AMP Phosphodiesterase Activity Research Articles

Adenosine 3':5'-monophosphate and hormone interrelationships in the mammary gland of the rat during pregnancy and lactation.

The activities of adenyl cyclase, the phosphodiesterases of low and high Km and the tissue content of adenosine 3′:5′‐monophosphate (3′: 5′‐AMP) in the mammary glands of rats at different stages of the lactation cycle have been measured. The effect of in vitro F−, Ca2+ and of the hormones progesterone, β‐estradiol, hydrocortisone, insulin and prolactin on the adenyl cyclase activity of membrane fractions isolated from the mammary glands of pregnant and lactating rats has been investigated. The results show that there is a coordinated change of the activities of the enzymes adenyl cyclase and 3′:5′‐AMP phosphodiesterase and the tissue content of 3′:5′‐AMP at different stages of the lactation cycle with the activity of adenyl cyclase and the level of the cyclic nucleotide rising to a peak at the end of pregnancy and then falling sharply after parturition. The reverse pattern was observed for the 3′:5′‐AMP phosphodiesterase activity. F−(1–10 mM) consistently produced an inhibition (40%) of adenyl cyclase activity of mammary glands from pregnant rats while having no effect on the enzyme from the lactating gland. Low concentrations of Ca2+ (1 μM) activated the enzyme from pregnant glands and from those of early lactation by about 40% but had little effect on the glands taken from rats of mid‐lactation. Increasing the Ca2+ concentration led to a decline of this effect and eventual inhibition at 5 mM, the effect being most marked on the fully lactating gland. Progesterone and β‐estradiol consistently caused a considerable stimulation (80 and 50%, respectively) of the adenyl cyclase activity of the pregnant gland; hydrocortisone, insulin and prolactin did not. None of the hormones tested activated the adenyl cyclase activity of the lactating gland and insulin appeared to inhibit it severely. The results, which suggest that the growth and development of the mammary gland is related to the tissue content of 3′: 5‐AMP while the initiation and scale of lactation may be related to its removal, are discussed in relation to development, lactogenesis and milk ejection and the complex endocrine control of these processes.

Effect of glucagon, dibutyryl adenosine 3′,5′-cyclic monophosphate and phosphodiesterase inhibitors on rat liver phosphorylase activity and adenosine 3′,5′-cyclic monophosphate levels

Theophylline, papaverine and 1-ethyl-4-(isopropylidenehydrazine)-1H-pyrazole-(3,4-b)-pyridine-5-carboxylic acid, ethyl ester HCl (SQ 20,009) were found to inhibit both high K m (200 μM) and low K m (3 μM) cyclic 3′,5′-AMP phosphodiesterase activity in 100,000 g supernatant fraction from rat liver. Theophylline was the least potent and SQ 20,009 the most potent inhibitor of both activities. In the liver slice preparation, papaverine (5 × 10 −4), theophylline (10 −3 M) and SQ 20,009 (10 −3 M) failed to elevate cyclic 3′,5′-AMP, while glucagon (10 −6 M) produced a significant (P < 0·05) elevation in the tissue levels of the cyclic nucleotide. All three phosphodiesterase inhibitors significantly (P < 0·05) augmented the ability of glucagon to elevate cyclic 3′,5′-AMP levels. Glucagon and papaverine stimulated phosphorylase activity, while theophylline inhibited both basal and glucagon-stimulated phosphorylase activity. SQ 20,009 was without effect. It is concluded that no cause and effect relationship can be assumed between an ability to inhibit phosphodiesterase and to alter phosphorylase activity.

Uncoupling of Catecholamine Activation of Pigeon Erythrocyte Membrane Adenylate Cyclase by Filipin

The interaction of the polyene antibiotic filipin with membrane cholesterol inhibits membranous enzyme systems differentially; catecholamine-activated adenylate cyclase in pigeon erythrocyte membranes was already inhibited at a filipin to cholesterol ratio of 0.3:1, while Mg2+-F- activation, phosphorylation of membrane components, ATPase and cyclic 3′ : 5′-AMP-phosphodiesterase activities were either much less inhibited or even enhanced. The binding of catecholamines to the receptor was not affected but the enhancement of catecholamine activation by GTP was even more sensitive to filipin as catecholamine activation itself. The filipin effect could be reversed on incubation of the membranes with β-sitosterol-lecithin vesicles. Another effective uncoupler of catecholamine (and GTP) activation of erythrocyte membrane adenylate cyclase was the amphipathic peptide melittin. The functional consequences of the cholesterol filipin reaction in erythrocyte membranes are heterogeneous. The relevant complex formation was shown not to involve lysis and disintegration and to occur in that part of the membrane which is probed by the hydrophobic dye perylene. The fluorescence of 8-anilino-1-naphthalenesulfonic acid which distributes itself in regions which are accessible to aqueous solvents was comparatively little affected. Differences in the degree of polarization of perylene in membranes and cholesterol-lecithin vesicles and of perylene bound to bovine serum albumin suggested that a part of the dye was bound to hydrophobic proteins. Filipin membrane interaction did not change fluorescence polarization of perylene suggesting that the microenvironment was little perturbed.

