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.     

Regulation of cyclic adenosine 3':5'-monophosphate phosphodiesterases: altered pattern in transformed myoblasts

Rat skeletal myoblasts, L6 and L8, have two major forms of phosphodiesterases, PDE II and PDE III. Only the former is activated by treatment with proteases. When the myoblasts are exposed to cAMP for 10-16 h, the activity of PDE III increases considerably. This increase is accompanied by a loss of activatability of PDE II by proteases. Leupeptin prevents the increase in the levels of PDE III suggesting that a protease in vivo may be responsible for the formation of PDE III from PDE II. Spontaneously or Rous sarcoma virus-transformed myoblasts, however, show altered regulation of the two forms of PDE. In the presence of cAMP in the medium, unlike the nontransformed cells, the levels of PDE III do not increase but the activity of PDE II rises. Simultaneously, PDE II becomes refractory to activation by proteases. The altered mode of PDE regulation in transformed cells is dominant in hybrids between normal and transformed myoblasts, which suggests that altered regulation is due to an "acquisition" of some new property by transformed cells.     

Isolation, characterization and Ca2+ and calmodulin regulation of cyclic nucleotide phosphodiesterases from human placentae of different ages

Two kinds of phosphodiesterases were isolated from human placenta by DEAE chromatography and characterized: one Ca2+ and calmodulin dependent, the other stimulated by Ca2+ but not by calmodulin. Both hydrolyzed cAMP and cGMP. The first one exhibited a higher affinity for cGMP. Half maximal activation by calmodulin was attained at 10(-8)M of calmodulin concentration independently of the hydrolyzed substrate (cGMP or cAMP). This phosphodiesterase appears to be almost homogeneous by molecular sieve chromatography on Ultragel AcA 34. The second phosphodiesterase exhibited similar affinities for cAMP and cGMP and could be resolved into three active isoforms with different molecular weight on Ultrogel AcA 34. Only minor differences were observed in the characteristics of these enzymes when the phosphodiesterases were prepared from placentae of 7-8 weeks of pregnancy or from normal term placenta.     

Multiple forms of cyclic nucleotide phosphotransferases in mouse thymocytes

The presence of cAMP- and cGMP- specific phosphodiesterases in mouse thymocytes has been demonstrated. Sucrose density gradient centrifugation revealed two peaks corresponding to cAMP-phosphodiesterase (3.6S and 6.0S) and one peak corresponding to cGMP-phosphodiesterase (6.6S). Gel filtration demonstrated high molecular forms of the enzymes; according to the rechromatography data, cAMP and cGMP phosphodiesterases are represented by oligomers containing subunits with Ms 45 000 and 160 000 respectively. Incubation of thymocyte lysates for 24-48 hours led to a decrease of the number of high molecular weight forms and an increase in the number of low molecular weight forms paralleled with a rise in V and Km values.     

A new cGMP phosphodiesterase isolated from bovine platelets is substrate for cAMP- and cGMP-dependent protein kinases: evidence for a key role in the process of platelet activation.

The biochemical differences among cGMP phosphodiesterases in platelets have not been thoroughly examined, primarily due to the lack of sufficient purified material. This report describes a simple method developed to isolate a specific bovine platelet cGMP phosphodiesterase. This enzyme is cytosolic in its native form and was purified to an apparent homogeneity by ion-exchange chromatography, affinity chromatography, and density gradient centrifugation. Cyclic GMP binds to a "pseudo-site" when the catalytic site is deprived of Mg++. The affinity for cGMP at alkaline pH in presence of EDTA and IBMX (Kd = 60 nM) suggests that the removal of Mg++ by EDTA converts the catalytic site to a binding site. A ligand affinity chromatography was designed to take advantage of these features. The core enzyme has a molecular weight 190,000 composed of 2 subunits (MW 95,000) and has a specific activity of 2.5 mumol/min/mg. Moreover, this enzyme was phosphorylated by cAMP- and cGMP-dependent protein kinases, suggesting that its activity could be indirectly regulated by cyclic nucleotides. Agents elevating cGMP and cAMP inhibit platelet activation by inhibiting protein kinase C and thrombin induced hydrolysis of phosphatidylinositol 4,5 diphosphate. The antiaggregating properties of some of these agents might therefore be attributed to the fact that they are inhibitors of phosphodiesterases.     

