Cyclic AMP Hydrolytic Activity Research Articles

Isolation and characterization of the rolipram-sensitive cyclic AMP-specific phosphodiesterase (type IV PDE) in human term myometrium

On the basis of the potencies of classical selective modulators of cyclic nucleotide phosphodiesterase (PDE) activities, five cyclic nucleotide PDE isoforms have been isolated and characterized in the cytosolic fraction of human term myometrium. By means of successive ion-exchange chromatographies, a calcium-calmodulin sensitive isoform, a cyclic GMP-stimulated isoform, a cyclic GMP-inhibited isoform, a rolipram-sensitive cyclic AMP-specific isoform and a cyclic GMP-specific isoform, corresponding to PDE I, PDE II, PDE III, PDE IV and PDE V, respectively, have been identified. We found that near term, human myometrium contains a higher proportion of the rolipram-sensitive type IV PDE isoform (about 50% of total cyclic AMP hydrolytic activity) than the type III cyclic GMP-inhibited PDE isoform (only 10%). Type IV PDE displays simple Michaelis-Menten kinetics with a high affinity for cyclic AMP ( K m ∼ 4.4 μM) and is selectively and competitively inhibited by rolipram ( K i ∼ 0.9 μM) and Ro 20-1724 ( K i ∼ 2.6 μM). The predominance of type IV PDE at the end of pregnancy suggests that this isoform contributes, via a modulation of the intracellular cyclic AMP level, to local control of uterine motility and thus could help the myometrium prepare for pronounced contractile activity at the time of parturition.

The effect of cyclic AMP and cyclic GMP phosphodiesterase inhibitors on the superoxide burst of guinea‐pig peritoneal macrophages

1. The cyclic nucleotide phosphodiesterase (PDE) activity of guinea-pig peritoneal macrophages was partially characterized and the effects of selective and non-selective inhibitors of adenosine 3':5'-cyclic monophosphate (cyclic AMP PDE) and guanosine 3':5'-cyclic monophosphate (cyclic GMP PDE) phosphodiesterases on superoxide generation were investigated using peritoneal macrophages from horse-serum pretreated guinea-pigs. 2. The non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX) and the PDE I/V selective inhibitor, zaprinast, inhibited spontaneous superoxide generation with IC50s of 30.7 +/- 11.3 microM and 145 +/- 17 microM respectively (n = 6 and 5). The concentration-response curves for the PDE IV selective inhibitors rolipram and Ro20-1724 were biphasic; mean maximum inhibitions were 56.9 +/- 5.9% and 66.8 +/- 10.5% respectively at 300 microM, but in 2 out of 6 (rolipram) and 2 out of 5 (Ro20-1724) experiments inhibition was < 50%. The PDE III inhibitor SK&F 94120 was without effect. Spontaneous superoxide generation was reduced 57 +/- 10% by 1 microM prostaglandin E2 (PGE2) and 62.6 +/- 3.76% by 1 microM salbutamol. 3. The increase in superoxide generation elicited by FMLP (10(-9)-10(-5)M) was unaffected by any of the PDE inhibitors studied. Inhibition of FMLP-stimulated superoxide generation by PGE2 was enhanced in the presence of 10 microM IBMX. 4. Macrophages were found to contain a predominantly membrane bound cyclic AMP PDE (90% of total activity) which was unaffected by cyclic GMP or calcium/calmodulin. The cyclic AMP PDE activity in the cytosolic fraction was enhanced in the presence of calcium/calmodulin. Selective inhibitors of PDE IV inhibited the particulate cyclic AMP PDE activity (IC50s rolipram 1.5 +/- 0.3 microM, Ro 20-17244.1 +/- 0.6 microm) as did the non-selective inhibitor IBMX (IC50 22 +/- 8 microM). The macrophage particulate PDE activity was resistant to inhibition by the PDE III inhibitor SK&F 94836 and the PDE I/V inhibitor, zaprinast. The cytosolic calcium/calmodulin stimulated cyclic AMP hydrolytic activity was inhibited by zaprinast (IC50 - calcium/calmodulin 123 +/- 39 microM; + calcium/calmodulin IC50 17.7 +/- 6.3 microM).5. The results indicate that guinea-pig peritoneal macrophages contain a type IV cyclic AMP PDE which is predominantly membrane associated and a predominantly cytosolic calcium/calmodulin stimulated cyclic AMP PDE. Functional studies suggest that both of these PDE activities contribute to cyclic AMP hydrolysis and regulation of superoxide generation in these cells. Inhibition of spontaneous superoxide generation, but not that stimulated by FMLP, suggests that the activity of PDE inhibitors is subject to functional antagonism but that this can be overcome by enhancing cyclic AMP formation.

