Functions of the N-terminal region of cyclic nucleotide phosphodiesterase 3 (PDE 3) isoforms

The N-terminal portion of phosphodiesterase (PDE) 3 was arbitrarily divided into region 1 (amino acids 1-300), which contains a large hydrophobic domain with six predicted transmembrane helices, and region 2 (amino acids 301-500), with a smaller hydrophobic domain ( approximately 50 residues). To analyze these regions, full-length human (H)PDE3A and mouse (M)PDE3B and a series of N-terminal truncated mutants were synthesized in Sf9 cells. Activities of HPDE3A, H3A-Delta189, MPDE3B, and M3B-Delta196, which retained all or part of the hydrophobic domain in region 1, were recovered almost entirely in particulate fractions. H3A-Delta321 and M3B-Delta302, containing region 2, were recovered essentially equally in particulate and cytosolic fractions. H3A-Delta397 and H3A-Delta457, lacking both hydrophobic domains, were predominantly cytosolic. H3A-Delta510 and M3B-Delta604, lacking both regions 1 and 2, were virtually completely cytosolic. M3B-Delta196 eluted as a large aggregated complex during gel filtration. With removal of greater amounts of N-terminal sequence, aggregation of PDE3 decreased, and H3A-Delta607, H3A-Delta721, and M3B-Delta604 eluted as dimers. Truncated HPDE3A proteins were more sensitive than full-length HPDE3A to inhibition by lixazinone. These results suggest that the hydrophobic domains in regions 1 and 2 contain structural determinants important for association of PDE3 with intracellular membranes, as well for self-association or aggregation during gel filtration and sensitivity to a specific inhibitor.     

Insulin-induced formation of macromolecular complexes involved in activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and its interaction with PKB

Fractionation of 3T3-L1 adipocyte membranes revealed that PDE3B (phosphodiesterase 3B) was associated with PM (plasma membrane) and ER (endoplasmic reticulum)/Golgi fractions, that insulin-induced phosphorylation/activation of PDE3B was greater in internal membranes than PM fractions, and that there was no significant translocation of PDE3B between membrane fractions. Insulin also induced formation of large macromolecular complexes, separated during gel filtration (Superose 6 columns) of solubilized membranes, which apparently contain phosphorylated/activated PDE3B and signalling molecules potentially involved in its activation by insulin, e.g. IRS-1 (insulin receptor substrate-1), IRS-2, PI3K p85 [p85-subunit of PI3K (phosphoinositide 3-kinase)], PKB (protein kinase B), HSP-90 (heat-shock protein 90) and 14-3-3. Expression of full-length recombinant FLAG-tagged murine (M) PDE3B and M3BDelta604 (MPDE3B lacking N-terminal 604 amino acids) indicated that the N-terminal region of MPDE3B was necessary for insulin-induced activation and recruitment of PDE3B. siRNA (small interfering RNA) knock-down of PDE3B indicated that PDE3B was not required for formation of insulin-induced complexes. Wortmannin inhibited insulin-induced assembly of macromolecular complexes, as well as phosphorylation/activation of PKB and PDE3B, and their co-immunoprecipitation. Another PI3K inhibitor, LY294002, and the tyrosine kinase inhibitor, Genistein, also inhibited insulin-induced activation of PDE3B and its co-immunoprecipitation with PKB. Confocal microscopy indicated co-localization of PDE3B and PKB. Recombinant MPDE3B co-immunoprecipitated, and co-eluted during Superose 12 chromatography, to a greater extent with recombinant pPKB (phosphorylated/activated PKB) than dephospho-PKB or p-DeltaPKB [pPKB lacking its PH domain (pleckstrin homology domain)]. Truncated recombinant MPDE3B proteins and pPKB did not efficiently co-immunoprecipitate, suggesting that structural determinants for their interaction reside in, or are regulated by, the N-terminal portion of MPDE3B. Recruitment of PDE3B in macromolecular complexes may be critical for regulation of specific cAMP pools and signalling pathways by insulin, e.g. lipolysis.     