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.     

Cardiac cyclic nucleotide phosphodiesterases: function, regulation, and therapeutic prospects

The second messengers cAMP and cGMP exist in multiple discrete compartments and regulate a variety of biological processes in the heart. The cyclic nucleotide phosphodiesterases, by catalyzing the hydrolysis of cAMP and cGMP, play crucial roles in controlling the amplitude, duration, and compartmentalization of cyclic nucleotide signaling. Over 60 phosphodiesterase isoforms, grouped into 11 families, have been discovered to date. In the heart, both cAMP- and cGMP-hydrolyzing phosphodiesterases play important roles in physiology and pathology. At least 7 of the 11 phosphodiesterase family members appear to be expressed in the myocardium, and evidence supports phosphodiesterase involvement in regulation of many processes important for normal cardiac function including pacemaking and contractility, as well as many pathological processes including remodeling and myocyte apoptosis. Pharmacological inhibitors for a number of phosphodiesterase families have also been used clinically or preclinically to treat several types of cardiovascular disease. In addition, phosphodiesterase inhibitors are also being considered for treatment of many forms of disease outside the cardiovascular system, raising the possibility of cardiovascular side effects of such agents. This review will discuss the roles of phosphodiesterases in the heart, in terms of expression patterns, regulation, and involvement in physiological and pathological functions. Additionally, the cardiac effects of various phosphodiesterase inhibitors, both potentially beneficial and detrimental, will be discussed.     

Changes in adenylate cyclase,cyclic AMP,and protein kinase levels in chick myoblasts,and their relationship to differentiation

Examination of components of the cAMP system in primary cultures of differentiating chick myoblasts revealed a basal intracellular cAMP level of 50–100 pmole/mg of DNA, which increased ten to fifteen-fold for approximately 1 hr between 37.5 and 39.5 hr of culture, only 5–6 hr before the initiation of myoblast fusion. Activities of the enzymes adenylate cyclase and protein kinase were examined during the initial stages of myoblast differentiation. Both the basal activity and the degree of NaF stimulation of adenylate cyclase increased during the time examined, the appearance of these changes coinciding in time of culture with the observed peak of cAMP. The protein kinase present was sensitive to cAMP, and its basal and cAMP stimulated activities increased throughout the prefusion period of culture. The results suggest a causal relationship between the increase in adenylate cyclase activities, the increase in intracellular cAMP, and the onset of fusion; and the possibility that intracellular cAMP levels control the expression of myoblast differentiation is discussed.     

cGMP-cAMP interplay in cardiac myocytes: a local affair with far-reaching consequences for heart function

cAMP and cGMP signalling pathways are common targets in the pharmacological treatment of heart failure, and often drugs that modulate the level of these second messengers are simultaneously administered to patients. cGMP can potentially affect cAMP levels by modulating the activity of PDEs (phosphodiesterases), the enzymes that degrade cyclic nucleotides. This biochemical cross-talk provides the means for drugs that increase cGMP to concomitantly affect cAMP signals. Recent studies using FRET (fluorescence resonance energy transfer) reporters and real-time imaging show that, in cardiac myocytes, the interplay between cGMP and cAMP has different outcomes depending on the specific location where the cross-modulation occurs. cGMP can either increase or decrease the cAMP response to catecholamines, based on the cyclase that generates it and on the PDEs associated with each subcellular compartment. cGMP-mediated modulation of cAMP signals has functional relevance as it affects protein phosphorylation downstream of protein kinase A and myocyte contractility. The physical separation of positive and negative modulation of cAMP levels by cGMP offers the previously unrecognized possibility to selectively modulate local cAMP signals to improve the efficacy of therapy.     

