Cyclic Nucleotide Phosphodiesterase Activity Research Articles

Modulation of the cardiac membrane-bound cyclic nucleotide phosphodiesterase: Inhibition due to a contamination of TLC purified SAdenosylL[methyl- 3H] methionine by Zn ++ ions

Cardiac membranes pretreated with SAdenosylL[methyl- 3H] methionine([ 3H] SAM) purified on TLC silica gel 60 F 254 plates exhibited a marked decrease in cyclic AMP and cyclic GMP phosphodiesterase activity. However, this inhibition did not appear when membranes were incubated with either [ 14C] SAM or unlabelled SAM. We showed that, during the TLC purification of [ 3H] SAM, which involved an acidic elution step, minute amounts of the fluorescent indicator F 254 (Zn sulfur) were eluted. The contaminating Zn ++ ions strongly inhibited cyclic nucleotide phosphodiesterase activity and phospholipid methylation with I 50 values in the micromolar range.

Increased cyclic nucleotide phosphodiesterase (PDE) and calmodulin activities in soluble fraction of Graves' thyroid: analysis of increase in Ca+2 dependence of PDE activities.

Phosphodiesterase (PDE) and calmodulin (CaM) activities were studied in soluble fractions of normal and Graves' thyroid tissue. Normal and Graves' thyroid tissues were obtained at thyroid surgery. PDE activities were assayed with cAMP and cGMP as substrates. CaM activity was measured as the ability to activate bovine thyroid CaM-dependent PDE. cAMP and cGMP PDE activities were increased 1.5- and 2.2-fold above normal in Graves' thyroid, respectively. The major cause of the increase in enzyme activities was their higher Ca+2 dependence. CaM activity also was 1.6-fold increased in Graves' thyroid, although it probably does not contribute to the increase in the Ca+2 dependence of PDE activities because of its relative sufficiency vs. PDE in normal thyroid. Three forms of cAMP and cGMP PDE activities were eluted from Sephadex G-200 columns, with mol wt of 280,000, 140,000, and 80,000-100,000, respectively. The first peak had little or no CaM dependence, the second peak had moderate or dominant CaM dependence, and the third peak revealed weak or dominant CaM dependence. The increase in the CaM-dependent form of the third peak of PDE activity in Graves' thyroid tissue may explain the increase in Ca+2 dependence of PDE activities.

Correlation between inhibition of a cyclic GMP phosphodiesterase and relaxation of canine tracheal smooth muscle

Inhibition of partially purified cyclic nucleotide phosphodiesterase activity as well as pharmacologically induced relaxation of respiratory airways smooth muscle was examined to determine whether any correlation between these two effects could be found. The phosphodiesterase in extracts of canine tracheal smooth muscle was chromatographed on a DEAE Bio-Gel A column and eluted with a sodium chloride gradient. The peak I activity hydrolyzed cGMP at a higher rate than cAMP although the apparent K m values for these two cyclic nucleotides were relatively close. Comparison of the K i values for alkylxanthine inhibitors of peak I activity correlated remarkably well with the ec 50 values of the same compounds as relaxants of canine tracheal smooth muscle strips. It is concluded that inhibition of the peak I enzyme may cause accumulation of an intracellular pool of cyclic nucleotide and thus produce or contribute to the muscle relaxant effects that were observed.

Evidence that the peripheral cyclic AMP phosphodiesterase of rat liver plasma membranes is a metalloenzyme.

Cyclic nucleotide phosphodiesterase activity in salt extracts of rat liver plasma membranes was progressively inactivated by treatment with the metal chelators 8-hydroxyquinoline and o-phenanthroline, but not the non-chelating m-phenanthroline isomer. Activity at 20 microM-cyclic AMP was lost more slowly than activity at 0.4 microM-cyclic AMP. The activity of treated preparations was partially restored by incubation with Zn2+ or Mn2+ ions (in the presence of 1 mM-MgCl2) but not with Ca2+, Cd2+, Co2+, Cu2+ or Fe2+ ions, nor by MgCl2 alone. The results suggest the presence in the membrane extracts of a cyclic AMP phosphodiesterase containing tightly bound metal, possibly Zn or Mn, that affects the enzyme's affinity for cyclic AMP.

Effects of thyroid status on membrane-bound low Km cyclic nucleotide phosphodiesterase activities in rat adipocytes.