Adenosine 3′,5′-monophosphate phosphodiesterase assay in tissue homogenates

1. 1. A fast, reliable and sensitive method for the assay of 3′,5′-AMP phosphodiesterase (adenosine 3′,5′-monophosphate phosphohydrolase, EC 3.1.4.c) is described, which is also applicable to homogenates and crude enzyme preparations containing enzymes catalyzing the breakdown of the reaction product 5′-AMP. The method has been worked out for rat pancreatic homogenate. 2. 2. The method is based on existing methods in which 3′,5′-[ 3H]AMP is used as substrate and in which the resulting 5′-AMP is dephosphorylated in a second step by means of added 5′-nucleotidase. 3. 3. It is shown that already during the first incubation step a mixture of radioactive products is formed: 5′-AMP, 5′-IMP, 3′,5′-IMP, adenosine, inosine, hypoxanthine and adenine. During the second incubation step 5′-AMP and 5′-IMP are converted into the corresponding nucleosides. 4. 4. The reaction mixture after the second incubation step is applied to a column of anionic exchange resin. All products of the phosphodiesterase reaction can be eluted with 0.1 M NaHCO 3, while the unchanged 3′,5′-AMP and the product of the side reaction 3′,5′-IMP, are retained. After liquid scintillation counting of the eluate, the 3′,5′-AMP phosphodiesterase activity can be calculated.

Studies on cyclic AMP levels and phosphodiesterase activity in developing sea urchin eggs

Abstract Cyclic adenosine monophosphate (CAMP) was measured in sea urchin eggs by the binding assay method of Gilman and with a radioimmune assay procedure. Intracellular concentrations of the nucleotide in unfertilized eggs were about 1.5 × 10−7 M and rose to about 3 times this value at first cleavage. Aminophylline, a known inhibitor of phosphodiesterase was shown to cause an increase in intracellular levels of CAMP by first cleavage and to inhibit phosphodiesterase activity in homogenates of both unfertilized and fertilized eggs. Puromycin and its purine component, 6-dimethylaminopurine (DMAP), did not cause an increase in intracellular CAMP levels and did not inhibit phosphodiesterase activity at concentrations an order of magnitude higher than those at which they inhibit cell division.

Cyclic 3',5'-AMP phosphodiesterase activity during cyclic AMP-induced differentiation of neuroblastoma cells in culture.

SummaryThe cyclic AMP phosphodiesterase (PDE) activity markedly increased in morphologically “differentiated” mouse neuroblastoma cells induced by dibutyryl cyclic AMP, prostaglandin E1 (stimulator of adenylate cyclase) and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (RO 20-1724, an inhibitor of PDE). Initially, RO 20-1724 decreased the PDE activity by about 50% of control, but later it increased the PDE activity. All of the above agents are known to increase the endogenous level of cyclic AMP, and therefore, in the differentiated cells, a high level of PDE correlated well with a high level of cyclic AMP. X-Irradiation which causes morphological “differentiation” similar to that by dibutyryl cyclic AMP did not increase the PDE activity. Sodium butyrate, 3′,5′-cyclic AMP, 5′-AMP and theophylline which inhibit cell division without causing morphological differentiation did not affect significantly the basal level of PDE. Cycloheximide markedly reduced the PDE activity, whereas actinomycin D did not. A wo...

Adenosine 3',5'-cyclic monophosphate and phosphodiesterase activities in isolated fetal and neonatal rat pancreatic islets.

Adenosine 3',5'-monophosphate (cyclic AMP) and phosphodiesterase activity were assessed in isolated maternal fetal and neonatal rat pancreatic islets. Two- to three-fold increases in cyclic AMP content and phosphodiesterase activity were observed in neonatal islets from pups 24, 48 and 72 hr following spontaneous delivery with a decrease to normal adult levels by the 13th day postpartum. Cyclic AMP content and phosphodiesterase activity in isolated female adult islets were unchanged at all time periods examined both pre- and postpartum. Islets from 21-day-old fetal rats were not significantly different from maternal islets. Islets from nonfed neonates had smaller, but statistically significant, increases in cyclic AMP and phosphodiesterase activity. The relationship of these changes to fetal islet cell unresponsiveness to glucose and to the alterations in the insulin release mechanism which occur in the neonate is discussed. (Endocrinology 92: 614, 1973)