Inhibition of myoblast differentiation in vitro by a protein isolated from liver cell medium

We have recently discovered that cells of Coon's Buffalo rat liver (BRL) line secrete a protein which is a potent inhibitor of skeletal myoblast differentiation in vitro. Using ion exchange and molecular exclusion chromatography, we have prepared this protein, which we designate "differentiation inhibitor" (DI), from the materials secreted by BRL cells maintained in serum-free medium. It is a relatively heat- stable protein which is inactivated by treatment with trypsin and mercaptoethanol and has an apparent molecular weight in the range 30,000--36,000. It exhibits no detectable mitogenic or lectin activity and differs from previously reported inhibitors of myoblast differentiation in several respects. It is active in all skeletal myoblast systems tested (Yaffe's L6 line as well as primary cultures of rat, chick, and Japanese quail myoblasts), and it blocks fusion, elevation of creatine kinase, and increased binding of alpha- bungarotoxin. Parallel fractionation of fetal bovine serum (FBS) and chick embryo extract (CEE) yields a peak of activity which similarly inhibits myoblast differentiation. We suggest that the differentiation inhibitor from BRL cells may correspond to the differentiation- inhibiting component(s) of FBS and CEE, and we call attention to the possibility that such a substance could play a role in embryonic growth of myoblasts and in satellite cell formation.     

Rat sperm enzymes during epididymal transit

The activities of cAMP and cGMP phosphodiesterases (EC 3.1.4.1), adenylate cyclase (EC 4.6.1.1) and protein carboxyl-methylase (EC 2.1.1.24) were measured in the particulate and soluble (105 000 g supernatant) fractions of washed spermatozoa isolated from five segments of the adult rat epididymis. The activities of both phosphodiesterases decreased during epididymal transit, whereas adenylate cyclase and protein carboxyl-methylase underwent a progressive increase, the latter showing the most marked alteration. Both cAMP and cGMP phosphodiesterases as well as the adenylate cyclase were all associated primarily with the particulate fraction, and the extent to which these enzymes were associated with the membranes increased as the spermatozoa passed through the epididymis. Sperm protein carboxyl-methylase activity was, on the other hand, predominantly soluble in all segments of the epididymis. Adenylate cyclase, cAMP phosphodiesterase and protein carboxyl-methylase activities were found predominantly in the sperm tails, whereas cGMP phosphodiesterase was equally distributed between heads and tails. These observations imply that the acknowledged increase in intracellular cAMP levels which occurs in spermatozoa during epididymal transit may be a consequence of both increased synthesis (adenylate cyclase) and reduced hydrolysis (phosphodiesterase).     

Reconstitution of cells by fusion of cell fragments : I. Myogenic expression after fusion of minicells from rat myoblasts (L6) with mouse fibroblast (A9) cytoplasm

Rat L6 myoblasts and mouse A9 fibroblasts (HGPRT-) were enucleated by centrifugation of monolayers in the presence of cytochalasin B. Intact cells were reconstituted by Sendai virus mediated fusion of nuclei (minicells) from enucleated rat myoblasts and cytoplasms from enucleated mouse fibroblasts. Colonies arising from proliferating reconstituted cells were distinguished from intact parental cell types on the basis of nuclear and cytoplasmic markers. In five replicate experiments, approx. 70% of all colonies found after fusion were derived from reconstituted cells, 30% arose from intact rat myoblasts contaminating the minicell preparations, and two colonies were identified as hybrids between the parental cell types. Clones derived from reconstituted cells formed myotubes which produced myosin and developed the cross-striated pattern typical of skeletal muscle. The myogenic program of the rat myoblast thus can persist through the enucleation and reconstitution procedures, and is not obviously altered by a period of exposure to mouse fibroblast cytoplasm.     

Substrate and effector specificity of a guanosine 3':5'-monophosphate phosphodiesterase from rat liver.