Human bronchial cyclic nucleotide phosphodiesterase isoenzymes: biochemical and pharmacological analysis using selective inhibitors.

1 The aims of the present study were to characterize the cyclic nucleotide phosphodiesterase (PDE) isoenzyme activities present in human bronchi and to examine the ability of selective isoenzyme inhibitors to relax histamine and methacholine precontracted preparations of human bronchi. 2 Three separations of pooled human bronchial tissue samples were performed. Ion-exchange chromatography showed that the soluble fraction of human bronchial preparations contains PDE I, II, III, IV and V isoenzyme activities. Multiple forms of PDE I and PDE IV were observed and PDE IV was the main cyclic AMP hydrolytic activity. 3 3-Isobutyl-l-methylxanthine (IBMX) non-selectively inhibited all separated isoenzyme activities. Zaprinast selectively inhibited PDE V, but also effectively inhibited one of the two PDE I isoforms identified. The PDE IV selective inhibitors rolipram and RO-201724, inhibited the PDE IV activities as did the dual PDE III/IV inhibitor, Org 30029. Org 9935, a PDE III selective inhibitor, potently attenuated part of the PDE IV activity peak in one of three separations performed, indicating that some PDE III activity may co-elute with PDE IV under the experimental conditions employed. 4 PDE IV-selective (rolipram), PDE III-selective (Org 9935) and dual PDE III/IV (Org 30029) inhibitors were effective relaxants of human bronchial smooth muscle. The PDE V/PDE I inhibitor, zaprinast was relatively ineffective. 5 The present study demonstrates in human bronchi, as in animal airways smooth muscle, that inhibitors of PDE III, PDEIV and dual PDE III/IV have potentially useful bronchodilator activity and are worthy of further consideration as anti-asthma drugs.

Pharmacological and biochemical effects of the cardiotonic agent Org10325 in isolated cardiac and vascular tissue preparations

1. The pharmacological and biochemical effects of a novel cardiotonic agent, Org10325 have been studied in isolated cardiac and vascular tissue preparations. 2. Org10325 produced concentration-dependent (0.15-4.8 mM) positive inotropic, positive chronotropic and vascular relaxant responses in rabbit isolated papillary, atrial and aortic preparations, respectively. The maximal chronotropic effect (45%) was significantly less than the isoprenaline maximum. The inotropic effects of Org10325 were not modified by alpha- or beta-adrenoceptor blockade or by pretreatment with reserpine. Org10325 was at least 23 times more potent at relaxing aortic strips pre-contracted with phenylephrine than with KCl. 3. Org10325 (74 microM) potentiated (10-14 fold) the positive inotropic effects of isoprenaline in rabbit isolated papillary muscles. Carbachol inhibited the positive inotropic effect of Org10325. Both the positive inotropic and vasorelaxant effects of Org10325 were accompanied by increases in cyclic AMP but not cyclic GMP. 4. In rat perfused heart preparation Org10325 increased phosphorylase a, cyclic AMP-dependent protein kinase activities and stimulated phosphorylation of contractile proteins (troponin-I and C-protein). 5. Org10325 selectively inhibited the cyclic AMP hydrolytic activity of cyclic AMP high affinity cyclic nucleotide phosphodiesterase (PDE) isoenzymes, PDE III (IC50 65 microM) and PDE IV (IC50 71 microM), from rabbit cardiac ventricle. Weak inhibition (IC50 greater than 250 microM) of PDE I and PDE II was observed. 6. The results show that the cardiac and vascular effects of Org10325 are mediated by an increase in cellular cyclic AMP due to inhibition of PDE III and PDE IV activities. However, in contrast to other PDE-inhibitors OrglO325 produced a marked increase in relaxation time of isolated papillary muscle suggesting the involvement of additional cyclic AMP-independent mechanisms of action.