Long-term regulation of cyclic nucleotide phosphodiesterase type 3B and 4 in 3T3-L1 adipocytes

Phosphodiesterase 3B (PDE3B), is known to play an important role in acute insulin and cAMP-mediated regulation of lipid metabolism, and PDE4 are the main PDE types expressed in adipocytes. Here, we show that members of all PDE4 isoforms are expressed in 3T3-L1 and primary mouse adipocytes. Long-term treatment of 3T3-L1 adipocytes with insulin induced up-regulation of PDE3B and PDE4D in a phosphatidylinositol 3-kinase-dependent manner whereas long-term treatment with beta-adrenergic agonists induced down-regulation of PDE3B and up-regulation of PDE4D. Thus, PDE3B and PDE4D can be added to the list of genes regulated by insulin and cAMP-increasing hormones. Altered expression of PDE3B and PDE4D in response to long-term treatment with insulin and catecholamines may contribute to altered regulation of metabolism in diabetes.     

Calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1) splice variants from bovine cardiac muscle

Calmodulin-dependent cyclic nucleotide phopshodiesterase (PDE1) has been extensively characterized and is a key enzyme involved in the complex interaction between cyclic nucleotide and Ca(2+) second-messenger systems. It is well established that PDE1 exists in different isozymes. For example, bovine brain tissue has two PDE1 isozymes (PDE1A2 and PDE1B1) whereas only one form (PDE1A1) is reported in bovine cardiac tissue. In this study, we report the cloning of two cDNA splice variants of PDE1: PDE1-small and PDE1-large, from bovine cardiac tissue. Their amino acid sequence similarity to PDE1 sequences from other mammalian species showed that all are very conserved, suggesting their importance in cellular functions. Interestingly, compared to other mammalian species, bovine PDE1A, PDE-small and PDE-large show a deletion at the C-terminal end of the catalytic domain of the gene. Although the significance of this deletion at this crucial location of the gene is not known, we have successfully over-expressed both PDE1-small and PDE1-large splice variants in E. coli and these splice variants are characterized in terms of Western blot, biotinylated calmodulin overlay and peptide mass fingerprinting. Results from these studies suggested that these two splice variants belong to the PDE1 superfamily. To our knowledge, this is the first report on cloning and characterization of these cDNA variants from bovine cardiac tissue. Since there are at least two isoforms of PDE1 in bovine heart tissue, this merits further in-depth study.     

Regulation by insulin of cyclic nucleotide phosphodiesterase (PDE) from rat luteal cells

The effect of insulin on cyclic nucleotide phosphodiesterase (PDE) in rat luteal cells was studied. Cells were obtained from PMSG/hCG primed rats and further incubated or not with insulin. The hormone produced an increase of enzyme activity after a 10 min incubation of intact cells. Maximal stimulation was achieved at 0.2 nM of insulin. Two peaks of cyclic nucleotide phosphodiesterase activity were resolved after chromatography of cell cytosolic extracts on DEAE-cellulose. These peaks (I and II) were active with cAMP as substrate but only peak I was active with cGMP. The enzyme activity of both peaks was increased in cells treated with insulin. Phosphodiesterase activity in the two peaks show two kinetic components for cAMP hydrolysis, one of high affinity (Km 2-4 microM) and the other of low affinity (47-56 microM). Treatment of the cells with insulin produced a 2 to 8 fold increase of the Vmax of these peaks. In addition after stimulation with insulin, the activation of peak I phosphodiesterase by calmodulin was less effective.     

Plasma membrane-bound cyclic AMP phosphodiesterase activity in 3T3-L1 adipocytes

Plasma membranes were isolated from 3T3-L1 adipocytes. Plasma membrane phosphodiesterase (PM-PDE) was measured in the presence of 5 microM cilostamide. Time course and cAMP dose response ranging from 0 to 2 microM were measured. PM-PDE remained linear up to 20 min. Non-linear curve fitting analysis showed that the low Km cAMP dose data fit a two component curve significantly better than a one component curve, indicating that there are two iso-forms of PDE in the plasma membrane of 3T3-L1 adipocytes, similar to swine adipocytes. The Km and Vmax values for this two component curve were Km1=0.12 microM, Vmax1=3.08 pmol min(-1) mg(-1) protein, and Km2=3.67 microM, Vmax2=83.8 pmol min(-1) mg(-1) protein. Inhibitors of PDE1, PDE2 and PDE5 failed to inhibit PM-PDE, as observed in swine adipocyte plasma membranes. However, PDE4 inhibitors were three-fold more effective at inhibiting PDE in 3T3-L1 PM compared to swine adipocyte PM. One mM 1, 3-dipropyl-8-p-sulfophenylxanthine (DPSPX) inhibited PM-PDE by approximately 75% in both preparations. These data demonstrate that PM-PDE is distinct from microsomal membrane PDE and may be responsible for extracellular cAMP metabolism to AMP in 3T3-L1 adipocytes.     