Adipocyte membranes from hypothyroid rats showed increased low Km cAMP phosphodiesterase activity compared to normals, provided that the subcellular fractionations were done in isotonic, as opposed to hypotonic, buffers. The enhanced cAMP phosphodiesterase activity in hypothyroid membranes was nearly normalized by incubation with a 10-fold excess of cGMP. Preincubation of hypothyroid adipocytes with cGMP also restored to normal the blunted lipolytic response to micromolar concentrations of epinephrine. DEAE-Sephacel chromatography of detergent-solubilized membrane-bound cAMP phosphodiesterase showed a 2.5-fold enhancement in hypothyroid membranes of a form of the enzyme that was completely inhibited by cGMP; the enzymatic elution profiles of the soluble fractions showed no difference between normal and hypothyroid fat pads. The results suggest a possible regulatory role of cGMP in adipocytes in the hypothyroid state.

Cyclic 3':5'-nucleotide phosphodiesterase in human myometrium at the end of pregnancy: partial purification and characterization of the different soluble isoenzymes.

Most of the cyclic nucleotide phosphodiesterase (PDE) activity of pregnant human myometrium was found in the soluble fraction. Chromatography of this fraction on DEAE-cellulose resolved two peaks of PDE activities. Peak I hydrolyzed both cAMP and cGMP and was activated by the Ca2+-calmodulin complex. Peak II, insensitive to this complex, hydrolyzed specifically cAMP. Sucrose gradient centrifugation of the soluble fraction resolved three peaks (A, B, C) of cAMP PDE activities, and only the first two peaks (A, B) were active towards cGMP. A subsequent sucrose gradient centrifugation of peak I, previously determined by DEAE-cellulose, allowed us to restore two peaks A and B identical to those directly obtained from the soluble fraction: peak A hydrolyzes both substrates, while peak B is specific to cGMP hydrolysis. For peak II, a single large cAMP PDE activity peak is generated. Considering Ca2+ and cAMP as intracellular messengers in the control of uterine motility, the characterization of the different forms of PDE in pregnant human myometrium will be of importance in developing improved tocolytic therapy.

18 Effects of steroid hormones on soluble cyclic nucleotide phosphodiesterase (PDE) activity in the human pregnant myometrium

18 Effects of steroid hormones on soluble cyclic nucleotide phosphodiesterase (PDE) activity in the human pregnant myometrium

Effect of cold exposure on liver and muscle cAMP content and cAMP phosphodiesterase activity.

Adenosine 3',5'-cyclic monophosphate (cAMP) concentration and 3',5'-cyclic-nucleotide phosphodiesterase (PDE) activity were measured in skeletal muscle, heart, and liver of rats exposed to 1, 3, 5, and 7 days of cold. Cyclic nucleotide concentration increased in fast-twitch red muscle at the same time that PDE activity was decreasing. Nucleotide concentration and enzyme activity of slow-twitch red muscle were not altered by the cold exposure. The PDE activity of fast-twitch white muscle was elevated approximately 50% above control after 1 and 3 days of cold exposure. By the 5th day in the cold, white muscle PDE activity had returned to control levels and remained there through the 7th day of experimentation. cAMP concentration in hearts of cold-exposed rats was significantly (P less than 0.01) elevated above control at all time points measured. Myocardial PDE activity was elevated above control (P less than 0.05) at 1 and 3 days of cold exposure but returned to control levels by the 5th day in the cold. Hepatic cAMP and PDE activity were elevated above control at all time points analyzed. These data suggest that changes in cyclic nucleotide metabolism play a role in attaining homeostasis during acute cold exposure.

Mutants of PC12 cells with altered cyclic AMP responses.

PC12 cells, derived from a rat pheochromocytoma, were mutagenized and selected in media containing agents known to elevate intracellular concentrations of cyclic AMP (cAMP). More than 40 clones were isolated by selection with cholera toxin or 2-chloroadenosine or both. The variants that were deficient in accumulating cAMP were obtained by using a protocol in which 1 microM 8-bromo-cAMP was included in addition to the agonist. Certain of these variants were partially characterized with respect to the site of altered cAMP metabolism. The profiles of adenylate cyclase activity responsiveness of certain variants to guanosine-5'-(beta, gamma-imido) triphosphate and to forskolin resembled those of UNC and cyc phenotypes of S49 lymphoma cells, which are functionally deficient in the GTP-sensitive coupling protein, Ns. Other variants were characterized by increased cyclic nucleotide phosphodiesterase activity at low substrate concentration. Diverse morphological traits were observed among the variants, but it was not possible to assign them to a particular cAMP phenotype. Two revertants of a PC12 mutant were isolated and observed to have regained a cellular cAMP response to 2-chloroadenosine and to forskolin. It is hoped that these PC12 mutants will have utility for defining cAMP-mediated functions, including any links to the action of nerve growth factor, in cells derived from the neural crest.

