Cyclic nucleotide phosphodiesterase. Partial purification and characterization of a high affinity enzyme activity from human lung tissue.

The rates of enzymatic hydrolysis of cyclic AMP, cyclic GMP, cyclic IMP, cyclic UMP, cyclic CMP and cyclic dTMP (all 3′:5′‐phosphates) have been assayed in several rat tissues. Subcellular distribution studies indicated that, in all tissues except brain and heart, most of the enzymatic activity is confined to the soluble cell fraction.The purine cyclic nucleotides are the preferred substrates for soluble 3′:5′‐cyclic nucleotide phosphodiesterase in all tissues. Cyclic AMP is a slightly better substrate than cyclic GMP and cyclic IMP in most tissues. However, spleen, lung and erythrocytes hydrolyse cyclic AMP 2–3 times faster than the other purine cyclic nucleotides. Hydrolysis of the cyclic pyrimidine nucleotides was very low or not detected in most tissues. The exceptions were cyclic UMP and cyclic dTMP hydrolysis by brain, and cyclic dTMP hydrolysis by liver.Polyacrylamide gel electrophoresis resolved the soluble cyclic nucleotide phosphodiesterase activity into a number of components. Two components, designated B and C, arc present in all tissues except brain although there are quantitative differences between tisues. These two components appear to be specific for purine cyclic nucleotides. A third form, A, is present in spleen, lung and erythrocytes. It hydrolyses all the cyclic nucleotides except cyclic CMP, although cyclic AMP is the best substrate. However, this component also has 5′‐nucleotidase activity which may indicate that A is not specific for 3′:5′‐cyclic nucleotides. A fourth component D, detected only in liver, hydrolyses cyclic AMP and cyclic dTMP at comparable rates.The electrophoretic patterns obtained for brain phosphodiesterase differ from those of all other tissues investigated and suggest that the enzyme(s) probably has markedly different properties from the cyclic nucleotide phosphodiesterases of other tissues.

The adenosine 3′,5′-monophosphate and guanosine 3′,5′-monophosphate phosphodiesterases from human lung tissue

Cyclic nucleotide phosphodiesterase activity in mammary tissue from rats in midlactation was resolved by DEAE-cellulose chromatography into three functionally distinct fractions: a Ca2+/calmodulin-stimulated cyclic GMP phosphodiesterase, a cyclic GMP-stimulated low-affinity cyclic nucleotide phosphodiesterase, and a high-affinity cyclic AMP-specific phosphodiesterase. The absolute activities and relative proportions of high- and low-affinity enzymes resemble those found, for example, in liver, as distinct from those in excitable tissues. Three functional characteristics are described which are peculiar to mammary-tissue phosphodiesterases. Firstly, the concentration of free Ca2+ required to achieve half-maximal activation of the Ca2+/calmodulin-stimulated phosphodiesterase is somewhat higher than for the analogous enzyme in other tissues; secondly, the activity of this enzyme towards cyclic AMP relative to that towards cyclic GMP is unusually low, and thirdly, the low-affinity cyclic nucleotide phosphodiesterase is inhibited by low concentrations of free Ca2+.

Comparison of cyclic nucleotide specificity of guanosine 3′,5′-monophosphate-dependent protein kinase and adenosine 3′,5′-monophosphate-dependent protein kinase

A cyclic GMP-stimulated cyclic nucleotide phosphodiesterase gene is highly expressed in the limbic system of the rat brain

Monocytes are immune cells that can differentiate into a number of cell types including macrophages, dendritic cells, and osteoclasts upon exposure to various cytokines. The phenotypes of these differentiated cells are highly heterogeneous and their differentiation can be affected by the cyclic nucleotides, 3'-5'-cyclic adenosine monophosphate (cAMP) and 3'-5'-cyclic guanosine monophosphate (cGMP). The intracellular levels of cAMP and cGMP are controlled through regulation of production by adenylyl and guanylyl cyclases and through degradation by cyclic nucleotide phosphodiesterases (PDEs). PDE inhibition and subsequent changes in cyclic nucleotide levels can alter the final phenotype of a differentiating monocyte with regards to surface marker expression, gene expression, or changes in secreted chemokine and cytokine levels. The differentiation process itself can also be either inhibited or augmented by changes in cyclic nucleotide levels, depending on the system being studied and the timing of cyclic nucleotide elevation. This chapter explores the effects of PDE inhibition and increases in cGMP and cAMP on monocytic differentiation into osteoclasts, dendritic cells, and macrophages.