Insulin and isoproterenol induce phosphorylation of the particulate cyclic GMP-inhibited, low Km cyclic AMP phosphodiesterase (cGI PDE) in 3T3-L1 adipocytes.

The cGI PDE in particulate fractions of differentiated adipocytes (but not control 3T3-L1 fibroblasts) was cross-reactive with a polyclonal antibody raised against the bovine adipose cGI PDE. The 3T3-L1 adipocyte cGI PDE is a 135 kDa protein which is phosphorylated in 32P-labeled cells in response to beta-agonist or insulin. These results indicate that the 3T3-L1 cGI PDE is similar in structure and hormonal regulation to the analogous enzyme in the rat adipocyte.     

Visual detection of AMP and real-time monitoring of cyclic nucleotide phosphodiesterase (PDE) activity in neutral aqueous solution. Chemosensor-coupled assay of PDE and PDE inhibitors

A phosphate chemosensor ensemble, [Zn(H-bpmp)(pyrocatechol violet)](+) is useful for a real-time assay of phosphodiesterases (PDEs) in a neutral aqueous solution. In addition, a simple and convenient screen and assay procedures for inhibitors of PDEs have been demonstrated using IBMX, a nonselective PDE inhibitor as an example.     

Triacylglycerol is synthesized in a specific subclass of caveolae in primary adipocytes

A principal metabolic function of adipocytes is to synthesize triacylglycerol (TG) from exogenous fatty acids. The level of fatty acids has to be tightly controlled in the adipocyte, as they can act as detergents that rapidly dissolve the plasma membrane, causing cell lysis if allowed to accumulate. Fatty acids therefore have to be efficiently converted to TG and stored in the central lipid droplet. We report that in intact primary adipocytes exogenous oleic acid was taken up and directly converted to TG in the plasma membrane, in a novel subclass of caveolae that specifically contains the protein perilipin. Isolated caveolae catalyzed de novo TG synthesis from oleic acid and glycerol 3-phosphate. Electron microscopy revealed the presence of caveolin and perilipin in caveolae and in lipid-laden bulbs in the plasma membrane, and fluorescence microscopy demonstrated colocalization of fatty acids/TG with caveolin and perilipin at the plasma membrane. A second caveolae fraction was isolated, which lacked perilipin and the triacylglycerol synthesizing enzymes. Both caveolae fractions contained caveolin-1 and the insulin receptor. The findings demonstrate that specific subclasses of caveolae carry out specific functions in cell metabolism. In particular, triacylglycerol is synthesized at the site of fatty acid entry in one of these caveolae classes.     

Protein kinase A-dependent activation of PDE4 (cAMP-specific cyclic nucleotide phosphodiesterase) in cultured bovine vascular smooth muscle cells

Incubation of cultured bovine vascular smooth muscle cells (VSMC) with forskolin increased cAMP as measured by an increase in cAMP-dependent protein kinase (PKA) activation (PKA ratio). Forskolin also produced a concentration- and time-dependent increase in activity (3–5-fold within 15 min) of a PDE4 (cAMP-specific cyclic nucleotide phosphodiesterase). The increase in PDE4 activity was not affected by cycloheximide and thus not likely due to increased synthesis of the enzyme. Activation, which was preserved during partial purification of the enzyme by chromatography on Sephacryl S-200 and MonoQ, was most likely due to a covalent modification. Incubation of cell homogenates with the catalytic subunit of PKA (PKA_c) induced a ∼5-fold activation of PDE4 with a time course similar to that in intact cells after forskolin addition. The forskolin-mediated activation was reversed during incubation of homogenates at room temperature for two hours. Addition of PKA_c resulted in rapid reactivation of PDE4. These data are consistent with the hypothesis that rapid, reversible activation of PDE4 in cultured VSMC is mediated by PKA.     