Mutants of PC12 cells with altered cyclic AMP responses.

PC12 cells, derived from a rat pheochromocytoma, were mutagenized and selected in media containing agents known to elevate intracellular concentrations of cyclic AMP (cAMP). More than 40 clones were isolated by selection with cholera toxin or 2-chloroadenosine or both. The variants that were deficient in accumulating cAMP were obtained by using a protocol in which 1 microM 8-bromo-cAMP was included in addition to the agonist. Certain of these variants were partially characterized with respect to the site of altered cAMP metabolism. The profiles of adenylate cyclase activity responsiveness of certain variants to guanosine-5'-(beta, gamma-imido) triphosphate and to forskolin resembled those of UNC and cyc phenotypes of S49 lymphoma cells, which are functionally deficient in the GTP-sensitive coupling protein, Ns. Other variants were characterized by increased cyclic nucleotide phosphodiesterase activity at low substrate concentration. Diverse morphological traits were observed among the variants, but it was not possible to assign them to a particular cAMP phenotype. Two revertants of a PC12 mutant were isolated and observed to have regained a cellular cAMP response to 2-chloroadenosine and to forskolin. It is hoped that these PC12 mutants will have utility for defining cAMP-mediated functions, including any links to the action of nerve growth factor, in cells derived from the neural crest.

Dibutyryl cyclic AMP increases phosphodiesterase activity in the rat heart.

The influence of increasing the in vivo concentration of cyclic AMP on the activity of cyclic nucleotide phosphodiesterase (PDE) in rat heart was investigated. One, three, and five hourly injections of 5.0 mg dibutyryl (Bt2) cyclic AMP significantly increased the activity of PDE in the supernatant fraction of rat heart using 1.0 microM cyclic AMP as the assay substrate concentration. When 100 microM cyclic AMP was used in the assay reaction, increases in enzymes activity were seen following five and eight nucleotide injections. The nucleotide-induced increase in PDE activity was dose dependent. When the five-injection protocol was used, PDE activity remained elevated for at least 4 h, while activity had returned to control levels within this time when two hourly injections were used. The nucleotide stimulation of PDE activity was blocked by cycloheximide. Five hourly infections of Bt2 cyclic AMP increased PDE activity in the liver and fast-twitch red muscle. A reduction in PDE activity in fast-twitch white muscle was seen following nucleotide injections. These findings are consistent with the hypothesis that prolonged elevations in the intracellular concentration of cyclic AMP cause an elevation in myocardial PDE activity. The increased activity seems to be the result of protein synthesis. These data suggest that cyclic AMP contributes significantly in regulating its own metabolism in the rat heart.

Vasoactive-intestinal-polypeptide-stimulated adenosine 3',5'-cyclic monophosphate accumulation in GH3 pituitary tumour cells. Reversal of desensitization by forskolin.

The interaction between forskolin and vasoactive intestinal polypeptide (VIP) in the regulation of cyclic AMP production in GH3 pituitary tumour cells was investigated. Both forskolin (10nM-10 microns) and VIP (10pM-10nM) increased the cyclic AMP content of GH3 cells. Forskolin (50-100nM) was additive with VIP in stimulating cyclic AMP accumulation when low concentrations (less than 1 nM) of the peptide were used, but exhibited a synergistic interaction with higher VIP concentrations (10-100 nM). These effects on cyclic AMP accumulation were reflected in a leftward shift in the concentration-response curve for VIP-stimulated prolactin release from GH3 cells, a process known to be regulated by intracellular cyclic AMP concentrations. The synergy observed did not appear to be related to changes in cyclic nucleotide phosphodiesterase activity, since it was even more marked in the presence of isobutylmethylxanthine, a phosphodiesterase inhibitor. Studies of the time-course of VIP-induced changes in GH3-cell cyclic AMP content revealed that, with high concentrations of VIP, production ceased within 2 min of addition. This attenuation of cyclic AMP synthesis was still observed in the presence of isobutylmethylxanthine, but was markedly inhibited by low concentrations of forskolin (50-100nM). The results suggest that VIP-induced cyclic AMP production rapidly becomes desensitized. This process, which is prevented by forskolin, may be related to changes in the ability of the guanine nucleotide regulatory protein to couple receptor occupancy to activation of adenylate cyclase.

