Metabolism of cyclic adenosine 3':5'-monophosphate in somatic cell hybrids

A somatic cell hybrid has been isolated between Chinese hamster fibroblasts (CH 23) and a mouse lymphoma (P388 F-36) cell line using nonselective pressure. The hybrid cell line PCM has a marked enhanced response to prostaglandin E₁, in terms of cyclic AMP production, when compared to the parental cells. The activity of cyclic nucleotide phosphodiesterase in both parental cells is higher than in the hybrid cells. Although this may contribute to the enhanced response in the hybrid cells, desensitization experiments suggest modification of the PGE₁ receptor in the hybrid cells.     

Calcium ion effects on cyclic adenosine 3':5'-monophosphate bindings to the plasma membrane of Dictyostelium discoideum.

The study of cell surface cyclic adenosine 3':5'-monophosphate binding to Dictyostelium discoideum amoebae indicates that Ca2+ increases the number of binding sites without significantly affecting their affinity constant(s). The effects of the ion are observed immediately (within 4 s after addition) and appear to be readily reversible. Ca2+ effects are observed at various temperatures and pH values and are not blocked by the presence of various metabolic inhibitors. Increases, and decreases, in the apparent number of cyclic nucleotide binding sites could also be effected by concanavalin A treatments which respectively stimulate, and inhibit cell differentiation.     

Fluctuations in cyclic adenosine 3':5'-monophosphate and cyclic guanosine 3':5'-monophosphate during the mitotic cycle of the acellular slime mould Physarum polycephalum.

Cyclic adenosine 3′:5′-monophosphate (cyclic AMP) and cyclic guanosine 3′:5′-monophosphate (cyclic GMP) have been determined at half-hourly intervals throughout the mitotic cycle of Physarum polycephalum. Cyclic AMP was constant at 1pmole/mg protein throughout except for a transient peak of 17pmoles/mg protein in the last quarter of G2. Cyclic GMP was more variable (2–4pmole/mg protein) rising to 9.5pmole/mg protein during the 3 hour S period and to 7pmole/mg protein during the last hour of G2. The significance of these changes is discussed.     

The effects of hormones on cyclic adenosine 3':5'-monophosphate accumulation in transitional epithelium of the urinary bladder.

Transitional epithelium lining rabbit urinary bladders was isolated and studied in vitro. The homogeneity of the isolated epithelium was demonstrated by light and electron microscopical monitoring as well as cell culture studies. Transitional epithelium responded to epinephrine and prostaglandin E1 (PGE1) in the presence of 2mM 1-methyl, 3-isobutylxanthine (MIX) with increases in intracellular levels of cyclic adenosine 3':5'-monophosphate (cyclic AMP). Corticotropin, aldosterone, insulin, parathyroid hormone and vasopressin were slightly but significantly stimulatory under similar conditions. Glucagon and oxytocin were not stimulatory at the concentrations tested. The effects of epinephrine and PGE1 were potentiated by 2mM MIX 20-fold or greater. The cells were slightly more sensitive to PGE1 then to epinephrine. The prostaglandin produced a noticeable response at about 10nM, while effects of epinephrine were discernible at 0.1muM. Maximal responses to both effectors were seen at about 10muM. The action of 10muM epinephrine, but not 10muM PGE1, was completely abolished by 0.1mM propranolol. Responses to combinations of epinephrine and PGE1 were additive. Cyclic AMP accumulated in the incubation medium of transitional epithelial cells exposed to epinephrine, PGE1, MIX, or combinations of the agonists. The appearance of cyclic AMP in the medium was slow compared to the rate of intracellular accumulation, but reached significant levels following prolonged stimulation.     

Protein inhibitor of cyclic adenosine 3':5'-monophosphate phosphodiesterase in retina.

A protein acting as inhibitor of cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.1.) activity was found in the ox retina tissue. An inhibitor from one tissue (ox retina) effectively cross-inhibited a phosphodiesterase from another tissue (rat brain), indicating a lack of tissue specificity. Kinetic analysis showed that inhibition was independent of the time of preliminary incubation of the inhibitor with enzyme but dependent on its concentration in the reaction mixture. An inhibitor decreased the V of the enzyme and had no effect on its Km for cyclic adenosine-3':5'-monophosphate. The inhibitory effect was more pronounced with cyclic adenosine-3':5'-monophosphate than with cyclic guanosine-3':5'-monophosphate used as substrates of the reaction. The extractable form of the phosphodiesterase of the retina rod outer segments was much more sensitive to the inhibitory action than the membrane-bound one. The binding of labeled cyclic adenosine-3':5'-monophosphate to the inhibitory protein was shown not to occur. The inhibitor was sensitive to trypsin treatment, indicating that it was a proten attempt was mode to purify the inhibitory factor. Gel filtration indicated that the inhibitor had a molecular weight of 38 000.     