A newly identified 50 kDa protein, which is associated with phosphodiesterase 3B, is phosphorylated by insulin in rat adipocytes

Phosphodiesterase 3B (PDE3B), a major PDE isoform in adipocytes, plays a pivotal role in the anti-lipolytic action of insulin. Insulin phosphorylates and activates PDE3B in a phosphatidylinositol 3-kinase-dependent manner. We identified a new 50 kDa protein that is phosphorylated by insulin and is co-immunoprecipitated with PDE3B by anti-PDE3B antibodies in rat adipocytes. The insulin-induced phosphorylation of the 50 kDa protein was also detected in a cell free system against the N-terminal and the catalytic regions, which are more than 700 amino acids apart recognize the 50 kDa protein, suggesting that it is not a proteolytic product, but an associated protein with PDE3B. Phosphoamino acid analysis indicated that both serine and threonine residues in the 50 kDa protein were phosphorylated, but only serine residues in PDE3B were phosphorylated. Therefore, it appears likely that this is a new protein which is associated with PDE3B.     

Analysis of cyclic nucleotide phosphodiesterase(s) by radioimmunoassay

A high-affinity form of cyclic AMP phosphodiesterase, purified to apparent homogeneity from dog kidney, was labeled with ¹²⁵I using a solid-state lactoperoxidaseglucose oxidase system and its purity confirmed by acrylamide gel electrophoresis and isoelectric focusing. Sheep anti-cyclic AMP phosphodiesterase immunoglobulin fraction was analyzed for ¹²⁵I-enzyme binding and covalently bound to agarose A 1.5m for isotopically labeled antigen displacement. Anti-phosphodiesterase antiserum was purified by Sepharose 4B-cAPDE affinity chromatography and used for a radioimmunoassay employing second-antibody precipitation. The specificity of the anti-cyclic AMP phosphodiesterase antibody was established by its use as a covalently bound affinity ligand for cyclic AMP phosphodiesterase purification and analysis of sodium dodecyl sulfate-gel extracts of partially purified and purified dog kidney supernatants. Radioimmunoassay using a monospecific antibody preparation demonstrated the similarity of high-affinity cyclic AMP phosphodiesterase forms of different tissues and species that had been separated by DEAE-cellulose chromatography. Various purified preparations of calmodulin, as well as brain calcineurin, did not cross-react in the high-affinity cyclic AMP phosphodiesterase radioimmunoassay. However, higher molecular weight cyclic GMP/lower affinity cyclic AMP phosphodiesterase enzyme forms, partially purified by anion-exchange chromatography, gel filtration, and Cibacron blue adsorption, were shown to cross-react in the high-affinity cAMP phosphodiesterase radioimmunoassay. These studies suggest immunological similarities between the major forms of this enzyme system and the possibility of higher molecular weight complexes containing both cyclic GMP and cyclic AMP hydrolytic sites.     

Membrane localization of cyclic nucleotide phosphodiesterase 3 (PDE3). Two N-terminal domains are required for the efficient targeting to, and association of, PDE3 with endoplasmic reticulum

Subcellular localization of cyclic nucleotide phosphodiesterases (PDEs) may be important in compartmentalization of cAMP/cGMP signaling responses. In 3T3-L1 adipocytes, mouse (M) PDE3B was associated with the endoplasmic reticulum (ER) as indicated by its immunofluorescent colocalization with the ER protein BiP and subcellular fractionation studies. In transfected NIH 3006 or COS-7 cells, recombinant wild-type PDE3A and PDE3B isoforms were both found almost exclusively in the ER. The N-terminal portion of PDE3 can be arbitrarily divided into region 1 (aa 1-300), which contains a large hydrophobic domain with six predicted transmembrane helices, followed by region 2 (aa 301-500) containing a smaller hydrophobic domain (of approximately 50 aa). To investigate the role of regions 1 and 2 in membrane association, we examined the subcellular localization of a series of catalytically active, Flag-tagged N-terminal-truncated human (H) PDE3A and MPDE3B recombinants, as well as a series of fragments from regions 1 and 2 of MPDE3B synthesized as enhanced green fluorescent (EGFP) fusion proteins in COS-7 cells. In COS-7 cells, the localization of a mutant HPDE3A, lacking the first 189 amino acids (aa) and therefore four of the six predicted transmembrane helices (H3A-Delta189), was virtually identical to that of the wild type. M3B-Delta302 (lacking region 1) and H3A-Delta397 (lacking region 1 as well as part of region 2) retained, to different degrees, the ability to associate with membranes, albeit less efficiently than H3A-Delta189. Proteins that lacked both regions 1 and 2, H3A-Delta510 and M3B-Delta604, did not associate with membranes. Consistent with these findings, region 1 EGFP-MPDE3B fusion proteins colocalized with the ER, whereas region 2 EGFP fusion proteins were diffusely distributed. Thus, some portion of the N-terminal hydrophobic domain in region 1 plus a second domain in region 2 are important for efficient membrane association/targeting of PDE3.     

