhibited cAMP phosphodiesterase (PDE3A). We previously showed that millimolar concentrations of the hydrolyzable 8-[(4-bromo-2,3-dioxobutyl)thioadenosine 3',5'-cyclic monophosphate (8-BDB-TcAMP) inactivate PDE3A. We have now synthesized a nonhydrolyzable affinity label to probe the active site of PDE3A. The nonhydrolyzable adenosine 3',5'-cyclic monophosphorothioates, Sp-cAMPS and Rp-cAMPS, function as competitive inhibitors of PDE3A with K(i) = 47.6 and 4400 microM, respectively. We therefore coupled Sp-cAMPS with 1,4-dibromobutanedione to yield (Sp)-adenosine-3',5'-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate, [Sp-cAMPS-(BDB)]. Sp-cAMPS-(BDB) inactivates PDE3A in a time-dependent, irreversible reaction with k(max) = 0.0116 min(-1) and K(I) = 10.1 microM. The order of effectiveness of protectants in decreasing the rate of inactivation (with K(d) in microM) is: Sp-cAMPS (24) > Rp-cGMPS (1360), Sp-cGMPS (1460) > GMP (4250), AMP (10600), Rp-cAMPS (22170). These results suggest that the inactivation of PDE3A by Sp-cAMPS-(BDB) is a consequence of reaction at the overlap of the cAMP and cGMP binding regions in the active site.     

Dissecting the cofactor-dependent and independent bindings of PDE4 inhibitors

Type 4 phosphodiesterases (PDE4s) are metallohydrolases that catalyze the hydrolysis of cAMP to AMP. At the bottom of its active site lie two divalent metal ions in a binuclear motif which are involved in both cAMP binding and catalysis [(2000) Science 288, 1822-1825; (2000) Biochemistry 39, 6449-6458]. Using a SPA-based equilibrium [(3)H]rolipram binding assay, we have determined that Mg(2+), Mn(2+), and Co(2+) all mediated a high-affinity (K(d) between 3 and 8 nM) and near stoichiometric (R)-rolipram binding to PDE4. In their absence, (R)-rolipram binds stoichiometrically to the metal ion-free apoenzyme with a K(d) of approximately 150 nM. The divalent cation dose responses in mediating the high-affinity rolipram/PDE4 interaction mirror their efficacy in catalysis, suggesting that both metal ions of the holoenzyme are involved in mediating the high-affinity (R)-rolipram/PDE4 interaction. The specific rolipram binding to the apo- and holoenzyme is differentially displaced by cAMP, AMP, and other inhibitors, providing a robust tool to dissect the components of metal ion-dependent and independent PDE4/ligand interactions. cAMP binds to the holoenzyme with a K(s) of 1.9 microM and nonproductively to the apoenzyme with a K(d) of 179 microM. In comparison, AMP binds to the holo- and apoenzyme with K(d) values of 7 and 11 mM, respectively. The diminished Mg(2+)-dependent component of AMP binding to PDE4 suggests that most of the Mg(2+)/phosphate interaction in the cAMP/PDE4 complex is disrupted upon the hydrolysis of the cyclic phosphoester bond, leading to the rapid release of AMP.     

The role of cyclic nucleotide phosphodiesterases in the regulation of adipocyte lipolysis

This study assessed the effects of selective inhibitors of 3',5'-cyclic nucleotide phosphodiesterases (PDEs) on adipocyte lipolysis. IC224, a selective inhibitor of type 1 phosphodiesterase (PDE1), suppressed lipolysis in murine 3T3-L1 adipocytes (69.6 +/- 5.4% of vehicle control) but had no effect in human adipocytes. IC933, a selective inhibitor of PDE2, had no effect on lipolysis in either cultured murine 3T3-L1 adipocytes or human adipocytes. Inhibition of PDE3 with cilostamide moderately stimulated lipolysis in murine 3T3-L1 and rat adipocytes (397 +/- 25% and 235 +/- 26% of control, respectively) and markedly stimulated lipolysis in human adipocytes (932 +/- 7.6% of control). Inhibition of PDE4 with rolipram moderately stimulated lipolysis in murine 3T3-L1 adipocytes (291 +/- 13% of control) and weakly stimulated lipolysis in rat adipocytes (149 +/- 7.0% of control) but had no effect on lipolysis in human adipocytes. Cultured adipocytes also responded differently to a combination of PDE3 and PDE4 inhibitors. Simultaneous exposure to cilostamide and rolipram had a synergistic effect on lipolysis in murine 3T3-L1 and rat adipocytes but not in human adipocytes. Hence, the relative importance of PDE3 and PDE4 in regulating lipolysis differed in cultured murine, rat, and human adipocytes.     

Studies of the molecular mechanism of discrimination between cGMP and cAMP in the allosteric sites of the cGMP-binding cGMP-specific phosphodiesterase (PDE5).

The regulatory domain of the cGMP-binding cGMP-specific 3':5'-cyclic nucleotide phosphodiesterase (PDE5) contains two homologous segments of amino acid sequence that encode allosteric cyclic nucleotide-binding sites, referred to as site a and site b, which are highly selective for cGMP over cAMP. The possibility that the state of protonation in these sites contributes to cyclic nucleotide selectivity was investigated. The binding of cGMP or cAMP was determined using saturation and competition kinetics at pH values between 5.2 and 9.5. The total cGMP binding by PDE5 was unchanged by variation in pH, but the relative affinity for cGMP versus cAMP progressively decreased as the pH was lowered. Using site-directed mutagenesis, a conserved residue, Asp-289, in site a of PDE5 has been identified as being important for cyclic nucleotide discrimination in this site. It is proposed that deprotonation of Asp-289 enhances the number and strength of bonds formed with cGMP, while concomitantly decreasing the interactions with cAMP.     