Guanosine 3':5'-monophosphate phosphodiesterases, which appear to be under allosteric control, have been partially purified from rat liver supernatant and particulate fractions. The preferred substrate for both phosphodiesterases was cGMP (Km values: cGMP less than cIMP less than cAMP). At subsaturating concentrations of substrate, the phosphodiesterases were stimulated by purine cyclic nucleotides. The order of effectiveness for activation of cyclic nucleotide hydrolysis was cGMP greater than cIMP greater than cAMP greater than cXMP. Using cAMP derivatives as activators of cIMP hydrolysis, modifications in the ribose, cyclic phosphate, and purine moieties were shown to alter the ability of the cyclic nucleotide to activate the supernatant enzyme. cGMP, at concentrations that stimulated cyclic nucleotide hydrolysis, enhanced chymotryptic inactivation of the supernatant phosphodiesterase. At similar concentrations, cAMP was not effective. It appears that on interaction with appropriate cyclic nucleotides, this phosphodiesterase undergoes conformational changes that are associated with increased catalytic activity and enhanced susceptibility to proteolytic attack. Divalent cation may not be required for the nucleotide-phosphodiesterase interaction and resultant change in conformation.     

Pattern of polypeptide synthesis in myoblast hybrids

We have examined cell hybrids derived from L6J1 rat myoblasts and A9 mouse fibroblastic cells for expression of the myogenic phenotype. Initial results showed that hybrid cells were no longer able to form myotubes and hence showed extinction of the myogenic phenotype. We then proceeded to characterize the pattern of protein synthesis in these cells using two-dimensional gel electrophoresis. Although we did detect extinction of synthesis of a small number of myoblast polypeptides in the hybrids these did not appear to be rat myoblast specific. Instead they correlated well with polypeptides lost upon viral transformation in another rat cell line. Analysis of the ability of parental cells and hybrids to grow in soft agar confirmed that both A9 cells and hybrids were more transformed than the parental L6J1 cells. The results are consistent with the interpretation that extinction of the ability to form myotubes is due to either transformation and/or a disrupted cell organization but is unlikely to be due to specific extinction of myoblast specific polypeptides, at least at the level detectable by 2D gel electrophoresis.     

New messages from old messengers: cAMP and mycobacteria

Cyclic nucleotides are ancient second messengers, and the enzymes that synthesize cAMP and cGMP [cyclic nucleotide monophosphates (cNMPs)] are encoded in the genomes of several bacteria. We focus here on recent biochemical and structural information on the proteins that make and break cyclic nucleotides in mycobacteria, namely the nucleotide cyclases and phosphodiesterases, respectively. The presence of these enzymes along with putative cNMP-binding proteins suggests an intricate regulation of cAMP metabolism and utilization by these organisms. It is anticipated that future research will be directed towards identifying cellular processes that are regulated by cAMP in mycobacteria and deciphering the cross-talk between mycobacterial pathogens and their eukaryotic host.     

Seven Dictyostelium discoideum phosphodiesterases degrade three pools of cAMP and cGMP

The Dictyostelium discoideum genome uncovers seven cyclic nucleotide PDEs (phosphodiesterases), of which six have been characterized previously and the seventh is characterized in the present paper. Three enzymes belong to the ubiquitous class I PDEs, common in all eukaryotes, whereas four enzymes belong to the rare class II PDEs that are present in bacteria and lower eukaryotes. Since all D. discoideum PDEs are now characterized we have calculated the contribution of each enzyme in the degradation of the three important pools of cyclic nucleotides: (i) extracellular cAMP that induces chemotaxis during aggregation and differentiation in slugs; (ii) intracellular cAMP that mediates development; and (iii) intracellular cGMP that mediates chemotaxis. It appears that each cyclic nucleotide pool is degraded by a combination of enzymes that have different affinities, allowing a broad range of substrate concentrations to be degraded with first-order kinetics. Extracellular cAMP is degraded predominantly by the class II high-affinity enzyme DdPDE1 and its close homologue DdPDE7, and in the multicellular stage also by the low-affinity transmembrane class I enzyme DdPDE4. Intracellular cAMP is degraded by the DdPDE2, a class I enzyme regulated by histidine kinase/phospho-relay, and by the cAMP-/cGMP-stimulated class II DdPDE6. Finally, basal intracellular cGMP is degraded predominantly by the high-affinity class I DdPDE3, while the elevated cGMP levels that arise after receptor stimulation are degraded predominantly by a cGMP-stimulated cGMP-specific class II DdPDE5. The analysis shows that the combination of enzymes is tuned to keep the concentration and lifetime of the substrate within a functional range.     

