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.     

Increasing synaptic noradrenaline, serotonin and histamine enhances in vivo binding of phosphodiesterase-4 inhibitor (R)-[11C]rolipram in rat brain, lung and heart

Phosphodiesterase-4 (PDE4) is one of the main enzymes that specifically terminate the action of cAMP, thereby contributing to intracellular signaling following stimulation of various G protein-coupled receptors. PDE4 expression and activity are modulated by agents affecting cAMP levels. The selective PDE4 inhibitor (R)-rolipram labeled with C-11 was tested in vivo in rats to analyze changes in PDE4 levels following drug treatments that increase synaptic noradrenaline (NA), serotonin (5HT), histamine (HA) and dopamine (DA) levels. We hypothesized that increasing synaptic neurotransmitter levels and subsequent cAMP-mediated signaling would significantly enhance (R)-[(11)C]rolipram retention and specific binding to PDE4 in vivo. Pre-treatments were performed 3 h prior to tracer injection, and rats were sacrificed 45 min later. Biodistribution studies revealed a dose-dependent increase in (R)-[(11)C]rolipram uptake following administration of the monoamine oxidase (MAO) inhibitor tranylcypromine, NA and 5HT reuptake inhibitors (desipramine [DMI], maprotiline; and fluoxetine, sertraline, respectively), and the HA H(3) receptor antagonist (thioperamide), but not with DA transporter blockers GBR 12909, cocaine or DA D(1) agonist SKF81297. Significant increases in rat brain and heart reflect changes in PDE4 specific binding (total-non-specific binding [coinjection with saturating dose of (R)-rolipram]). These results demonstrate that acute treatments elevating synaptic NA, 5HT and HA, but not DA levels, significantly enhance (R)-[(11)C]rolipram binding. Use of (R)-[(11)C]rolipram and positron emission tomography as an index of PDE4 activity could provide insight into understanding disease states with altered NA, 5HT and HA concentrations.     

The identification of a new cyclic nucleotide phosphodiesterase activity in human and guinea-pig cardiac ventricle. Implications for the mechanism of action of selective phosphodiesterase inhibitors.

Four cyclic nucleotide phosphodiesterase (PDE) activities were separated from low-speed supernatants of homogenates of human cardiac ventricle by DEAE-Sepharose chromatography, and designated PDE I-PDE IV in order of elution with an increasing salt gradient. PDE I was a Ca2+/calmodulin-stimulated activity, and PDE II was an activity with a high Km for cyclic AMP which was stimulated by low concentrations of cyclic GMP. Human ventricle PDE III had Km values of 0.14 microM (cyclic AMP) and 4 microM (cyclic GMP), and showed simple Michaelis-Menten kinetics with both substrates. PDE IV is a previously unrecognized activity in cardiac muscle, the human enzyme having Km values of 2 microM (cyclic AMP) and 50 microM (cyclic GMP). PDE III and PDE IV were not activated by cyclic nucleotides or calmodulin. Four PDE activities were also isolated from guinea-pig ventricle, and had very similar kinetic properties. By gel filtration, the Mr of PDE III was 60,000, and that of PDE IV 45,000. The drug SK&F 94120 selectively and competitively inhibited PDE III with a Ki value of 0.8 microM (human), showing simple hyperbolic inhibition kinetics. Rolipram (Schering ZK 62711) and Ro 20-1724 (Roche), which have previously been reported to inhibit PDE III-like activities strongly, were shown to be weak inhibitors of human and guinea-pig PDE III enzymes (Ki values greater than 25 microM), but potent inhibitors of PDE IV [Ki values 2.4 microM (Rolipram) and 3.1 microM (Ro 20-1724) with human PDE IV]. The inhibition in all cases demonstrated simple hyperbolic competition. These observations suggest that the previously reported complex inhibition of PDE III-type activities from cardiac muscle was caused by incomplete separation of the PDE III from other enzymes, particularly PDE IV.     

