Demonstration, in leukemia L-1210 cells, of a phosphodiesterase acting on 3':5'-cyclic CMP but not on 3':5'-cyclic AMP or 3':5'-cyclic GMP.

cCMP-specific phosphodiesterase activity was demonstrated in the 80 to 100% ammonium sulfate fraction obtained from disrupted leukemia L-1210 cells. The activity was linear with time (up to 60 min), was a function of protein concentration, and was markedly stimulated by Mg2+ and by ammonium sulfate. Under identical assay conditions, no significant hydrolysis of cAMP or cGMP was observed, although these cyclic nucleotides served as substrates for phosphodiesterase(s) present in all the fractions obtained by less than 80% ammonium sulfate saturation. This is the first demonstration of a cCMP-specific phosphodiesterase.     

An effect of glucagon on 3', 5'-cyclic AMP phosphodiesterase activity in isolated rat hepatocytes.

Glucagon was found to activate the low Km form of 3′,5′-cyclic AMP phosphodiesterase in intact isolated rat hepatocytes while the high Km phosphodiesterase was unaltered. Activation was concentration dependent and occurred at the same concentration required to observe an increase in 3′,5′-cyclic AMP levels in the cell. The maximal increase in activity occurred within 5 minutes of incubation with glucagon and was sustained for the 35 minutes assayed.     

Content of 3',5' cyclic AMP and cyclic AMP phosphodiesterase in dormant and activated tissues of Jerusalem artichoke tubers

Using an highly sensitive and specific radioimmunoassay for 3′,5′ cyclic AMP, we have detected this cyclic nucleotide in partially purified extracts obtained from tuber tissues of Jerusalem artichoke. The level of cyclic AMP found in dormant tubers was very high compared with those of sprouting tubers and rapidly declines when dormant tissue slices are activated by incubation in aerated water. Cyclic AMP phosphodiesterase seems to play an important role in these changes of cyclic AMP content.     

Differences and similarities between guanosine 3',5'-cyclic monophosphate phosphodiesterase and adenosine 3',5'-cyclic monophosphate phosphodiesterase activities in neuroblastoma cells in culture.

There are phosphodiesterase activities in both particulate and supernatant fractions which hydrolyze guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) with an apparent Km of 2-8 muM and with an apparent Km of 44-222 muM. 4-(3-Butoxy-4-methoxybenzyl-2-imidazolidinone (RO20-1724) did not inhibit cGMP phosphodiesterase activity in homogenates of mouse neuroblastoma cells, but markedly inhibited cAMP phosphodiesterase activity. Papaverine and theophylline inhibited both cGMP and cAMP phosphodiesterase activities to about the same extent. The former was more potent than the latter. The specific activity of cGMP phosphodiesterase as a function of protein concentrations first increased and then decreased. The specific activity of cAMP phosphodiesterase decreased under a similar experimental condition.     

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.     

Chlorpropamide and tolbutamide inhibition of adenosine 3'5' cyclic monophosphate phosphodiesterase

These sulfonylurea agents inhibit the cyclic AMP phosphodiesterase, and thereby could increase the steady state level of cyclic AMP in various tissues, depending upon the tissue concentrations achieved after oral or parental administration.     

Zinc-mediated inhibition of cyclic nucleotide phosphodiesterase activity and expression suppresses TNF-alpha and IL-1 beta production in monocytes by elevation of guanosine 3',5'-cyclic monophosphate

The trace element zinc affects several aspects of immune function, such as the release of proinflammatory cytokines from monocytes. We investigated the role of cyclic nucleotide signaling in zinc inhibition of LPS-induced TNF-alpha and IL-1beta release from primary human monocytes and the monocytic cell line Mono Mac1. Zinc reversibly inhibited enzyme activity of phosphodiesterase-1 (PDE-1), PDE-3, and PDE-4 in cellular lysate. It additionally reduced mRNA expression of PDE-1C, PDE-4A, and PDE-4B in intact cells. Although these PDE can also hydrolyze cAMP, only the cellular level of cGMP was increased after incubation with zinc, whereas cAMP was found to be even slightly reduced due to inhibition of its synthesis. To investigate whether an increase in cGMP alone is sufficient to inhibit cytokine release, the cGMP analogues 8-bromo-cGMP and dibutyryl cGMP as well as the NO donor S-nitrosocysteine were used. All three treatments inhibited TNF-alpha and IL-1beta release after stimulation with LPS. Inhibition of soluble guanylate cyclase-mediated cGMP synthesis with LY83583 reversed the inhibitory effect of zinc on LPS-induced cytokine release. In conclusion, inhibition of PDE by zinc abrogates the LPS-induced release of TNF-alpha and IL-1beta by increasing intracellular cGMP levels.     