Characterization of phosphodiesterase catalytic sites by means of cyclic nucleotide derivatives

Cyclic nucleotide derivatives have been used as a tool to characterize distinct catalytic sites on phosphodiesterase enzyme forms: the cGMP-stimulated enzyme from rat liver and the calmodulin-sensitive enzyme from rat or bovine brain. Under appropriate assay conditions, the analogues showed linear competitive inhibition with respect to cAMP (adenosine 3',5'-monophosphate) as substrate. The inhibition sequence of the fully activated cGMP-stimulated phosphodiesterase was identical to the inhibition sequence of the desensitized enzyme, i.e. the enzyme which has lost its ability to be stimulated by cGMP. The inhibition pattern could, therefore, not be attributed to competition with cGMP at an allosteric-activating site. Also, the inhibition sequence of the calmodulin-sensitive phosphodiesterase was maintained whether activity was basal or fully stimulated by calmodulin. When cAMP and cGMP, with identical chemical ligands substituted at the same position, were compared as inhibitors of the calmodulin-sensitive phosphodiesterase, the cGMP analogues were always the more potent suggesting that, for that enzyme, the catalytic site was sensitive to a guanine-type cyclic nucleotide structure. Comparing the two phosphodiesterases, it was possible to establish both similar and specific inhibitor potencies of cyclic nucleotide derivatives. In particular, the two enzymes exhibited large differences in analogue specificity modified at C-6, 6-chloropurine 3',5'-monophosphate or purine 3',5'-monophosphate.     

An intracellular modulator of the 3'5'-cGMP hydrolysis in growing Dictyostelium discoideum amoebae

A heat-stable and acid-stable macromolecular factor present in the cytosol of growing Dictyostelium discoideum amoebae affects specifically the intracellular cGMP phosphodiesterase. It decreases the V of the enzyme but does not alter its Km. It has no effect on the cAMP or cGMP hydrolysis catalyzed by the intracellular cAMP-cGMP phosphodiesterases or by the extracellular phosphodiesterase. It is also expressed in a mutant (HPX235), defective in the synthesis of the cAMP-cGMP phosphodiesterases but capable of intracellular transduction of the chemotactic signal. This factor is resistant to several nucleases, proteases and phospholipases, and has an apparent molecular weight between 3500-10000. In contrast, the protein phosphodiesterase inhibitor secreted by the amoebae exerts an opposite inhibition on the intracellular phosphodiesterases. These two inhibitory factors may regulate intracellular cGMP hydrolysis during the chemotactic response.     

Characterization of soluble uterine cyclic nucleotide phosphodiesterase.

Soluble cyclic nucleotide phosphodiesterase of rat uterus displays distinct structural and regulatory properties. Like phosphodiesterases from many mammalian sources the soluble uterine enzyme system exhibits nonlinear Lineweaver--Burk kinetics with cyclic adenosine 3':5'-monophosphate (cAMP) as substrate (apparent Kms congruent to 3 and 20 micron) and linear kinetics with cyclic guanosine 3':5'-monophosphate (cGMP) as substrate (apparent Km congruent to 3 micron). Unlike most other mammalian phosphodiesterases, however, numerous separation procedures reveal only a single form of uterine phosphodiesterase which catalyzes the hydrolysis of both cAMP and cGMP. A single form of the enzyme is observed upon sucrose gradient centrifugation (7.9 S), agarose gel filtration, and DEAE-cellulose chromatography at either pH 8.0 OR 6.0. Heat denaturation (50 degrees C) of soluble uterine phosphodiesterase causes the loss of both cAMP and cGMP hydrolytic activities at the same rate. Isoelectric focusing reveals major (pI = 5.2) and minor forms (pI = 5.8) of phosphodiesterase which both catalyze the hydrolysis of the two cyclic nucleotide substrates. In vivo administration of estradiol produces identical decreases in the activities of cAMP and cGMP phosphodiesterase. These results raise the possibility that the uterus contains a single form of soluble phosphodiesterase which catalyzes the hydrolysis of both cAMP and cGMP.