Three-dimensional structures of PDE4D in complex with roliprams and implication on inhibitor selectivity

Selective inhibitors against the 11 families of cyclic nucleotide phosphodiesterases (PDEs) are used to treat various human diseases. How the inhibitors selectively bind the conserved PDE catalytic domains is unknown. The crystal structures of the PDE4D2 catalytic domain in complex with (R)- or (R,S)-rolipram suggest that inhibitor selectivity is determined by the chemical nature of amino acids and subtle conformational changes of the binding pockets. The conformational states of Gln369 in PDE4D2 may play a key role in inhibitor recognition. The corresponding Y329S mutation in PDE7 may lead to loss of the hydrogen bonds between rolipram and Gln369 and is thus a possible reason explaining PDE7's insensitivity to rolipram inhibition. Docking of the PDE5 inhibitor sildenafil into the PDE4 catalytic pocket further helps understand inhibitor selectivity.     

CDP840

We present the in vitro characterization of a novel phosphodiesterase type 4 inhibitor, CDP840 (R-[+]-4-[2-{3-cyclopentyloxy-4-methoxyphenyl}-2-phenylethyl]pyridine), which has shown efficacy in a phase II allergen challenge study in asthmatics without adverse effects. CDP840 potently inhibits PDE-4 isoenzymes (IC₅₀ 2–30 nM) without any effect on PDE-1, 2, 3, 5, and 7 (IC₅₀>100 μM). It exhibited no significant selectivity in inhibiting human recombinant isoenzymes PDE-4A, B, C or D and was equally active against the isoenzymes lacking UCR1 (PDE-4B2 and PDE-4D2). In contrast to rolipram, CDP840 acted as a simple competitive inhibitor of all PDE-4 isoenzymes. Studies with rolipram indicated a heterogeneity within all the preparations of PDE-4 isoenzymes, indicative of rolipram inhibiting the catalytic activity of PDE-4 with both a low or high affinity. These observations were confirmed by the use of a PDE-4A variant, PDE-4A_330–886, which rolipram inhibited with low affinity (IC₅₀=1022 nM). CDP840 in contrast inhibited this PDE-4A variant with similar potency (IC₅₀=3.9 nM), which was in good agreement with theK _d of 4.8 nM obtained from [³H]-CDP840 binding studies. Both CDP840 and rolipram inhibited the high-affinity binding of [³H]-rolipram binding to PDE-4A, B, C and D with similarK _{d app} (7–19 nM and 3–5 nM, respectively). Thus, the activity of CDP840 at the [³H]-rolipram binding site was in agreement with the inhibitor’s activity at the catalytic site. However, rolipram was ∼100-fold more potent than CDP840 at inhibiting the binding of [³H]-rolipram to mouse brain in vivo. These data clearly demonstrate that CDP840 is a potent selective inhibitor of all PDE-4 isoenzymes. In contrast to rolipram, CDP840 was well-tolerated in humans. This difference, however, cannot at present be attributed to either isoenzyme selectivity or lack of activity in vitro at the high-affinity rolipram binding site (Sr).     

Type III phosphodiesterase plays a necessary role in the growth-promoting actions of insulin, insulin-like growth factor-I, and Ha p21ras in Xenopus laevis oocytes.

Three phosphodiesterase (PDE) type III inhibitors were tested and found to inhibit Xenopus oocyte maturation induced by insulin with apparent IC50 values of 2.2 +/- 0.2 microM Cl-930, 25 +/- 3 microM imazodan (Cl-914), and 786 +/- 237 microM piroximone (MDL 19,205). The same rank order of potencies was observed for inhibition of insulin-like growth factor-I (IGF-I)-induced oocyte maturation, with IC50 values of 5.5 +/- 0.9 microM Cl-930, 54 +/- 4 microM imazodan, and 1190 +/- 395 microM piroximone. Oocyte maturation induced by microinjection of Ha p21ras was also inhibited by pretreatment of oocytes with Cl-930 or imazodan, with IC50 values of 4.3 +/- 1.2 and 59 +/- 4 microM, respectively. Progesterone-induced maturation was not affected by PDE III inhibitor action; and, neither type IV PDE inhibitors (Ro 20, 1724 or rolipram) nor dipyridamole (a type V PDE inhibitor) inhibited cell division induced by IGF-I or microinjected Ha p21ras. In addition, while insulin-stimulated oocyte PDE activity measured in vivo after microinjection of 200 microM [3H] cAMP was inhibited by nonselective and type III-specific drugs (with IC50 values of 4.2 +/- 1.8 microM Cl-930 and 26 +/- 6 microM imazodan), type IV and type V inhibitors did not inhibit hormone-stimulated enzyme activity. This pharmacological evidence demonstrates a necessary role for PDE III in insulin-, IGF-I-, and p21ras-induced meiotic cell division in Xenopus laevis oocytes.     