Cyclic nucleotide phosphodiesterase activity in bovine brain coated vesicles.

Cyclic AMP phosphodiesterase activity in bovine brain coated vesicles displayed a Km of approximately 22 microM for cyclic AMP, a Vmax of 3.2 nmol/min/mg protein, and a Hill coefficient of 1.5, suggesting positive cooperativity. The enzyme activity was stimulated by cyclic GMP with maximal indexes of stimulation ranging between 40 and 300%. Both basal and stimulated phosphodiesterase activities were immunotitrated with polyclonal antibodies against clathrin attached to heat-inactivated, formaldehyde-fixed Staphylococcus aureus cells. The main form of phosphodiesterase activity present in the immunoprecipitated brain coated vesicle preparation also is stimulated by cyclic GMP. The allosteric behavior was modulated by cyclic GMP. All of these properties are typical of type II or cyclic GMP-sensitive phosphodiesterases in addition to their calcium and calmodulin independence. Competition experiments with a series of phosphodiesterase inhibitors, papaverine, 1-methyl-3-isobutylxanthine, and theophylline, showed inhibition of cyclic AMP hydrolysis. Trifluoperazine was inactive at the highest concentration used, 100 microM. These compounds also inhibited the cyclic GMP-stimulated cyclic AMP hydrolysis with trifluoperazine practically inactive. At 5 microM cyclic AMP none of the inhibitors was seen to stimulate the cyclic AMP hydrolytic activity. The presence of an enzyme for the breakdown of cyclic nucleotides in brain coated vesicles may suggest a role for these second messengers in the in vivo functions of this organelle.

3′:5′-Cyclic-nucleotide phosphodiesterase in the bovine pituitary gland

Cyclic-AMP phosphodiesterase activity in the homogenate of the anterior pituitary gland was 2-fold higher than that in the homogenate of the posterior pituitary, whereas cyclic-GMP phosphodiesterase activity was dominant in the posterior homogenate. There were two peaks of cyclic-AMP phosphodiesterase activity with different isoelectric points of 4.3 and 5.2. Fraction I had a molecular weight of 240 000 and a sedimentation coefficient of 6.2 S; fraction II had a molecular weight of 180 000 and a sedimentation coefficient of 3.1 S. Cyclic AMP hydrolytic activity in the supernatant of the posterior lobe corresponded to fraction I in the anterior lobe. Cyclic GMP hydrolytic activity in both the anterior and posterior lobes (activated by Ca 2+ / calmodulin) had an isoelecteric point of 5.2, a molecular weight of 240 000 and a sedimentation coefficient of 6.2 S. Cyclic AMP and GMP hydrolytic activities in both the anterior and posterior lobes appeared in fraction I and did not separate when the preparations were mixed before electric focusing or sucrose density gradient procedures. Cyclic AMP hydrolytic activity in fraction II could be separated from cyclic GMP hydrolytic activity.