Bovine aortic endothelial cells contain cyclic GMP-stimulated phosphodiesterase (PDE) regulating intracellular cyclic AMP and cyclic GMP levels. To investigate the roles of this PDE isoform for cyclic AMP hydrolysis in intact endothelial cells (EC), we used an adenine prelabeling method to determine cyclic AMP accumulation in response to agents that might produce effects that increase cyclic GMP levels. Atrial natriuretic peptide (ANP), which dramatically increased cyclic GMP accumulation, reduced cyclic AMP in cultured EC from bovine aorta with or without addition of L-isoproterenol (L-ISO), whereas sodium nitroprusside (SNP) and 8-bromo cyclic GMP had no effect. The reduction in cyclic AMP by ANP was dose dependent (> or = 1.0 nM) and rapid (significant reduction was induced in < or = 15 s) and was abolished when EC were preincubated with 3-isobutyl-1-methylxanthine (IBMX) and HL-725, both nonselective PDE inhibitors. ANP had no effects on adenylate cyclase activity, nor any direct effects on the activities of partially purified cyclic GMP-stimulated PDE isoform or cyclic AMP-specific isoform. However, cyclic AMP hydrolyzing activities of EC were enhanced when EC were pretreated with 0.1 microM ANP. ANP activates cyclic GMP-stimulated PDE and induces reduction of cyclic AMP accumulation in intact EC, which may modify cyclic GMP-dependent endothelial function involved in ANP.

1. The 3':5'-cyclic AMP phosphodiesterase in the microsomal fraction of baker's yeast is highly specific for cyclic AMP, and not inhibited by cyclic GMP, cyclic IMP or cyclic UMP. Catalytic activity is abolished by 30 micrometer-EDTA. At 30 degrees C and pH8.1, the Km is 0.17 micrometer, and theophylline is a simple competitive inhibitor with Ki 0.7 micrometer. The pH optimum is about 7.8 at 0.25 micrometer-cyclic AMP, so that over the physiological range of pH in yeast the activity changes in the opposite direction to that of adenylate cyclase [PH optimum about 6.2; Londesborough & Nurminen (1972) Acta Chem. Scand. 26, 3396-3398].2. At pH 7.2, dissociation of the enzyme from dilute microsomal suspensions increased with ionic strength and was almost complete at 0.3 M-KCl. MgCl2 caused more dissociation than did KCl or NaCl at the same ionic strength, but at low KCl concentrations binding required small amounts of free bivalent metal ions. In 0.1 M-KCl the binding decreased between pH 4.7 and 9.3. At pH 7.2 the binding was independent of temperature between 5 and 20 degrees C. These observations suggest that the binding is electrostatic rather than hydrophobic. 3. The proportion of bound activity increased with the concentration of the microsomal fraction, and at 22 mg of protein/ml and pH 7.2 was 70% at I0.18, and 35% at I0.26. Presumably a substantial amount of the enzyme is particle-bound in vivo. 4. At 5 degrees C in 10 mM-potassium phosphate, pH 7.2, the apparent molecular weight of KCl-solubilized enzyme decreased with enzyme concentration from about 200 000 to 40 000. In the presence of 0.5M-KCl, a constant mol.wt. of about 55 000 was observed over a 20-fold range of enzyme concentrations.

The immunologic release of histamine and slow reacting substance of anaphylaxis (SRS-A) from human lung tissue can be enhanced by stimulation with either alpha adrenergic agents (phenylephrine or norepinephrine in the presence of propranolol) or cholinergic agents (acetylcholine or Carbachol). The finding that atropine prevents cholinergic but not comparable alpha adrenergic enhancement is consistent with the view that cholinergic and alpha adrenergic agonists interact with separate receptor sites on the target cells involved in the immunologic release of chemical mediators. The consistent qualitative relationship between the antigen-induced release of mediators and the level of cyclic adenosine monophosphate (cyclic AMP) as measured by the isolation of 14C-labeled cyclic AMP after incorporation of adenine-14C into the tissues or by the cyclic AMP binding protein assay suggests that changes in the level of this cyclic nucleotide mediate adrenergic modulation of the release of histamine and SRS-A. The addition of 8-bromo-cyclic guanosine monophosphate (cyclic GMP) produces an enhancement of the immunologic release of mediators while dibutyryl cyclic AMP is inhibitory. As cholinergic-induced enhancement was not associated with a measurable change in the levels of cyclic AMP, the possibility is suggested that cyclic GMP may be the intracellular mediator of cholinergic-induced enhancement of the immunologic release of histamine and SRS-A.

Partial purification of adenosine 3′,5′-cyclic monophosphate phosphodiesterases from rat pancreas in the presence of excess protease inhibitors