Tyrosine kinase-independent inhibition of cyclic-AMP phosphodiesterase by genistein and tyrphostin 51.

The phosphodiesterase activity in the HT4.7 neural cell line was pharmacologically characterized, and phosphodiesterase isozyme 4 (PDE4) was found to be the predominant isozyme. The Km for cAMP was 1-2 microM, indicative of a "low Km" phosphodiesterase, and the activity was inhibited by PDE4-selective inhibitors rolipram and Ro20-1724, but not PDE3- or PDE2-selective inhibitors. Calcium, calmodulin, and cGMP, regulators of PDE1, PDE2, and PDE3, had no effect on cAMP hydrolysis. The protein tyrosine kinase inhibitor, genistein, inhibited HT4.7 cAMP phosphodiesterase activity by 85-95% with an IC50 of 4 microM; whereas daidzein, an inactive structural analog of genistein, had little effect on phosphodiesterase activity. This is a common pharmacological criterion used to implicate the regulation by a tyrosine kinase. However, genistein still inhibited phosphodiesterase activity with a mixed pattern of inhibition even when ion-exchange chromatography was used to partially purify phosphodiesterase away from the tyrosine kinase activity. Moreover, tyrphostin 51, another tyrosine kinase inhibitor, was found to also inhibit partially purified phosphodiesterase activity noncompetitively. These data suggest that HT4.7 phosphodiesterase activity is dominated by PDE4 and can be regulated by genistein and tyrphostin 51 by a tyrosine kinase-independent mechanism.     

Phosphorylation of phosphodiesterase-5 by cyclic nucleotide-dependent protein kinase alters its catalytic and allosteric cGMP-binding activities.

In addition to its cGMP-selective catalytic site, cGMP-binding cGMP-specific phosphodiesterase (PDE5) contains two allosteric cGMP-binding sites and at least one phosphorylation site (Ser92) on each subunit [Thomas, M.K., Francis, S.H. & Corbin, J.D. (1990) J. Biol. Chem. 265, 14971-14978]. In the present study, prior incubation of recombinant bovine PDE5 with a phosphorylation reaction mixture [cGMP-dependent protein kinase (PKG) or catalytic subunit of cAMP-dependent protein kinase (PKA), MgATP, cGMP, 3-isobutyl-1-methylxanthine], shown earlier to produce Ser92 phosphorylation, caused a 50-70% increase in enzyme activity and also increased the affinity of cGMP binding to the allosteric cGMP-binding sites. Both effects were associated with increases in its phosphate content up to 0.6 mol per PDE5 subunit. Omission of any one of the preincubation components caused loss of stimulation of catalytic activity. Addition of the phosphorylation reaction mixture to a crude bovine lung extract, which contains PDE5, also produced a significant increase in cGMP PDE catalytic activity. The increase in recombinant PDE5 catalytic activity brought about by phosphorylation was time-dependent and was obtained with 0.2-0.5 microM PKG subunit, which is approximately the cellular level of this enzyme in vascular smooth muscle. Significantly greater stimulation was observed using cGMP substrate concentrations below the Km value for PDE5, although stimulation was also seen at high cGMP concentrations. Considerably higher concentration of the catalytic subunit of PKA than of PKG was required for activation. There was no detectable difference between phosphorylated and unphosphorylated PDE5 in median inhibitory concentration for the PDE5 inhibitors, sildenafil, or zaprinast 3-isobutyl-1-methylxanthine. Phosphorylation reduced the cGMP concentration required for half-maximum binding to the allosteric cGMP-binding sites from 0.13 to 0.03 microM. The mechanism by which phosphorylation of PDE5 by PKG could be involved in physiological negative-feedback regulation of cGMP levels is discussed.     