Specificity of the cyclic adenosine 3',5'-monophosphate signal in granulosa cell function

Cyclic AMP signaling is involved in most aspects of differentiation and maturation of the granulosa cells in the ovarian follicle. As the genetic programs activated at different stages of follicle growth maturation are being elucidated, it is becoming increasingly difficult to reconcile the simplicity of the cAMP cascade with the complexity and the divergent patterns of gene expression activated in these cells. To account for these divergent outcomes of the cAMP signal, three aspects of this signaling cascade in granulosa cells will be reviewed. We will discuss the evidence for gonadotropin receptors coupling to different G proteins and effectors. Next, we will explore the possibility that the temporal and spatial dimensions of the cAMP signal itself may contribute to the diverse outcomes. Finally, we will summarize available data showing that the cAMP signal is distributed through several cascades of kinase activation. It is hoped this compendium will provide a framework with which to understand how the initial signals activated by gonadotropins control the complex patterns of gene expression that are required for follicle maturation and ovulation.     

Compartmentation of cyclic adenosine 3',5'-monophosphate signaling in caveolae.

The cAMP-signaling pathway is composed of multiple components ranging from receptors, G proteins, and adenylyl cyclase to protein kinase A. A common view of the molecular interaction between them is that these molecules are disseminated on the plasma lipid membrane and random collide with each other to transmit signals. A limitation to this idea, however, is that a signaling cascade involving multiple components may not occur rapidly. Caveolae and their principal component, caveolin, have been implicated in transmembrane signaling, particularly in G protein-coupled signaling. We examined whether caveolin interacts with adenylyl cyclase, the membrane-bound enzyme that catalyzes the conversion of ATP to cAMP. When overexpressed in insect cells, types III, IV, and V adenylyl cyclase were localized in caveolin-enriched membrane fractions. Caveolin was coimmunoprecipitated with adenylyl cyclase in tissue homogenates and copurified with a polyhistidine-tagged form of adenylyl cyclase by Ninitrilotriacetic acid resin chromatography in insect cells, suggesting the colocalization of adenylyl cyclase and caveolin in the same microdomain. Further, the regulatory subunit of protein kinase A (RIIalpha, but not RIalpha) was also enriched in the same fraction as caveolin. Gsalpha was found in both caveolin-enriched and non-caveolin-enriched membrane fractions. Our data suggest that the cAMP-signaling cascade occurs within a restricted microdomain of the plasma membrane in a highly organized manner.     

Effect of treadmill exercise on plasma and urinary cyclic adenosine 3'5'-monophosphate.

The effect of treadmill exercise on plasma and urinary cyclic adenosine 3'5' monophosphate levels (cyclic AMP) was studied in twelve healthy subjects. Plasma cyclic AMP levels were found to be markedly elevated without significant changes in urinary cyclic AMP or cyclic AMP/creatine ratio. Most likely altered plasma glucagon and catecholamine levels were responsible for these changes.     

Studies on the presence of adenosine cyclic 3':5'-monophosphate in oat coleoptiles

The incorporation of adenosine-8-¹⁴C into adenosine cyclic 3′:5′-monophosphate in coleoptile-first leaf segments of Avena sativa L. was investigated. Homogenates of segments incubated in adenosine-8-¹⁴C for either 4 or 10 hours were partially purified by thin layer chromatography followed by paper electrophoresis. A radioactive fraction, less than 0.06% of the ¹⁴C present in the original homogenate, migrated as adenosine cyclic 3′:5′-monophosphate during electrophoresis. Upon treatment with cyclic nucleotide phosphodiesterase, however, less than 10% of this radioactive fraction appeared as 5′-AMP. Deamination with NaNO₂ as well as further chromatographical purification also suggested that only a small fraction of the ¹⁴C in the partially purified samples could be in adenosine cyclic 3′:5′-monophosphate. The data suggest that levels of this nucleotide can probably be no greater than 7 to 11 picomoles per gram of fresh weight in oat coleoptiles. Treatment of such coleoptiles with physiologically active concentrations of indoleacetic acid, furthermore, had no significant effect on the ¹⁴C radioactivity in marker adenosine cyclic 3′:5′-monophosphate-containing fractions at any stage of purification during several experiments.     

Calcium-dependent cyclic nucleotide phosphodiesterase from brain: comparison of adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate as substrates.