Calmodulin Activity in Corticosteroid-induced Osteopenia

A reproducible high-pressure liquid chromatography assay of calmodulin, a multifunctional calcium-dependent modulating protein, was developed for cartilage and bone by using cyclic nucleotide phosphodiesterase activity as the basis for standard curve determination. Calmodulin activity was then measured in rabbits that were made osteopenic by prednisolone injection in an effort to characterize in vivo cellular events. A significant rise in bone calmodulin levels was noted when this data was correlated with osteocyte and osteoblast content by quantitative histomorphometry. This suggests that calmodulin mediates steroid effects on the collagen matrix as well as on calcium homeostasis. Diminished calmodulin levels in weight-bearing cartilage of steroid-treated animals has as yet unresolved significance. Further characterization of calmodulin activity appears warranted in the study of osteopenic states at the subcellular level.

Phosphorylation and activation of calmodulin-sensitive cyclic nucleotide phosphodiesterase by a brain Ca 2+, calmodulin-dependent protein kinase

A Ca 2+, calmodulin-dependent protein kinase from rat brain with a MW of 640,000 phosphorylated calmodulin-sensitive phosphodiesterase from the brain cytosol. The Km of the enzyme for the phosphodiesterase was 5.0 μM and the Vmax was 212 nmol/mg/min. The amount of phosphate incorporated into the phosphodiesterase was 0.7 mol/mol subunit. Phosphorylation of the phosphodiesterase enhanced the enzyme activity by about 20% for hydrolysis of a higher concentration of cyclic AMP.

Heat-stable calmodulin-binding protein in rat testis. Inhibition of calmodulin-stimulated cyclic nucleotide phosphodiesterase activity.

An inhibitor of procine brain calmodulin-dependent cyclic nucleotide phosphodiesterase was purified about 940-fold from rat testis. This inhibitor inhibited the calmodulin-induced activation of the enzyme without affecting its basal activity. The inhibitor activity was counteracted by a high concentration of calmodulin, but was not by a high concentration of Ca2+. The analysis on polyacrylamide disc gel electrophoresis demonstrated that the inhibitor and calmodulin form a complex in the presence of Ca2+ but not in the presence of excess amount of EGTA. This inhibitor also inhibited the calmodulin-induced activation of Ca2+, Mg2+ -ATPase of human erythrocytes. The inhibitor appeared to be a heat-stable protein, since the inhibitor activity was not attenuated by boiling up to 9 min but was completely abolished by tryptic or chymotryptic digestion. The molecular weights of the inhibitor determined by linear polyacrylamide gradient gel electrophoresis under nondenaturing conditions and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 40,000 and 32,000, respectively. Thus, the inhibitor is suggested to be a calmodulin-binding protein composed of a monomer which has unique properties different from those of other tissues.

Cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase activities of rat mammary 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+.

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A series of xanthine derivatives and papaverine have been studied to determine their abilities to alter tissue levels of cyclic AMP and cyclic GMP, inhibit phosphodiesterase activities, and cause relaxation of pig coronary arteries. The agents selected for this study exhibited a wide range of potencies to inhibit phosphodiesterase activities in the coronary artery supernatant fraction. In addition, some of these agents were up to 10 times more potent as inhibitors of cyclic GMP hydrolysis than of cyclic AMP hydrolysis, while others were 2-4 times more potent as inhibitors of cyclic AMP than of cyclic GMP hydrolysis. The rank order of potencies of these agents to cause relaxation of coronary artery strips was similar to the rank order of potencies to inhibit cyclic nucleotide phosphodiesterase activities. There were, however, some notable exceptions to the correlation between inhibition of cyclic nucleotide phosphodiesterase activities and relaxation. 1-Isoamyl-3-isobutylxanthine was a more potent relaxing agent than might be expected from its relatively low potency to inhibit cyclic nucleotide hydrolysis in tissue extracts. On the other hand, 1-methyl-3-isobutyl-7-(3-chlorobenzyl)-xanthine was one of the more potent inhibitors of cyclic nucleotide hydrolysis but was not as potent in causing relaxation as might have been expected. Exposure of the coronary artery strips to inhibitors caused increases in tissue levels of cyclic AMP and cyclic GMP and there was a statistically significant multiple linear regression of cyclic AMP and cyclic GMP levels on percent relaxation after 5 min of exposure to the agents. Cyclic AMP and cyclic GMP levels made approximately equal contributions to the regression of changes in percent relaxation, as determined by analysis of variance methods. While 1-isoamyl-3-isobutylxanthine did not fit the correlation between phosphodiesterase inhibition and potency to relax the arterial strips as well as the other agents, this agent also was found to cause unexpectedly large increases in cyclic AMP levels. Some agents caused relaxation accompanied by significant elevation of cyclic GMP levels and no significant change in cyclic AMP levels while other agents caused relaxation accompanied by significant increases in cyclic AMP but not cyclic GMP. These data offer some support for a hypothesis that both cyclic AMP and cyclic GMP are involved in the relaxation processes of pig coronary arteries.