3',5'-cyclic nucleotide phosphodiesterase from human brain

3':5'-Cyclic nucleotide phosphodiesterase was isolated from human brain and characterized. After the first stage of purification on phenyl-Sepharose, the enzyme activity was stimulated by Ca2+ and micromolar concentrations of cGMP. High pressure liquid chromatography on a DEAE-TSK-3SW column permitted to identify three ranges of enzymatic activity designated as PDE I, PDE II and PDE III. Neither of the three enzymes possessed a high selectivity for cAMP and cGMP substrates. The catalytic activity of PDE I and PDE II increased in the presence of Ca2+-calmodulin (up to 6-fold); the degradation of cAMP was decreased by cGMP. The Ca2+-calmodulin stimulated PDE I and PDE II activity was decreased by W-7. PDE I and PDE II can thus be classified as Ca2+-calmodulin-dependent phosphodiesterases. With cAMP as substrate, the PDE III activity increased in the presence of micromolar concentrations of cGMP (up to 10-fold), Ca2+ and endogenous calmodulin (up to 2-3-fold). No additivity in the effects of saturating concentrations of these compounds on PDE III was observed. Ca2+ did not influence the rate of cGMP hydrolysis catalyzed by PDE III. In comparison with PDE I and PDE II, the inhibition of PDE III was observed at higher concentrations of W-7 and was not limited by the basal level of the enzyme. These results do not provide any evidence in favour of the existence of several forms of the enzyme in the PDE III fraction. The double regulation of PDE III creates some difficulties for its classification.     

An update view on the substrate recognition mechanism of phosphodiesterases: a computational study of PDE10 and PDE4 bound with cyclic nucleotides

Early studies strongly implied that the specificity of cyclic nucleotide phosphodiesterases (PDEs) toward its endogenous substrates can be uniquely determined by the amido orientation of the invariant glutamine locating in the binding pocket of the enzyme. However, recently solved crystal structures of PDE4 (cAMP specific) and PDE10 (dual specific) in the presence of endogenous substrates have revealed that their invariant glutamine orientations are very similar despite exhibiting different substrate specificities proven physiologically. To understand this subtle specificity issue in the PDE family, here several experimentally inaccessible PDE-substrate complex models have been studied computationally, and the results are juxtaposed and compared in detail. Modeling results show that PDE10 in fact favors cAMP energetically but still can bind to cGMP owing to the robust hydrogen-bond network in the vicinity of the invariant glutamine side chain. PDE4 fails to accommodate cGMP is correlated to the weakening of this same hydrogen-bond network but not owing to any steric strain in the binding pocket. An Asn residue in the binding pocket of PDE4 has enhanced the specificity of the binding to cAMP sideway as observed in our computer simulation. Further to the previously studied syn- versus anti-conformational specificity of cAMP in PDE10, the unexpected substrate-binding mode in PDE10 versus PDE4 as reported here strongly suggested that there are remaining uncertainties in the substrate orientation and recognition mechanism in the PDE families. The molecular details of the binding pocket observed in this study provide hints for more optimal PDE4 and PDE10 inhibitor design.     

Crystal structures of phosphodiesterases 4 and 5 in complex with inhibitor 3-isobutyl-1-methylxanthine suggest a conformation determinant of inhibitor selectivity

Cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of enzymes controlling cellular concentrations of the second messengers cAMP and cGMP. Crystal structures of the catalytic domains of cGMP-specific PDE5A1 and cAMP-specific PDE4D2 in complex with the nonselective inhibitor 3-isobutyl-1-methylxanthine have been determined at medium resolution. The catalytic domain of PDE5A1 has the same topological folding as that of PDE4D2, but three regions show different tertiary structures, including residues 79-113, 208-224 (H-loop), and 341-364 (M-loop) in PDE4D2 or 535-566, 661-676, and 787-812 in PDE5A1, respectively. Because H- and M-loops are involved in binding of the selective inhibitors, the different conformations of the loops, thus the distinct shapes of the active sites, will be a determinant of inhibitor selectivity in PDEs. IBMX binds to a subpocket that comprises key residues Ile-336, Phe-340, Gln-369, and Phe-372 of PDE4D2 or Val-782, Phe-786, Gln-817, and Phe-820 of PDE5A1. This subpocket may be a common site for binding nonselective inhibitors of PDEs.     

Aggregation states of cyclic nucleotide phosphodiesterase of murine thymocytes

In murine thymocytes cyclic nucleotide phosphodiesterase is represented by cAMP- and cGMP-specific forms. cAMP and cGMP phosphodiesterase activities showed anomalous kinetic behaviour indicative of 'low' and 'high' affinity enzyme forms. Sucrose density gradient centrifugation resolved only 'low' affinity forms of cAMP and cGMP phosphodiesterases. Gel filtration on Ultragel Aca 34 column showed that cAMP and cGMP phosphodiesterases are probably oligomeric enzymes. Storage of enzyme preparation at 4 degrees C for 24-48 h led to a decrease of higher molecular weight form and enhancement of cAMP and cGMP phosphodiesterase activities.