A Ca²⁺-dependent cyclic nucleotide phosphodiesterase has been partially purified from extracts of porcine brain by column chromatography on Sepharose 6 B containing covalently linked protamine residues, ammonium sulfate salt fractionation, and ECTEOLA-cellulose column chromatography. The resultant preparation contained a single form of cyclic nucleotide phosphodiesterase activity by the criteria of isoelectric focusing, gel filtration chromatography on Sephadex G-200, and electrophoretic migration on polyacrylamide gels. When fully activated by the addition of Ca²⁺ and microgram quantities of a purified Ca²⁺-binding protein (CDR), the phosphodiesterase hydrolyzed both adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP), with apparent K_m values of 180 and 8 μm, respectively. Approximately 15% of the total enzymic activity was present in the absence of added CDR and Ca²⁺. This activity exhibited apparent K_m values for the two nucleotides identical to those observed for the maximally activated enzyme. Competitive substrate kinetics and heat destabilization studies demonstrated that both cyclic nucleotides were hydrolyzed by the same phosphodiesterase. The purified enzyme was identical to a Ca²⁺-dependent phosphodiesterase present in crude extract by the criteria of gel filtration chromatography, polyacrylamide-gel electrophoresis, and kinetic behavior.Apparent K_m values of the Ca²⁺-dependent phosphodiesterase for cyclic AMP and cyclic GMP were lowered more than 20-fold as CDR quantities in the assay were increased to microgram amounts, whereas the respective maximal velocities remained constant. The apparent K_m for Mg²⁺ was lowered more than 50-fold as CDR was increased to microgram amounts. Half-maximal activation of the phosphodiesterase occurred with lower amounts of CDR as a function of either increasing degrees of substrate saturation or increasing concentrations of Mg²⁺. At low cyclic nucleotide substrate concentrations i.e., 2.5 μm, cyclic GMP was hydrolyzed at a fourfold greater velocity than cyclic AMP. At high substrate concentrations (millimolar range) cyclic AMP was hydrolyzed at a threefold greater rate than cyclic GMP.     

The lack of effect of cyclic adenosine 3':5'-monophosphate on Avena coleoptile growth

The effects of cyclic adenosine 3':5'-monophosphate (cAMP) on the growth of Avena coleoptile segments over 4 to 10 hours were monitored with a position sensing transducer. At pH 6, cAMP (0.1 mm with and without 2.5 mm glucose; or 2 mm alone) or dibutyryl cAMP (0.1 mm) was added at the beginning of the experiment, or after about 1 hour or after about 6 or 7 hours. Under all conditions tested, cAMP compounds had little or no effect on coleoptile segment elongation. Inasmuch as cAMP does not duplicate the rapid and vigorous elongation obtained with 2 mum auxin, the hypothesis that cAMP is a mediator of auxin activity is not supported by experimental evidence in this system. This conclusion is dependent upon the assumption that the cAMP compounds penetrated the tissue.     

Regulation of galactose operon expression: glucose effects and role of cyclic adenosine 3',5'-monophosphate.

We studied the following two aspects of the glucose effect on galactose operon expression in Escherichia coli K-12: catabolite repression and inducer exclusion. Using both inducible and constitutive strains and measuring the rate of promoter-proximal enzyme synthesis, we found that the galactose operon did not seem to exhibit catabolite repression. The only glucose effect on galactose operon expression which we observed was inducer exclusion, as shown by the existence of diauxic growth in the presence of glucose and galactose. This diauxie was not relieved by cyclic adenosine 3',5'-monophosphate. Cyclic adenosine 3',5'-monophosphate did not seem to be an antagonist of any glucose effect on galactose operon expression; its only effect was to stimulate promoter-distal gene expression.     

Variations in the levels of cyclic adenosine 3':5'-monophosphate and in the activities of adenylate cyclase and cyclic adenosine 3':5'-monophosphate phosphodiesterase during aerobic morphogenesis of Mucor rouxii

Particulate cell fractions of mycelium of Mucor rouxii contain adenylate cyclase activity which can be partially solubilized by 2% Lubrol PX. The enzyme requires Mn²⁺ and its activity is not modified by NaF or guanosine nucleotides. Mycelial extracts also contain cyclic adenosine 3′:5′-monophosphate phosphodiesterase activity, 60% of which is soluble. This activity shows characteristic low K_m (1 μm) for cyclic AMP and does not hydrolyze cyclic guanosine 3′:5′-monophosphate. It requires Mn²⁺ ions for maximal activity and is not inhibited by methylxanthines or activated by imidazole. Both enzymatic activities vary during the aerobic life cycle of the fungus. The spores have the highest levels of adenylate cyclase and cAMP phosphodiesterase, which decrease during the aerobic development. At the round cell stage, phosphodiesterase activity reaches 40% of the activity of the spores and varies only slightly thereafter. At this stage the specific activity of adenylate cyclase is 25% of the activity of ungerminated spores, and from this stage on, the activity increases up to the end of the logarithmic phase. Intracellular levels of cyclic AMP have been measured during aerobic germination. The variations of the intracellular level are tentatively explained by unequal variations in the activities of adenylate cyclase and cyclic AMP phosphodiesterase. A continuous increase of the extracellular cyclic AMP level during aerobic development has also been found, which cannot be accounted for solely by variations in the cyclase and diesterase activities.