Quantitative estimation of 3'5' cyclic AMP phosphodiesterase using anion exchange resin in a batch process.

An improved method for the preparation of adenosine 3′-phosphate 5′-phosphosulfate (PAPS) is described which includes: enzymes from Chlorella for PAPS synthesis; conversion of ATP to AMP after PAPS formation with hexokinase (EC 2.7.1.1) and myokinase (EC 2.7.4.3); and separation of PAPS on DEAE-Sephadex using triethylammonium bicarbonate buffers. Any specific activity can be obtained by using appropriate concentrations of carrier-free ³⁵S and nonradioactive sulfate in the incubations. Between 300 and 2000 μmol of PAPS per batch can be obtained depending on the scale of the preparation. The PAPS is over 95% pure radiochemically and shows only one ultraviolet-absorbing spot on paper electrophoresis at pH 5.8. Adenosine 5′-phosphosulfate (APS) is prepared by incubating PAPS with a 3′-nucleotidase (EC 3.1.3.6) from rye grass. Quantitative conversion of PAPS to APS is obtained, and the APS is purified by column chromatography in the same manner as for PAPS. The APS obtained is better than 95% pure radiochemically and shows only one uv-absorbing spot on paper electrophoresis at pH 5.8.     

Insulin-sensitive adenosine 3',5'-cyclic monophosphate phosphodiesterase of hepatocyte plasma membrane.

Adenosine 3',5'-cyclic monophosphate phosphodiesterase (EC 3.1.4.17) has been investigated in rat liver as to its insulin sensitivity. Hormone action has been assayed in vitro on a liver homogenate purified by DEAE-cellulose column chromatography, on isolated hepatocytes, on isolated plasma membranes. The DEAE-cellulose chromatography purified homogenate showed no sensitivity to insulin, whereas isolated hepatocytes incubated in presence of insulin showed increased phosphodiesterase activity in a plasma membrane-containing fraction. The plasma membrane-bound enzyme, which shows both high and low affinity components, was significantly stimulated after hormonal treatment; this effect being dependent on a V increase of the low Km form.     

Inhibition of adenosine-3',5'-cyclic monophosphate phosphodiesterase by potential antiallergic compounds.

The potential antiallergic compounds doxantrazole (3- (5-tetrazolyl)-thioxanthone 10, 10-dioxide) and CTD (3- carboxythioxanthone 10, 10-dioxide) are inhibitors of the phosphodiesterases of human and guinea pig lung and beef heart. Disodium cromoglycate is a weak inhibitor of all these enzymes. It is suggested that the antiallergic activity of doxantrazole and CTD is due, at least in part, to their ability to elevate intracellular cAMP levels by inhibiting phosphodiesterase activity.     

Cyclic 3'.5'-nucleotide phosphodiesterase. Effect of binding protein on the hydrolysis of cyclic AMP

The rate of cyclic AMP hydrolysis by a cyclic 3′,5′-nucleotide phosphodiesterase was diminished by the presence of a cyclic AMP binding protein in the reaction mixture. The reduction was proportional to the concentration of the binding protein; and was more pronounced at 0° than at 30°, presumably because the affinity of cyclic AMP to the binding protein was greater at 0° (“apparent dissociation constant” = 3 × 10⁻⁸ M) than at 30° (“apparent dissociation constant” = 4 × 10⁻⁷ M). These experiments indicate that cyclic AMP bound to the binding protein is not susceptible to the action of phosphodiesterase. It is hydrolyzed only when dissociated from the protein, and the rate of dissociation appears to be the limiting factor. The possible physiological significance of these results is discussed.     

Stereochemistry of hydrolysis of adenosine 3':5'-cyclic phosphorothioate by the cyclic phosphodiesterase from beef heart.

Adenosine 3':5'-cyclic phosphorothioate, Sp-diastereomer was hydrolyzed by cyclic phosphodiesterase from beef heart in the presence of [18O]water to [18O]adenosine 5'-phosphorothioate. This was phosphorylated by myokinase and pyruvate kinase to [18O]adenosine 5'-(1-thiotriphosphate),Sp-diastereomer. The position of 18O was determined to be in a nonbridging position. This result indicates that the hydrolysis proceeded with inversion of configuration at phosphorus.