Occupancy of the catalytic site of the PDE4A4 cyclic AMP phosphodiesterase by rolipram triggers the dynamic redistribution of this specific isoform in living cells through a cyclic AMP independent process

In cells transfected to express wild-type PDE4A4 cAMP phosphodiesterase (PDE), the PDE4 selective inhibitor rolipram caused PDE4A4 to relocalise so as to form accretion foci. This process was followed in detail in living cells using a PDE4A4 chimera formed with Green Fluorescent Protein (GFP). The same pattern of behaviour was also seen in chimeras of PDE4A4 formed with various proteins and peptides, including LimK, RhoC, FRB and the V5-6His tag. Maximal PDE4A4 foci formation, occurred over a period of about 10 h, was dose-dependent on rolipram and was reversible upon washout of rolipram. Inhibition of protein synthesis, using cycloheximide, but not PKA activity with H89, inhibited foci generation. Foci formation was elicited by Ro20-1724 and RS25344 but not by either Ariflo or RP73401, showing that not all PDE4 selective inhibitors had this effect. Ariflo and RP73401 dose-dependently antagonised rolipram-induced foci formation and dispersed rolipram pre-formed foci as did the adenylyl cyclase activator, forskolin. Foci formation showed specificity for PDE4A4 and its rodent homologue, PDE4A5, as it was not triggered in living cells expressing the PDE4B2, PDE4C2, PDE4D3 and PDE4D5 isoforms as GFP chimeras. Altered foci formation was seen in the Deltab-LR2-PDE4A4 construct, which deleted a region within LRZ, showing that appropriate linkage between the N-terminal portion of PDE4A4 and the catalytic unit of PDE4A4 was needed for foci formation. Certain single point mutations within the PDE4A4 catalytic site (His505Asn, His506Asn and Val475Asp) were shown to ablate foci formation but still allow rolipram inhibition of PDE4A4 catalytic activity. We suggest that the binding of certain, but not all, PDE4 selective inhibitors to PDE4A4 induces a conformational change in this isoform by 'inside-out' signalling that causes it to redistribute in the cell. Displacing foci-forming inhibitors with either cAMP or inhibitors that do not form foci can antagonise this effect. Specificity of this effect for PDE4A4 and its homologue PDE4A5 suggests that interplay between the catalytic site and the unique N-terminal region of these isoforms is required. Thus, certain PDE4 selective inhibitors may exert effects on PDE4A4 that extend beyond simple catalytic inhibition. These require protein synthesis and may lead to redistribution of PDE4A4 and any associated proteins. Foci formation of PDE4A4 may be of use in probing for conformational changes in this isoform and for sub-categorising PDE4 selective inhibitors.     

Conformational difference between PDE4 apoenzyme and holoenzyme

The type 4 cAMP-specific phosphodiesterases (PDE4s) are Mg(2+)-dependent hydrolases that catalyze the hydrolysis of 3', 5'-cAMP to AMP. Previous studies indicate that PDE4 exists in two conformations that bind the inhibitor rolipram with affinities differing by more than 100-fold. Here we report that these two conformations are the consequence of PDE4 binding to its metal cofactor such as Mg(2+). Using a fluorescence resonance energy transfer (FRET)-based equilibrium binding assay, we identified that L-791,760, a fluorescent inhibitor, binds to the apoenzyme (free enzyme) and the holoenzyme (enzyme bound to Mg(2+)) with comparable affinities (K(d) approximately 30 nM). By measuring the displacement of the bound L-791,760, we have also identified that other inhibitors bind differentially with the apoenzyme and the holoenzyme depending upon their structure. CDP-840, SB-207499, and RP-73401 bind preferentially to the holoenzyme. The conformational-sensitive inhibitor (R)-rolipram binds to the holoenzyme and apoenzyme with affinities (K(d)) of 5 and 300 nM, respectively. In contrast to its high affinity (K(d) approximately 2 microM) and active holoenzyme complex, cAMP binds to the apoenzyme nonproductively with a reduced affinity (K(d) approximately 170 microM). These results demonstrate that cofactor binding to PDE4 is responsible for eliciting its high-affinity interaction with cAMP and the activation of catalysis.     

Evaluation of the pentose phosphate pathway from 14CO2 data. Fallibility of a classic equation when applied to non-homogeneous tissues.