Ca2+-dependent cyclic nucleotide phosphodiesterase is activated by poly(L-aspartic acid)

Ca2+-dependent cyclic nucleotide phosphodiesterase (Ca2+-PDE) activity was stimulated by poly(L-aspartic acid) but not by poly(L-glutamic acid), poly(L-arginine), poly(L-lysine), and poly(L-proline). This activation was Ca2+ independent and did not further enhance the activation of Ca2+-PDE by Ca2+-calmodulin (CaM). Poly(L-aspartic acid) produced an increase in the Vmax of the phosphodiesterase, associated with a decrease in the apparent Km for the substrate, such being similar to results obtained with Ca2+-CaM. Poly(L-aspartic acid) did not significantly stimulate myosin light chain kinase and other types of cyclic nucleotide phosphodiesterase. CaM antagonists such as N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), trifluoperazine, and chlorpromazine selectively antagonized activation of the enzyme by poly(L-aspartic acid). Kinetic analysis of W-7-induced inhibition of activation of phosphodiesterase by poly(L-aspartic acid) was in a competitive fashion, and the Ki value was 0.19 mM. On the other hand, prenylamine, another type of calmodulin antagonist that binds to CaM at sites different from the W-7 binding sites, did not inhibit the poly(L-aspartic acid)-induced activation of Ca2+-dependent cyclic nucleotide phosphodiesterase. These results imply that poly(L-aspartic acid) is a calcium-independent activator of Ca2+-dependent phosphodiesterase and that aspartic acids in the CaM molecule may play an important role in the activation of Ca2+-PDE.     

Targeting cyclic nucleotide phosphodiesterase 5 (PDE5) in brain: Toward the development of a PET radioligand labeled with fluorine-18

With the aim to develop a specific radioligand for imaging the cyclic nucleotide phosphodiesterase 5 (PDE5) in brain by positron emission tomography (PET), seven new fluorinated inhibitors (3–9) were synthesized on the basis of a quinoline core. The inhibitory activity for PDE5 together with a panel of other PDEs was determined in vitro and two derivatives were selected for IC₅₀ value determination. The most promising compound 7 (IC₅₀ = 5.92 nM for PDE5A), containing a 3-fluoroazetidine moiety, was further radiolabeled by aliphatic nucleophilic substitution of two different leaving groups (nosylate and tosylate) using [¹⁸F]fluoride. The use of the nosylate precursor and tetra-n-butyl ammonium [¹⁸F]fluoride ([¹⁸F]TBAF) in 3-methyl-3-pentanol combined with the addition of a small amount of water proved to be the best radiolabeling conditions achieving a RCY of 4.9 ± 1.5% in an automated procedure. Preliminary biological investigations in vitro and in vivo were performed to characterize this new PDE5 radioligand. Metabolism studies of [¹⁸F]7 in mice revealed a fast metabolic degradation with the formation of radiometabolites which have been detected in the brain.     

Interaction of caspase-3 with the cyclic GMP binding cyclic GMP specific phosphodiesterase (PDE5a1).

