Cyclic nucleotide phosphodiesterase activity in Phaseolus vulgaris

Centrifugal fractionation showed that 70% of the cyclic nucleotide phosphodiesterase activity of Phaseolus vulgaris seedlings is recovered in the 1     

The molecular basis for different recognition of substrates by phosphodiesterase families 4 and 10

Phosphodiesterases (PDEs) are key enzymes that control the cellular concentrations of the second messengers cAMP and cGMP. The mechanism for selective recognition of substrates cAMP and cGMP by individual PDE families remains a puzzle. To understand the mechanism for substrate recognition by PDE enzymes, the crystal structure of the catalytic domain of an inactive D201N mutant of PDE4D2 in complex with substrate cAMP has been determined at 1.56 A resolution. The structure shows that Gln369 forms only one hydrogen bond with the adenine of cAMP. This finding provides experimental evidence against the hypothesis of two hydrogen bonds between the invariant glutamine and the substrate cAMP in PDE4, and thus suggests that the widely circulated "glutamine switch" model is unlikely the mechanism for substrate recognition by PDEs. A structure comparison between PDE4D2-cAMP and PDE10A2-cAMP reveals an anti configuration of cAMP in PDE4D2 but syn in PDE10A2, in addition to different contact patterns of cAMP in these two structures. These observations imply that individual PDE families have their characteristic mechanisms for substrate recognition.     

mRNA expression and antilipolytic role of phosphodiesterase 4 in rat adipocytes in vitro

Adipocyte lipolysis is dependent on an increase in the intracellular concentration of cAMP. Intracellular phosphodiesterases (PDEs) hydrolyze cAMP and limit stimulation of lipolysis. In the present study, the mRNA expression of PDE4 subtypes and the antilipolytic role of PDE4 in rat adipocytes were investigated. Fragments encoding PDE4A (233 bp), PDE4B (786 bp), PDE4C (539 bp), and PDE4D (262 bp) sequences were amplified by RT-PCR. The mRNA expression of PDE4 subtypes (A, B, C, D) determined by real-time quantitative PCR was 7, 18.7, 18.9, and 7.2% relative to PDE3B. Inhibition of PDE4 by rolipram increased basal lipolysis and reversed in part prostaglandin E2 antilipolysis. The combination of PDE3 and PDE4 inhibitors synergistically reversed both prostaglandin E2 and phenylisopropyl adenosine antilipolysis. Stimulation of adipocytes with prostaglandin E2 increased total PDE activity and PDE3 activity measured by hydrolysis of 3[H]cAMP by the particulate fraction of adipocytes. The present study confirmed that mRNAs for all four PDE4 subtypes were expressed in rat adipocytes, with PDE4B and PDE4C predominant. Moreover, PDE4 not only limits the rate of basal lipolysis but also contributes to prostaglandin E2 antilipolysis in rat adipocytes.     

Molecular cloning and expression pattern of rpr-1, a resiniferatoxin-binding, phosphotriesterase-related protein, expressed in rat kidney tubules

Bacterial phosphotriesterases are enzymes that hydrolyse phosphotriester-containing organophosphate pesticides. Resiniferatoxin is a vanilloid that desensitises nociceptive neurons. By screening a rat cDNA library with labelled resiniferatoxin, we unexpectedly isolated a novel rat phosphotriesterase homologue, here named rpr-1, that encodes a 349 amino acid, 39 kDa protein (confirmed by in vitro translation). Northern blotting and in situ hybridisation show expression primarily in proximal tubules of the kidney, in which rpr-1 distribution correlates with resiniferatoxin-binding activity. These results suggest an unsuspected link between the phosphotriesterase enzyme family and resiniferatoxin toxicity and pharmacology.     

Developmental and regional quantitation of glycerophosphorylcholine phosphodiesterase activities in rat brain

The activity of glycerophosphorylcholine cholinephosphodiesterase was quantified in the diencephalon, mesencephalon, cerebral hemispheres, olfactory bulb and cerebellum postnatally for P5 until P70 of rat brain. The initially low activities gradually increase to adult levels by P30. The patterns of regional development are reminescent of those previously described for choline acetyltransferase activity. It is suggested that these may be functionally linked in neuronal cells. The activity of glycerophosphorylcholine phosphocholine phosphodiesterase was also determined and found to be similar although only one half as active as the enzyme liberating choline. The present experiments show that both the GPC phosphocholine phosphodiesterase and the GPC choline phosphodiesterase are regionally and developmentally regulated in rat brain.     