Cyclic 3′,5′-AMP phosphodiesterase of Saccharomyces carlsbergensis Inhibition by adenosine 5′-triphosphate, inorganic pyrophosphate and inorganic polyphosphate

1. 1. Cyclic 3′,5′-AMP phosphodiesterase activity can be demonstrated in Saccharomyces carlsbergensis by measuring the 5′-AMP formation from cyclic 3′,5′-AMP. 2. 2. Enzymic activity was optimal at pH 8.5 and shows a 2-fold stimulation in the presence of 4 mM Mn 2+; Mg 2+, Ca 2+ and EDTA hardly show any effect. 3. 3. ATP, inorganic polyphosphate and pyrophosphate are effective inhibitors of the phosphodiesterase. The nature of these inhibitions was found to be of the mixed type. 4. 4. The possible physiological significance of this inhibition is discussed.

Adenyl cyclase activity in the rabbit ovary.

An assay for adenyl cyclase activity in homogenates of ovarian interstitial tissue from pseudopregnant rabbits is described and evaluated. The assay is based on the estimation of [14C] adenosine-3′,5′-(cyclic) monophosphate ([UC] 3′,5′-AMP) formation from 8-[14C] ATP. 3′,5′-AMP phosphodiesterase activity is estimated simultaneously by the loss of [3H] 3′,5′-AMP from the incubation mixture. Ovine LH and HCG added in vitro stimulated the adenyl cyclase activity, whereas ovine FSH and porcine ACTH were ineffective. LH had no effect on phosphodiesterase activity. (Endocrinology 84: 989, 1969)

Influence of insulin on cyclic 3?,5?-AMP phosphodiesterase activity in liver, skeletal muscle, adipose tissue, and kidney

Influence of insulin on liver glycogen metabolism and on lipolysis appears to be mediated by a decreased intracellular 3′,5′-AMP concentration. Reduced formation of 3′,5′-AMP had been shown in adipose tissue incubated with insulin. The influence of insulin on 3′,5′-AMP degradation has been investigated. — 3′,5′-AMP phosphodiesterase (PDE) activity was reduced in liver, adipose tissue and, insignificantly, in skeletal muscle of insulin deficient, i.e. alloxan diabetic or starved rats. I.V. injection of a low dose of insulin (0.5 U/kg) or stimulation of endogenous insulin secretion by injection of glucose led to a rapid increase of PDE activity in these tissues. 15 min after insulin injection liver PDE activity was increased. The maximal effect occurred after 30–45 min. Renal PDE activity was not decreased in alloxan diabetes, insulin injection has been found ineffective. —In vitro, there was an activating effect of crystalline insulin on PDE purified from beef heart. Insulin concentration required for duplication of enzyme activity was of the order of 2 · 10−5 M. Treatment with actinomycin D nearly prevented stimulation of liver PDE by insulin. This may indicate that the action of insulin on PDE activity is essentially based on an increased enzyme synthesis. — Owing to the influence of insulin secretion on liver and adipose tissue 3′,5′-AMP concentration, glycogen metabolism and lipolysis can be quickly adapted to food intake.

Effects of glucocorticoids and insulin on 3',5'-AMP phosphodiesterase activity in adrenalectomized rats.

Glucocorticoids stimulate the rate of lipolysis which is reduced in adrenalectomized animals. This hormonal action is antagonized by insulin. The antilipolytic action of insulin appears to be mediated by a reduced intracellular concentration of 3′,5′-AMP. This reduction can partly be attributed to an insulin-induced acceleration of 3′,5′-AMP degradation. — It is shown that the stimulatory influence of glucocorticoids on lipolysis is due to a reduction of 3′,5′-AMP phosphodiesterase (PDE) activity, which is increased by adrenalectomy. PDE activity was also increased in liver, skeletal muscle and kidney of adrenalectomized rats; treatment with a glucoeorticoid prevented this increase.In vitro, PDE purified from beef heart was inhibited by glucocorticoids in high concentrations (Ki=1.1 · 10−3 M for 6 α-methylprednisolone hemisuccinate,K i=1.6 · 10−3 M for prednisolone succinate).In vivo, the glucocorticoid-induced decrease of PDE activity (with retarded onset as shown in liver), may essentially be attributed to a decreased enzyme synthesis. — Studies on the interaction of insulin and glucocorticoids on PDE activity were performed in the liver. In adrenalectomized, alloxan diabetic rats insulin stimulated PDE activity suppressed by treatment with a glucoeorticoid, unsuppressed PDE activity was not increased by insulin. In contrast, the action of glucocorticoids on PDE activity was independent of the presence or the effectiveness of insulin.