Here, we show that recombinant bovine PDE5A1 is proteolysed by recombinant caspase-3 in in vitro and transfected Cos-7 cells. In addition, the treatment of PDE5A1-transfected Cos-7 and PC12 cells with staurosporine, an apoptotic agent that activates endogenous caspase-3, also induced proteolysis and inactivation of PDE5A1. These findings suggest that there is specificity in the interaction between caspase-3 and PDE5A1 that requires application of an apoptotic stimulus. The potential proteolysis of the [778]DQGD[781] site in PDE5A1 by caspase-3 might affect cGMP's hydrolyzing activity as this is within the boundary of the active site. We therefore created a truncated D781 mutant corresponding exactly to the potential cleavage product. This mutant was expressed equally well compared with the wild-type enzyme in transfected Cos-7 cells and was inactive. Inactivity of the truncated mutant was not due to potential misfolding of the enzyme as it eluted from gel filtration chromatography in the same fraction as the wild-type enzyme. Homology model comparison with the catalytic domain of PDE4B2 was used to probe a functional role for the region in PDE5A1 that might be cleaved by caspase-3. From this, we can predict that a caspase-3-mediated cleavage of the [778]DQGD[781] motif would result in removal of the C-terminal tail containing Q807 and F810, which are potentially important amino acids required for substrate binding.     

Identification of rat cyclic nucleotide phosphodiesterase 11A (PDE11A): comparison of rat and human PDE11A splicing variants.

We have isolated and characterized rat cyclic nucleotide phosphodiesterase (PDE)11A, which exhibits properties of a dual-substrate PDE, and its splice variants (RNPDE11A2, RNPDE11A3, and RNPDE11A4). The deduced amino-acid sequence of the longest form of rat PDE11A splice variant, RNPDE11A4, was 94% identical with that of the human variant (HSPDE11A4). Rat PDE11A splice variants were expressed in a tissue-specific manner. RNPDE11A4 showed unique tissue distribution distinct from HSPDE11A4, which is specifically expressed in the prostate. Rat PDE11A splice variants were expressed in COS-7 cells, and their enzymatic characteristics were compared. Although the Km values for cAMP and cGMP were similar for all of them (1.3-1.6 and 2.1-3.9 microM, respectively), the Vmax values differed significantly (RNPDE11A4 > RNPDE11A2 > RNPDE11A3). Human PDE11A variants also displayed very similar Km values and significantly different Vmax values (HSPDE11A4 > HSPDE11A2 > HSPDE11A3 > HSPDE11A1). The Vmax values of HSPDE11A4 for cAMP and cGMP were at least 100 times higher than those of HSPDE11A1. These observations indicate unique characteristics of PDE11A splicing variants.     

Disease progression in MRL/lpr lupus-prone mice is reduced by NCS 613, a specific cyclic nucleotide phosphodiesterase type 4 (PDE4) inhibitor

Systemic lupus erythematosus is a polymorphic and multigenic inflammatory autoimmune disease. Cyclic AMP (cAMP) modulates inflammation and the inhibition of cyclic nucleotide phosphodiesterase type 4 (PDE4), which specifically hydrolyzes cAMP, inhibits TNFα secretion. This study was aimed at investigating the evolution of PDE activity and expression levels during the course of the disease in MRL/lpr lupus-prone mice, and to evaluate in these mice the biological and clinical effects of treatments with pentoxifylline, denbufylline and NCS 613 PDE inhibitors. This study reveals that compared to CBA/J control mice, kidney PDE4 activity of MRL/lpr mice increases with the disease progression. Furthermore, it showed that the most potent and selective PDE4 inhibitor NCS 613 is also the most effective molecule in decreasing proteinuria and increasing survival rate of MRL/lpr mice. NCS 613 is a potent inhibitor, which is more selective for the PDE4C subtype (IC₅₀ = 1.4 nM) than the other subtypes (PDE4A, IC₅₀ = 44 nM; PDE4B, IC₅₀ = 48 nM; and PDE4D, IC₅₀ = 14 nM). Interestingly, its affinity for the High Affinity Rolipram Binding Site is relatively low (K_i = 148 nM) in comparison to rolipram (K_i = 3 nM). Finally, as also observed using MRL/lpr peripheral blood lymphocytes (PBLs), NCS 613 inhibits basal and LPS-induced TNFα secretion from PBLs of lupus patients, suggesting a therapeutic potential of NCS 613 in systemic lupus. This study reveals that PDE4 represent a potential therapeutic target in lupus disease.