Cyclic 3′:5′-nucleotide phosphodiesterase of rabbit sinoatrial node

The sinoatrial node contains pacemaker cells which control heart rate. Changes of heart rate correlate closely to cyclic adenosine 3′:5′-monophosphate (cyclic AMP) levels in sinoatrial node. In order to elucidate the mechanism of regulation of cyclic AMP level in sinoatrial node, we investigated cyclic 3′:5′-nucleotide phosphodiesterase (3′:5′-cyclic-nucleotide 5′-nucleotidohydrolase, EC 3.1.4.17) of isolated rabbit sinoatrial node. Sinoatrial node of rabbit heart contains at least three kinetically distinct forms of cyclic nucleotide phosphodiesterase (F I, F II and F III) which were clearly separated by DEAE-cellulose column chromatography. Although phosphodiesterase F I and F II hydrolyzed cyclic GMP faster than cyclic AMP at low substrate concentration, phosphodiesterase F III hydrolyzed equally cyclic GMP and cyclic AMP at a low substrate concentration. Phosphodiesterase F I displayed normal kinetics both for cyclic AMP hydrolysis, with a K m of 8.3 μM, and for cyclic GMP hydrolysis, with a K m of 2.0 μM. Phosphodiesterase F II and F III displayed abnormal kinetics both for cyclic AMP, with a K m for 1.2 and 0.8 μM, respectively, and for cyclic GMP, with a K m of 2.0 and 0.1 μM, respectively. The effects of the activator, Ca 2+, cyclic AMP and cyclic GMP on these forms were also studied. The cyclic GMP and cyclic AMP hydrolytic activities in phosphodiesterase F I and F II were stimulated by micromolar concentration of Ca 2+ in the presence of the activator. Phosphodiesterase F III was not affected by addition of Ca 2+ in the presence of the activator. Cyclic AMP hydrolysis by phosphodiesterase F I and F II was activated by micromolar amounts of cyclic GMP, whereas cyclic GMP hydrolysis by these fractions was inhibited by micromolar amounts of cyclic AMP. Cyclic AMP hydrolytic activity in phosphodiesterase F III was inhibited by micromolar amounts of cyclic GMP, and cyclic GMP hydrolysis by phosphodiesterase F III was also inhibited by cyclic AMP.

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

Human lung tissue contains phosphodiesterase enzymes capable of hydrolyzing both adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP). The cyclic AMP enzyme exhibits three distinct binding affinities for its substrate (apparent Km = 0.4 μM, 3 μM, and 40 μM) while the cyclic GMP enzyme reveals only two affinities (Km = 5 μM and 40 μM). The pH optima for the cyclic AMP and cyclic GMP phosphodiesterase are similar (pH 7.6–7.8). Both are inhibited by known inhibitors of phosphodiesterase activity (aminophylline, caffeine, and 3-isobutyl-1-methylxanthine). The divalent cations Mg 2+ and Mn 2+ stimulate cyclic AMP phosphodiesterase activity (in the absence of Mg 2+) while Ca 2+, Ni 2+, and Cu 2+ inhibit the enzyme. Histamine and imidazole slightly stimulate cyclic AMP hydrolytic activity. Thus, human lung tissue does contain multiple forms of both the cyclic AMP and cyclic GMP phosphodiesterase which are influenced by a variety of effectors.

Human thyroid cyclic nucleotide phosphodiesterase: Its characterization and the effect of several hormones on the activity

Cyclic AMP and cyclic GMP phosphodiesterase activities (3′5′-cyclic AMP 5′nucleotidohydrolase, EC 3.1.4.17) were investigated in the human thyroid gland from patients with hyperthyroidism. Low substrate concentration (0.4 μM) was used. About 60% of the cyclic-AMP and 80% of the cyclic-GMP hydrolytic activities in the homogenate were obtained in the soluble fraction ( 105 000 × g supernatant). The thyroid gland contains two forms of cyclic-AMP phosphodiesterase, one with a K m of 1.3 · 10 −5 M and the second with a K m 2 · 10 −6 M. Cyclic-AMP and cyclic-GMP phosphodiesterase were purified by gel filtration on a Sepharose-6B column. Cyclic-AMP phosphodiesterase activities were found in a broad area corresponding to molecular weights ranging from approx. 200 000 to 250 000 and cyclic-GMP phosphodiesterase activity was found in a single area corresponding to a molecular weight of 260 000. Cyclic-AMP phosphodiesterase activities were stimulated by the protein activator which was found in human thyroid and this stimulation was dependent on Ca 2+. Stimulation of cyclic-AMP phosphodiesterase by the activator was not significant even in the presence of enough Ca 2+. The effect of d,l-triiodothyronine, d,l-thyroxine, l-diiodotyrosine, l-monoiodotyrosine, l-thyronine, l-diiodothyronine, thyrotropin, hydrocortisone, adrenocorticotropin, cyclic-AMP and cyclic-GMP on the phosphodiesterase activities was studied. Cyclic-AMP, cyclic-GMP, d,l-triiosothyronine, d,l-thyroxine, adrenocorticotropin and hydrocortisone were found to inhibit the phophodiesterase. Triiodothyronine and thyroxine inhibited cyclic-AMP phosphodiesterase more effectively than cyclic-GMP phosphodiesterase. Thyroxine was a more potent inhibitor than triiodothyronine. The concentration of cyclic AMP producing a 50% inhibition of clclic-GMP phosphodiesterase activity was 5 · 10 −5 M, while the concentration of cyclic GMP producing a 50% inhibition of cyclic-AMP phosphodiesterase was 3 · 10 −3 M. Both cyclic-AMP and cyclic-GMP phosphodiesterase activities in the homogenate of hyperthyroidism, thyroid carcinoma and adenoma were higher than in normal thyroid tissue, when assayed with a low concentration of the substrate (0.4 μM). When a higher concentration (1 mM) of cyclic nucleotides was used as the substrate, cyclic-AMP hydrolytic activity in adenoma tissue was similar to that of normal tissue, while the other activities were higher than normal.