Two cyclic nucleotide phosphodiesterase (PDE) activities were identified in pig aortic endothelial cells, a cyclic GMP-stimulated PDE and a cyclic AMP PDE. Cyclic GMP-stimulated PDE had Km values of 367 microM for cyclic AMP and 24 microM for cyclic GMP, and low concentrations (1 microM) of cyclic GMP increased the affinity of the enzyme for cyclic AMP (Km = 13 microM) without changing the Vmax. This isoenzyme was inhibited by trequinsin [IC50 (concn. giving 50% inhibition of substrate hydrolysis) = 0.6 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 0.6 microM for cyclic GMP hydrolysis] and dipyridamole (IC50 = 5 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 3 microM for cyclic GMP hydrolysis). Cyclic AMP PDE exhibited a Km of 2 microM for cyclic AMP and did not hydrolyse cyclic GMP. This activity was inhibited by trequinsin (IC50 = 0.2 microM), dipyridamole (IC50 = 6 microM) and, selectively, by rolipram (IC50 = 3 microM). Inhibitors of cyclic GMP PDE (M&B 22948) and of low Km (Type III) cyclic AMP PDE (SK&F 94120) only weakly inhibited the two endothelial PDEs. Incubation of intact cells with trequinsin and dipyridamole induced large increases in cyclic GMP, which were completely blocked by LY-83583. Rolipram, SK&F 94120 and M&B 22948 did not significantly influence cyclic GMP accumulation. Dipyridamole enhanced the increase in cyclic GMP induced by sodium nitroprusside. Cyclic AMP accumulation was stimulated by dipyridamole and trequinsin with and without forskolin. Rolipram, although without effect alone, increased cyclic AMP in the presence of forskolin, whereas M&B 22948 and SK&F 94120 had no effects on resting or forskolin-stimulated levels. These results suggest that cyclic GMP-stimulated PDE regulates cyclic GMP levels and that both endothelial PDE isoenzymes contribute to the control of cyclic AMP.     

Probing the Catalytic Sites and Activation Mechanism of Photoreceptor Phosphodiesterase Using Radiolabeled Phosphodiesterase Inhibitors

Retinal photoreceptor phosphodiesterase (PDE6) is unique among the phosphodiesterase enzyme family not only for its catalytic heterodimer but also for its regulatory γ-subunits (Pγ) whose inhibitory action is released upon binding to the G-protein transducin. It is generally assumed that during visual excitation both catalytic sites are relieved of Pγ inhibition upon binding of two activated transducin molecules. Because PDE6 shares structural and pharmacological similarities with PDE5, we utilized radiolabeled PDE5 inhibitors to probe the catalytic sites of PDE6. The membrane filtration assay we used to quantify [³H]vardenafil binding to PDE6 required histone II-AS to stabilize drug binding to the active site. Under these conditions, [³H]vardenafil binds stoichiometrically to both the α- and β-subunits of the activated PDE6 heterodimer. [³H]vardenafil fails to bind to either the PDE6 holoenzyme or the PDE6 catalytic dimer reconstituted with Pγ, consistent with Pγ blocking access to the drug-binding sites. Following transducin activation of membrane-associated PDE6 holoenzyme, [³H]vardenafil binding increases in proportion to the extent of PDE6 activation. Both [³H]vardenafil binding and hydrolytic activity of transducin-activated PDE6 fail to exceed 50% of the value for the PDE6 catalytic dimer. However, adding a 1000-fold excess of activated transducin can stimulate the hydrolytic activity of PDE6 to its maximum extent. These results demonstrate that both subunits of the PDE6 heterodimer are able to bind ligands to the enzyme active site. Furthermore, transducin relieves Pγ inhibition of PDE6 in a biphasic manner, with only one-half of the maximum PDE6 activity efficiently attained during visual excitation.     

Interactions between cAMP- and cGMP-dependent protein kinase inhibitors and phosphodiesterase IV inhibitors on arachidonate release from human monocytes

The effects of specific inhibitors of cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) on the inhibitory activity of phosphodiesterase (PDE) type IV inhibitors and of the cell permeable analogue of cAMP, db-cAMP, were investigated on fMLP-induced arachidonate release from human monocytes. When monocytes were preincubated with the combined PKA/PKG inhibitor H8 (10⁻⁶ to 10⁻⁴ M) or the selective PKG inhibitor Rp-8-cpt-cGMPs (10⁻⁶ to 10⁻⁴ M) a concentration-dependent reduction of the inhibitory effect of db-cAMP (10⁻ M), rolipram (10⁻⁵ M) and Ro 20-1724 (10⁻⁵ M) was noted. When monocytes were preincubated with the selective PKA inhibitor H89 (10⁻⁶ to 10⁻⁴ M), only a small inhibition of the effect of db-cAMP and no inhibition of the effects of rolipram and Ro 20–1724 were observed. The present data indicate that db-cAMP and PDE IV inhibitors elicit an in vitro anti-inflammatory activity by a PKA-independent mechanism, which do not appear to be mainly mediated via the PKG activation.     