1. The mechanism by which M&B 22,948, MY-5445, vinpocetine and 1-methyl-3-isobutyl-8-(methylamino)xanthine (MIMAX), which have been described as selective cyclic GMP phosphodiesterase (PDE) inhibitors, relax rat aorta was investigated. 2. Three cyclic nucleotide PDEs were identified in the soluble fraction of rat aorta; a Ca2+-insensitive form exhibiting substrate selectivity for cyclic GMP (cGMP PDE), a Ca2+/calmodulin-stimulated form which also preferentially hydrolyzed cyclic GMP (Ca2+ PDE), and a form demonstrating substrate selectivity for cyclic AMP (cAMP PDE). 3. M&B 22,948 and MIMAX inhibited cGMP PDE (Ki = 0.16 microM and 0.43 microM, respectively) and Ca2+ PDE (Ki = 9.9 microM and 0.55 microM, respectively), but exhibited weak activity against cAMP PDE (Ki = 249 microM and 42 microM, respectively). MY-5445 selectivity inhibited cGMP PDE (Ki = 1.3 microM) and vinpocetine selectively inhibited Ca2+ PDE (Ki = 14 microM). 4. M&B 22,948 and MIMAX induced dose-dependent increases in the accumulation of cyclic GMP, but not cyclic AMP, in rat aorta pieces. These effects were greatly reduced by endothelial denudation and by methylene blue (5 microM) which blocks the actions of endothelium-derived relaxant factor. MY-5445 and vinpocetine had no effect on rat aorta cyclic GMP or cyclic AMP accumulation. 5. All four compounds caused dose-related relaxation of 5-hydroxytryptamine (10 microM) contracted, endothelium-intact rat aorta, the effects of M&B 22,948 and MIMAX being greatly reduced by methylene blue (5 microM). Methylene blue also caused 10 fold and 100 fold rightward shifts in the dose-response curves of MY-5445 and vinpocetine, respectively. 6. The results are consistent with the smooth muscle relaxant actions of M&B 22,948 and MIMAX, but not vinpocetine and MY-5445, being mediated through a mechanism involving inhibition of cyclic GMP hydrolysis.

The type V cyclic GMP phosphodiesterase was partially purified from the high-speed supernatant of guinea-pig lung. The isoenzyme displayed linear kinetics for cyclic GMP hydrolysis, with Km = 2.2 +/- 0.2 microM and Vmax. = 1.2 +/- 0.08 nmol/min per mg. The selective type V phosphodiesterase inhibitor Zaprinast inhibited cyclic GMP hydrolysis with IC50 (concn. giving 50% inhibition) = 0.45 +/- 0.08 microM. Isobutylmethylxanthine promoted a 3-fold increase in the binding of cyclic GMP to the isoenzyme. The addition of the catalytic subunit of protein kinase A to an activation cocktail containing the partially purified type V phosphodiesterase resulted in a marked increase in Vmax. for cyclic GMP hydrolysis (approximately 10-fold at 40 units of protein kinase A). We have suggested that protein kinase A triggers phosphorylation of the phosphodiesterase, which results in activation of phosphodiesterase activity. In addition, the sensitivity to inhibition by Zaprinast is severely decreased (the IC50 for inhibition is 7.5 +/- 1.1 microM), suggesting that the potency of phosphodiesterase inhibitors is effected by phosphorylation of the enzyme.

Activation and dissociation of adenosine 3′,5′-monophosphate-dependent and guanosine 3′,5′-monophosphate-dependent protein kinases by various cyclic nucleotide analogs

Phosphodiesterase activities of horse (and dog) thyroid soluble fraction were compared with either cyclic AMP (adenosine 3':3'-monophosphate) or cyclic GMP (guanosine 3':5'-monophosphate) as substrate. Optimal activity for cyclic AMP hydrolysis was observed at pH 8, and at pH 7.6 for cyclic GMP. Increasing concentrations of ethyleneglycol bis(2-aminoethyl)-N,N'-tetraacetic acid inhibited both phosphodiesterase activities; in the presence of exogenous Ca2+, this effect was shifted to higher concentrations of the chelator. In a dialysed supernatant preparation, Ca2+ had no significant stimulatory effect, but both Mg2+ and Mn2+ increased cyclic nucleotides breakdown. Mn2+ promoted the hydrolysis of cyclic AMP more effectively than that of cyclic GMP. For both substrates, substrate velocity curves exhibited a two-slope pattern in a Hofstee plot. Cyclic GMP stimulated cyclic AMP hydrolysis, both nucleotides being at micromolar concentrations. Conversely, at no concentration had cyclic AMP any stimulatory effect on cyclic GMP hydrolysis. 1-Methyl-3-isobutylxanthine and theophylline blocked the activation by cyclic GMP of cyclic GMP of cyclic AMP hydrolysis, whereas Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone), a non-methylxanthine inhibitor of phosphodiesterases, did not alter this effect. In dog thyroid slices, carbamoylcholine, which promotes an accumulation of cyclic GMP, inhibits the thyrotropin-induced increase in cyclic AMP. This inhibitory effect of carbamoylcholine was blocked by theophylline and 1-methyl-3-isobutylxanthine, but not by Ro 20-1724. It is suggested that the cholinergic inhibitory effect on cyclic AMP accumulation is mediated by cyclic GMP, through a direct activation of phosphodiesterase activity.

Induction of cyclic AMP and cyclic GMP 3′:5′-cyclic nucleotide phosphodiesterase activities in neuroblastoma lines under differentiating conditions