Cyclic 3′, 5′-AMP phosphodiesterase of rabbit aorta

Cyclic AMP and cyclic GMP phosphodiesterase activities (3′ : 5′-cyclic AMP 5′-nucleotidohydrolase, EC 3.1.4.17) were demonstrated in the isolated intima, media, and adventitia of rabbit aorta. The activity for cyclic AMP hydrolysis in the intima was 2.7-fold higher than that for cyclic GMP hydrolysis. The activity for cyclic AMP hydrolysis in the media was approximately equal to that for cyclic GMP hydrolysis, but in the adventitia, cyclic GMP hydrolytic activity was 2.1-fold higher than cyclic AMP hydrolytic activity. Distribution of the activator of the phosphodiesterase was studied in the three layers. Each layer contained the activator. The activator was predominantly localized in the smooth muscle layer (the media). The effect of the activator and Ca 2+ on the media cyclic AMP and cyclic GMP phosphodiesterase was also briefly studied. The activity of the cyclic GMP phosphodiesterase was stimulated by micromolar concentration of Ca 2+ in the presence of the activator. However, the activity of the cyclic AMP Phosphodiesterase was not significantly stimulated by Ca 2+ up to 100 μM in the presence of the activator. Above 90% of cyclic nucleotide phosphodiesterase activity in the whole aorta was found to be derived from the media. A major portion (60–70%) of the media enzyme was found in 105 000 × g supernatant. Cyclic AMP phosphodiesterase in the supernatant was partially purified through Sepharose 6B column chromatography and partially separated from cyclic GMP phosphodiesterase. Using a partially purified preparation from the 105 000 × g supernatant the main kinetic parameters were specified as follows: 1. 1)|The pH optimum was found to be about 9.0 using Tris-maleate buffer. The maximum stimulation of the enzyme by Mg 2+ was achieved at 4 mM of MgCl 2. 2. 2)|High concentration of cyclic GMP (0.1 mM) inhibited noncompetitively the enzyme activity, and the activity was not stimulated at any tested concentration of cyclic GMP. 3. 3)|Activity-substrate concentration relationship revealed a high affinity ( K m = 1.0 μM ) and low affinity ( K m = 45 μM ) for cyclic AMP. The homogenate and 105 000 × g supernatant of the media also showed non-linear kinetics similar to the Sepharose 6B preparation and their apparent K m values for cyclic AMP hydrolysis were 1.2 μM and 36–40 μM and an enzyme extracted by sonication from 105 000 × g precipitate also exhibited non-linear kinetics ( K m = 5.1 μM and 70 μM . 4. 4)|Papaverine exhibited much stronger inhibition on the aorta cyclic AMP phosphodiesterase (50% inhibition of the intima enzyme, I 5 0 at 0.62 μM, I 5 0 of the media at 0.62 μM and 5 0 of the adventitia at 1.0 μM) than on the brain ( I 5 0 at 8.5 μM) and serum ( I 5 0 at 20 μM) cyclic AMP phosphodiesterase, while theophylline inhibited these enzymes similarly. However, cyclic GMP phosphodiesterases in all tissues examined were inhibited similarly, not only by theophylline but also by papaverine.