Calmodulin content of rat mammary tissue and isolated cells during pregnancy and lactation.

The calmodulin content of heat-treated extracts of rat mammary tissue and isolated cells was measured by using stimulation of cyclic nucleotide phosphodiesterase (PDE) activity and radioimmunoassay (r.i.a.) procedures. The calmodulin content of mammary tissue increased 2.5-fold near the time of parturition, remained at the elevated level during lactation, then, after the onset of involution, decreased to values similar to those measured from mammary tissue of pregnant rats. When tissue from 15 animals in different stages of pregnancy, lactation and involution were compared, the r.i.a. gave 2.6-fold higher results than the PDE assay. To investigate further the increase in calmodulin content of mammary tissue, secretory and myoepithelial cells were enzymically dissociated from rat mammary tissue during different stages of pregnancy, lactation and involution. Protein, DNA, lactose, glucose-6-phosphate dehydrogenase and alkaline phosphatase were assayed to characterize the cell fractions. By using r.i.a., the calmodulin content per mg of protein in isolated secretory-cell fractions was high near parturition, then decreased and remained relatively constant during lactation. The amount of calmodulin expressed per mg of DNA in secretory cells did not show a marked change near parturition, suggesting a constant amount of calmodulin per cell. The calmodulin content of myoepithelial cells dissociated from mammary tissue and measured by using r.i.a. was 6-fold lower than in secretory cells and remained relatively constant during the course of lactation. The changing levels of calmodulin in rat mammary tissue during development are suggested to be related to proliferation and destruction of secretory epithelial cells, events that occur near parturition and involution respectively.

Cyclic nucleotide content and phosphodiesterase activity in the rds mouse 020/A) retina

Cyclic nucleotide metabolism was examined in the retina and in the retinal pigment epithelium (RPE)-choroid complex of the rds mouse (020/A), a mutant in which discrete photoreceptor outer segment disc structures fail to develop. In retinas of both rds and control (Balb/c) mice, cyclic AMP levels peak at 10–15 days (20–25 pmol mg −1 protein). The level drops to about 10 pmol mg −1 at about one month in normal retinas but remains high in affected retinas. Cyclic GMP levels increase five-fold in Balb/c retinas as ROS develop whereas, in affected retinas, the levels remain constant and low (about 5 pmol mg −1). In RPE-choroid, cyclic nucleotide levels are similar in control and affected mice. Cyclic AMP phosphodiesterase (PDE) activity is somewhat higher in affected than in control retinas; conversely, cyclic GMP-PDE is lower. Both cyclic AMP-PDE and cyclic GMP-PDE activities are different in normal and affected RPE-choroid. Thus, although the rds (020/A) mouse belongs to the early-onset photoreceptor dysplasia group of hereditary retinal degenerations on a morphological basis, it does not exhibit high retinal cyclic GMP levels and thus appears to be distinct from other animals exhibiting early postnatal photoreceptor dysfunction.

Evidence for the presence of calmodulin in fish mucus.

Partly purified mucus collected from the skin of three species of fish contains a protein that, on sodium dodecyl sulphate/polyacrylamide gel electrophoresis, comigrates with bovine brain calmodulin and shows the same calcium-dependent shift in electrophoretic mobility as calmodulin. Fish mucus contains a heat-stable activator of cyclic nucleotide phosphodiesterase; activation is concentration dependent and sensitive to the specific calmodulin inhibitor calmidazolium (R 24571). The presence of calmodulin in fish mucus is further indicated by means of a specific radioimmunoassay. A drop in the calcium concentration of the water induces an increase in the immunoassayable calmodulin concentration of mucus, which indicates that the function of calmodulin in mucus is related to control of permeability of the skin epithelium to water and ions.