In vitro PKA phosphorylation-mediated human PDE4A4 activation

The PDE4 catalytic machinery comprises, in part, two divalent cations in a binuclear motif. Here we report that PDE4A4 expressed in Sf9 cells exhibits a biphasic Mg(2+) dose-response (EC(50) of 0.15 and >10 mM) in catalyzing cAMP hydrolysis. In vitro phosphorylation of PDE4A4 by the PKA-catalytic subunit increases the enzyme's sensitivity to Mg(2+), leading to 4-fold increased cAMP hydrolysis without affecting its K(m). The phosphorylation also increases the potencies of (R)- and (S)-rolipram without affecting CDP-840 and SB-207499. The results support that modulating the cofactor binding affinity of PDE4 represents a mechanism for regulating its activity.     

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.     

OPC-13013, a cyclic nucleotide phosphodiesterase type III, inhibitor, inhibits cell proliferation and transdifferentiation of cultured rat hepatic stellate cells.

Activated hepatic stellate cells (HSC; lipocytes; Ito cells) proliferate and are responsible for extracellular matrix synthesis during hepatic fibrogenesis. During activation, HSC undergo transdifferentiation into myofibroblasts expressing alpha-smooth muscle actin (alpha-SMA). Adenosine 3', 5'-cyclic monophosphate (cyclic AMP) is an ubiquitous intracellular signaling molecule, and is upregulated by the activation of adenylate cyclase and downregulated via hydrolysis by cyclic nucleotide phosphodiesterases (PDEs). Recently, increased intracellular cyclic AMP has been shown to inhibit HSC activation. The aim of the current study was to determine the effects of inhibition of PDEs on cell proliferation and transdifferentiation in cultured rat HSC. Cell proliferation was determined by [3H]thymidine incorporation, and Western blot analysis was performed for detection of alpha-SMA, a phenotypic marker of transdifferentiation into myofibroblast. When the cells were exposed to 3-isobutyl-1-methylxanthine (IBMX; 50-1000 microM), a nonselective PDE inhibitor, serum-stimulated [3H]thymidine incorporation was suppressed in a dose-dependent manner with a maximum inhibition of 66% at a concentration of 500 microM OPC-13013 (1-60 microM), a selective PDE III isoenzyme inhibitor, induced a dose-dependent inhibitory effect on serum-stimulated DNA synthesis that reached a maximum inhibition of 95% at a concentration of 60 microM, while neither 8-methoxymethyl-3-isobutyl-1-methylxanthine (8-MMX), a PDE I isoenzyme inhibitor, nor Ro-20-1724, a PDE IV isoenzyme inhibitor, had an inhibitory effect. Western blot analysis revealed that IBMX or OPC-13013 decreased alpha-SMA expression, while other selective PDE isoenzyme inhibitors did not have a suppressive effect. IBMX, OPC-13013 or Ro-20-1724, but not 8-MMX augmented forskolin-induced increase in intracellular cyclic AMP levels although cyclic AMP levels were not affected by treatment with any of these PDE inhibitors alone. These data indicate that inhibition of PDEs, especially PDE III isoenzyme, can produce an inhibitory effect on HSC activation. The PDE III isoenzyme may contribute to the regulation of HSC activation during fibrogenesis. In addition, OPC-13013 may have the potential to inhibit initiation and progression of hepatic fibrosis by interfering with HSC activation.     

Crystal structure of phosphodiesterase 4D and inhibitor complex(1)

Cyclic nucleotide phosphodiesterases (PDEs) regulate physiological processes by degrading intracellular second messengers, adenosine-3′,5′-cyclic phosphate or guanosine-3′,5′-cyclic phosphate. The first crystal structure of PDE4D catalytic domain and a bound inhibitor, zardaverine, was determined. Zardaverine binds to a highly conserved pocket that includes the catalytic metal binding site. Zardaverine fills only a portion of the active site pocket. More selective PDE4 inhibitors including rolipram, cilomilast and roflumilast have additional functional groups that can utilize the remaining empty space for increased binding energy and selectivity. In the crystal structure, the catalytic domain of PDE4D possesses an extensive dimerization interface containing residues that are highly conserved in PDE1, 3, 4, 8 and 9. Mutations of R358D or D322R among these interface residues prohibit dimerization of the PDE4D catalytic domain in solution.     