A further study on the regulation of cyclic nucleotide phosphodiesterase activity in neuroblastoma cells: Effect of growth

Summary Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) phosphodiesterase activity in mouse neuroblastoma cells in culture markedly increased during exponential growth and reached a maximal level at confluency; whereas guanosine 3′, 5′-cyclic monophosphate (cyclic GMP) phosphodiesterase activity only slightly but significantly increased under a similar experimental condition. The increase in cyclic AMP phosphodiesterase activity was blocked by both cycloheximide and dactinomycin, whereas the increase in cyclic GMP phosphodiesterase was blocked by only cycloheximide. When the confluent cells were replated at low density, the cyclic nucleotide phosphodiesterase activity decreased; however, when they were plated at high cell density which equaled confluency, the enzyme activity did not decrease. Unlike cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity did not change significantly in prostaglandin E₁-treated cells, but decreased in cells treated with the inhibitor of phosphodiesterase. Like cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity also did not change in cells treated with serum-free medium, X-irradiation, sodium butyrate and 6-thioguanine. This work was supported by USPHS NS-09230, and DRG-1273 from Damon Runyon-Walter Winchell Cancer Fund.     

Studies On Cyclic Nucleotide Metabolism In Tetrahymena Pyriformis: Partial Characterization of Cyclic Amp- and Cyclic Gmp-Dependent Phosphodiesterases*

SYNOPSIS. Cyclic nucleotide phosphodiesterase [EC 3.1.4.17] was examined in Tetrahymena pyriformis strain NT-1. Enzymic activity was associated with the soluble and the particulate fractions, whereas most of the cyclic GMP phosphodiesterase activity was localized in the soluble fraction: the activities were optimal at pH 8.0–9.0. Although very low activities were detected in the absence of divalent cations, they were significantly increased by the addition of either Mg²⁺ or Mn^2-. A kinetic analysis of the properties of the enzymes yielded 2 apparent K_III values ranging in concentration from 0.5 to 50 μM and from 0.1 to 62 μ M for cyclic AMP and GMP. respectively. A Ca²⁺-dependent activating factor for cyclic nucleotide phosphodiesterase was extracted from Tetrahymena cells, but this factor did not stimulate guanylate cyclase [EC 4.6.1.2] activity in this organism. On the other hand, Tetrahymena also contained a protein activator which stimulated guanylate cyclase in the presence of Ca²⁺, although this activator did not stimulate the phosphodiesterase. the results suggested that Tetrahymena might contain 2 types of Ca²⁺-dependent activators, one specific for phosphodiesterase and the other for guanylate cyclase.     

Cyclic adenosine 3',5'-monophosphate phosphodiesterase from Mucor rouxii: regulation of enzyme activity by phosphorylation and dephosphorylation.

Crude preparations of cyclic adenosine 3′, 5′-monophosphate phosphodiesterase were activated 1.5 to 2 fold by incubation with ATP, Mg²⁺ and cyclic AMP in a reaction which was both, time and temperature dependent. Cyclic AMP phosphodiesterase remained in an activated state upon filtration of the enzymatic preparation through Sephadex G-25 and ion-exchange chromatography. Activation of the enzyme in the presence of [γ ³²P]ATP resulted in a significant amount of [³²P] protein-bound radioactivity. Reversible deactivation of cyclic AMP phosphodiesterase was enhanced by Mg²⁺ and was accompanied by the release of [³²P] protein bound radioactivity. The evidence is consistent with a mechanism for controlling cyclic AMP phosphodiesterase through phosphorylation-dephosphorylation sequence.     

Rapid changes in the activities of the enzymes of cyclic AMP metabolism after addition of A23187 to macrophages

The ionophore A23187 stimulated adenylate cyclase activity in intact macrophages within 1 min. This action was blocked by pretreatment with indomethacin (25 μmol/l) suggesting the involvement of a prostaglandin (PG). PGE₂ (500 nmol/l) also stimulated adenylate cyclase activity in intact cells, but this was not prevented by indomethacin pretreatment. Colchicine (100 μmol/l) potentiated the increases in macrophage cyclic AMP production seen after addition of PGE₂ or A23187. The high affinity form of cyclic AMP phosphodiesterase (PDE) was activated within 1 min of the addition of A23187 to intact macrophages. The data suggest that the increase in macrophage cyclic AMP production after A23187 is a consequence of adenylate cyclase activation and not PDE inhibition. The endogenous production of a prostaglandin probably mediates this effect of A23187, emphasizing the importance of arachidonic acid metabolites in the regulation of macrophage functions.     

Synthesis and biological evaluation of new fluorinated and chlorinated indenoisoquinoline topoisomerase I poisons

Fluorine and chlorine are metabolically stable, but generally less active replacements for a nitro group at the 3-position of indenoisoquinoline topoisomerase IB (Top1) poisons. A number of strategies were employed in the present investigation to enhance the Top1 inhibitory potencies and cancer cell growth inhibitory activities of halogenated indenoisoquinolines. In several cases, the new compounds’ activities were found to rival or surpass those of similarly substituted 3-nitroindenoisoquinolines, and several unusually potent analogs were discovered through testing in human cancer cell cultures. A hydroxyethylaminopropyl side chain on the lactam nitrogen of two halogenated indenoisoquinoline Top1 inhibitors was found to also impart inhibitory activity against tyrosyl DNA phosphodiesterases 1 and 2 (TDP1 and TDP2), which are enzymes that participate in the repair of DNA damage induced by Top1 poisons.     

Optogenetic manipulation of cyclic guanosine monophosphate to probe phosphodiesterase activities in megakaryocytes

Cyclic guanosine monophosphate (cGMP) signalling plays a fundamental role in many cell types, including platelets. cGMP has been implicated in platelet formation, but mechanistic detail about its spatio-temporal regulation in megakaryocytes (MKs) is lacking. Optogenetics is a technique which allows spatio-temporal manipulation of molecular events in living cells or organisms. We took advantage of this method and expressed a photo-activated guanylyl cyclase, Blastocladiella emersonii Cyclase opsin (BeCyclop), after viral-mediated gene transfer in bone marrow (BM)-derived MKs to precisely light-modulate cGMP levels. BeCyclop-MKs showed a significantly increased cGMP concentration after illumination, which was strongly dependent on phosphodiesterase (PDE) 5 activity. This finding was corroborated by real-time imaging of cGMP signals which revealed that pharmacological PDE5 inhibition also potentiated nitric oxide-triggered cGMP generation in BM MKs. In summary, we established for the first-time optogenetics in primary MKs and show that PDE5 is the predominant PDE regulating cGMP levels in MKs. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology.     

磷酸二酯酶7及其抑制剂

磷酸二酯酶7(phosphodiesterase7, PDE7)作为体内特异性水解第二信使环腺苷一磷酸（cyclic adenosine monophosphate, cAMP）的一类蛋白水解酶,通过调 控组织细胞内cAMP水平及机体信号传导活动,参与了体内多种疾病的发生发展过 程.PDE7主要分布于前炎症细胞及免疫细胞.最新研究表明,PDE7作为一个新的潜在的抗动脉粥样硬化及抗炎症免疫类药物的新靶点,其抑制剂可能在研究及治疗动脉粥样硬化及慢性阻塞性肺病等免疫炎症类疾病方面具有重要意义. 本文简述PDE7基因结构、生物学功能以及其抑制剂研究,并重点就其与疾病之间联系做一综述.     

Phosphodiesterase activity of mouse sperm incubated under conditions that modulate fertilizing potential in vitro

Cyclic AMP has been implicated as a regulator of capacitation, but the control of its metabolism in sperm remains obscure. A recent study of mouse sperm has shown capacitation-related changes in the activities of both adenylate cyclase, which increased during incubation, and cyclic nucleotide phosphodiesterase, which decreased. The present study was conducted to extend these observations by measuring phosphodiesterase activity in sperm incubated in media with modified calcium and/or glucose content, conditions known to modulate fertilizing ability. Phosphodiesterase activity of sequential sperm samples, taken first when sperm are essentially uncapacitated and then when they are either partially or completely capacitated, decreased with time under all conditions, and in each case the greater fall in activity was seen in the medium that would support the greater change in fertilizing ability of the sperm population. Sperm washed by centrifugation to remove epididymal fluid also displayed a reduction in phosphodiesterase activity with time. The medium surrounding the sperm contained about half of the total phosphodiesterase activity, as well as 5′-nucleotidase and adenosine deaminase. The crude enzyme preparation showed complex kinetic behavior when assayed over a range of cAMP concentrations, but the reduction in activity with time was seen at all substrate levels. The observed changes in phosphodiesterase activity, together with the increased adenylate cyclase activity seen under these sperm incubation conditions, would increase cAMP availability with time, thus providing further evidence for a fundamental role for cAMP in controlling the events of capacitation.     