cGMP-activated phosphodiesterase from human brain: kinetic and regulatory properties]

The kinetic and regulatory properties of cGMP-activated phosphodiesterase (PDE) from human brain were studied. In double reciprocal plots the enzyme activity is characterized by a linear dependence of cAMP and a nonlinear one for cGMP. Micromolar concentrations of cGMP accelerate cAMP hydrolysis (7-14-fold) with Ka for cGMP of 0.36 microM. Stimulation of cAMP hydrolysis is accompanied by a decrease of Km with no changes in Vmax. With a rise in the cGMP concentration above 5 microM PDE activation is changed by its inhibition. Both substrates act as competitive inhibitors towards each other. The Ki value for both cGMP and cAMP is 30 microM. After the increase in the cAMP (Bt)2 concentration the activation of 5 microM cAMP hydrolysis is accompanied by the enzyme inhibition. Both analogs competitively inhibit cGMP hydrolysis with Ki of 10 and 1500 microM for cGMP(Bt)2 and cAMP(Bt)2, respectively. The data obtained point to the existence of two binding sites for cyclic nucleotides, namely, a regulatory site which is highly specific for cGMP and a catalytic site responsible for the hydrolysis of the both substrates which displays no apparent specificity either for cAMP or for cGMP. The different affinity of natural and synthetic cyclic nucleotides for these sites is determined, to a large extent, by the amino groups in the 2nd and 6th positions of the purine ring.     

Effects of Mg2+ ions on the plasma membrane [H+]-ATPase of Neurospora crassa. II. Kinetic studies

The rate of ATP hydrolysis by the Neurospora plasma membrane [H+]-ATPase has been measured over a wide range of Mg2+ and ATP concentrations, and on the basis of the results, a kinetic model for the enzyme has been developed. The model includes the following three binding sites: 1) a catalytic site at which MgATP serves as the true substrate, with free ATP as a weak competitive inhibitor; 2) a high affinity site for free Mg2+, which serves to activate the enzyme with an apparent K1/2 (termed KMgA) of about 15 microM; and 3) a separate low affinity site at which Mg2+ causes mixed type inhibition, lowering the Vmax while raising the KS for MgATP at the catalytic site. The Ki for Mg2+ at the low affinity site (termed KMgI) is about 3.5 mM. The model satisfactorily explains the activity of the enzyme as Mg2+ and ATP are varied, separately and together, over a wide range. It can also account for the previously reported effects of Mg2+ and ATP on the inhibition of the Neurospora [H+]-ATPase by N-ethylmaleimide (Brooker, R. J., and Slayman, C. W. (1982) J. Biol. Chem. 257, 12051-12055; Brooker, R. J., and Slayman, C. W. (1983) J. Biol. Chem. 258, 8827-8832).     

Characterization df a [3H]Glycine Recognition Site as a Modulatory Site of the N-Methyl-D-Aspartate Receptor Complex

A [3H]glycine recognition site in rat brain synaptic plasma membranes (SPM) has been identified, having characteristics expected of a modulatory component of the N-methyl-D-aspartate receptor complex. Incubation of SPM with [3H]glycine for 10 min at 2 degrees C results in saturable, reversible binding with a KD of 0.234 microM and a Bmax of 9.18 pmol/mg. A pharmacological analysis of this binding site indicates that D-serine (Ki = 0.27 microM), D-alanine (Ki = 1.02 microM), and D-cycloserine (Ki = 2.33 microM) are potent inhibitors of binding, whereas the corresponding L isomers have significantly less activity (Ki = 25.4 microM, 15.9 microM, and greater than 100 microM, respectively). Inactive at concentrations of up to 100 microM were strychnine, L-valine, N,N-dimethylglycine, aminomethylphosphonate, and aminomethylsulfonate. The active compounds were analyzed further for their ability to stimulate [3H]1-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) binding to Triton X-100-washed SPM. Results indicate that the affinity of the compounds for the [3H]glycine recognition site correlates with the ability of these analogues to stimulate [3H]TCP binding.