Involvement of both a Zn2+ site and an anionic binding site in the selective inhibition of a Zn2+-glycerophosphocholine cholinephosphodiesterase by thiols and tellurites

Inhibition of a Zn²⁺-glycerophosphocholine cholinephosphodiesterase by thiols or tellurites were examined mechanistically. Inactivation of the phosphodiesterase by thio-carboxylates, which was due to the removal of Zn²⁺ in the catalytic site, was enhanced by introduction of an amino group in the structure of thiols, suggesting the presence of an anionic site adjacent to a Zn²⁺ site. In support of the suggestion, it was found that thiols, associable with both a Zn²⁺ site and an anionic site, were more potent reversible inhibitors; dimethylaminoethanethiol (Ki, 17 M), diethylaminoethanethiol (Ki, 1.2 M) and thiocholine (Ki, 2.6 M). Meanwhile, the inhibition of the phosphodiesterase by tellurites is ascribed to the binding of tellurite anions to a Zn²⁺ site, based on the protective action of tellurite anions against the inactivation of the enzyme by EDTA. Moreover, the inhibition of the phosphodiesterase by tellurites was prevented by phosphate ions, which expressed the protective effect against EDTA inactivation. In further support, it was observed that gellurite and thiocholine appeared to interact with active site in an additive manner, in contrast to a synergistic action between tellurites and quaternary ammonium compounds such as acetylcholine or choline.     

Characterization of the Isoenzymes of cyclic nucleotide phosphodiesterase in human platelets and the effects of E4021

In extracts of human platelets, three isoenzymes of cyclic nucleotide phosphodiesterase (PDE), namely, PDE2, PDE3, and PDE5, were identified; activities of PDE1 and PDE4 were not detected. In human platelets, the cGMP-hydrolytic activity was about six times higher than the cAMP-hydrolytic activity, and PDE5 and PDE3 are the major phosphodiesterase isoenzymes that hydrolyze cGMP and CAMP, respectively. PDE5 exhibited organ-specific expression in humans, and platelets were among the tissues richest in PDE5. A novel inhibitor of PDE5, sodium 1-[6-chloro-4-(3,4-methylenedioxybenzyl)aminoquinazolin-2-yl] piperidine-4-carboxylate sesquihydrate (E4021), was a potent and highly selective inhibitor of human platelet PDE5. However, E4021 (up to 10 μM) did not inhibit 9,11-epithio-11,12-methano-thromboxane A₂-induced platelet aggregation, in vitro. E4021 plus SIN-1 (3-morpholino-sydnonimine), at concentrations that had little effect individually, inhibited aggregation. These results suggest the unique distribution of phosphodiesterase isoenzymes in human platelets and the PDE5 inhibitors might be useful as a new class of antiplatelet drugs.     

Effect of hydrocortisone on the desialylation of intestinal brush-border enzymes of the rat during postnatal development

High affinity cyclic AMP phosphodiesterase activity in preparations of acini isolated from mammary tissue of lactating rats is shown to be stimulated by the addition of physiological concentrations of insulin to incubations of acini in vitro. This effect is expressed specifically on membrane-associated phosphodiesterase and occurs in the absence of concurrent protein synthesis. The possible functional role of this aspect of insulin's action on mammary tissue is discussed and compared with the well-known reversal by this hormone of the effects of lipolytic agents in adipose tissue and liver.     

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.     

Rapid separation and quantitation of 3',5'-cyclic nucleotides and 5'-nucleotides in phosphodiesterase reaction mixtures using high-performance liquid chromatography

A simple, rapid high-performance liquid-chromatography system for the fractionation and direct quantitation of substrates and products in crude phosphodiesterase reaction mixtures is described. Phosphate buffers and a pellicular anion exchange resin are used at ambient temperature. The method is sensitive, measuring picomoles of products with ultraviolet detection and femtomoles with isotopic measurement, and offers several advantages over the more popular batch sorption and manual methods for measuring phosphodiesterase activity. The time required for analysis, less than 8 min for single substrate reaction mixtures, is a fraction of that required with other chromatographic systems, and precision is +/- 5%. Results of studies with an activatable form of phosphodiesterase demonstrate the accuracy, precision and utility of the procedure for biochemical analyses.