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.     

Effect of Ricinus communis toxin on cyclic adenosine 3':5'-monophosphate metabolism in Yoshida ascites sarcoma cells.

Prostaglandin E1 (2.5 mug/ml) enhanced the level of cyclic adenosine 3':5'-monophosphate (cyclic AMP) three to four times in Yoshida ascites sarcoma (YS) cells cultured in vitro. When Ricinus communis toxin (RC-toxin) was added 30 min after the addition of prostaglandin E1, the enhanced level of cyclic AMP in the YS cells decreased rapidly. Of RC-toxin, 0.2 mug/ml was enough to produce the maximum effect. By addition of 5 mM lactose with RC-toxin, approximately 60% of the RC-toxin effect on the levels of cyclic AMP was abolished. This indicates that the specific binding of RC-toxin on the surface membrane is largely responsible for the observed decrease of the cyclic AMP level. The toxin treatment did not induce either leakage of cyclic AMP from the cell or change in the activity of cyclic AMP phosphodiesterase. However, the treatment of YS cells with RC-toxin caused a decrease of adenylate cyclase activity when the activity was measured at a substrate concentration of 0.15 mM ATP. In contrast, there was little difference with the control when the activity was assayed at a higher ATP concentration, 0.24 mM. It was found that the K-m of adenylate cyclase for ATP was changed by RC-toxin from 0.1 to 0.25 mM, and that the Mg2+ activation of the enzyme observable in untreated cells disappeared. These results suggested that the decrease in the level of cyclic AMP in YS cells induced by RC-toxin can be explained in terms of the change in K-m of the adenylate cyclase activity.     

Self-phosphorylation of cyclic guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung. Effect of cyclic adenosine 3':5'-monophosphate, cyclic guanosine 3':5'-monophosphate and histone.

Incubation of purified cyclic guanosine 3':5'-monophospate-dependent protein kinase with [gamma-32P]ATP and Mg2+ led to formation of one 32P-labeled protein, Mr = 75,000, which corresponded to the single protein band detected after polyacrylamide gel electrophoresis in sodium dodecyl sulfate. When electrophoresis was performed without detergent, the labeled protein coincided with the position of cGMP-dependent protein kinase activity. Phosphorylation was enhanced severalfold by either histone or cAMP and was inhibited by the addition of cGMP. Low concentrations of cGMP blocked the stimulatory effects of cAMP or histone (or both). Since neither cAMP-dependent protein kinase nor cGMP-dependent phosphoprotein phosphatase activities were detected in the purified enzyme, we concluded that the cGMP-dependent protein kinase is a substrate for its own phosphotransferase activity and that other protein substrates (histone) and cyclic nucleotides modulate the process of self-phosphorylation.     

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.     

Modulation of agonist-induced inositol phosphate metabolism by cyclic adenosine 3',5'-monophosphate in adrenal glomerulosa cells

Activation of the cAMP messenger system was found to cause specific changes in angiotensin-II (All)-induced inositol phosphate production and metabolism in bovine adrenal glomerulosa cells. Pretreatment of [3H]inositol-labeled glomerulosa cells with 8-bromo-cAMP (8Br-cAMP) caused both short and long term changes in the inositol phosphate response to stimulation by All. Exposure to 8Br-cAMP initially caused dose-dependent enhancement (ED50 = 0.7 microM) of the stimulatory action of All (50 nM; 10 min) on the formation of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and its immediate metabolites. This effect of 8Br-cAMP was also observed in permeabilized [3H]inositol-labeled glomerulosa cells in which degradation of Ins(1,4,5)P3 was inhibited, consistent with increased activity of phospholipase-C. Continued exposure to 8Br-cAMP for 5-16 h caused selective enhancement of the All-induced increases in D-myo-inositol 1,3,4,6-tetrakisphosphate [Ins(1,3,4,6)P4] and myo-inositol 1,4,5,6-tetrakisphosphate. The long term effect of 8Br-cAMP on the 6-phosphorylated InsP4 isomers, but not the initial enhancement of Ins(1,4,5)P3 formation, was inhibited by cycloheximide. The characteristic biphasic kinetics of All-induced Ins(1,4,5)P3 formation were also changed by prolonged treatment with 8Br-cAMP to a monophasic response in which Ins(1,4,5)P3 increased rapidly and remained elevated during All stimulation. In permeabilized glomerulosa cells treated with 8Br-cAMP for 16 h, the conversion of D-myo-inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] to Ins(1,3,4,6)P4 was consistently increased, whereas dephosphorylation of Ins(1,4,5)P3 to D-myo-inositol 1,4-bisphosphate and of D-myo-inositol 1,3,4,5-tetrakisphosphate to Ins(1,3,4)P3, was reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of organotin compounds and hexachlorophene on brain adenosine cyclic 3',5'-monophosphate metabolism.

The effect of triethyltin (TET), triphenyltin (TPT), hexachlorophene (HCP) and cuprizone on adenosine cyclic 3',5'-monophosphate (cyclic AMP) production in rat brain was examined both in vitro and in vivo. TET and TPT inhibited basal adenylate cyclase activity of brain homogenate at a concentration as low as 1 microM in vitro but these compounds had no effect on norepinephrine (NE) and dopamine(DA)-stimluated enzyme activity. HCP and cuprizone failed to inhibit adenylate cyclase activity. In vivo TET given intravenously at a dose rate of 10 mg/kg decreased the cyclic AMP content of cerebrum, but not of medulla. TPT and HCP give intravenously and intraperitoneally respectively failed to decrease the cyclic AMP content of the cerebrum. In the case of TET the reduction in cyclic AMP content of the cerebrum was prevented by maintaining the rats normothermic after treatment. On the basis of these results the inhibition of adenylate cyclase produced by TET in brain homogenates in vitro would not appear to be involved in the development of nervous changes associated with acute TET toxicity, or in the production of progressive brain oedema caused by TET, HCP and cuprizone.     

An equilibrium study of the cooperative binding of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate to the adenosine cyclic 3',5'-monophosphate receptor protein from Escherichia coli

The binding of adenosine cyclic 3',5'-monophosphate (cAMP) and guanosine cyclic 3',5'-monophosphate (cGMP) to the adenosine cyclic 3',5'-monophosphate receptor protein (CRP) from Escherichia coli was investigated by equilibrium dialysis at pH 8.0 and 20 degrees C at different ionic strengths (0.05--0.60 M). Both cAMP and cGMP bind to CRP with a negative cooperativity that is progressively changed to positive as the ionic strength is increased. The binding data were analyzed with an interactive model for two identical sites and site/site interactions with the interaction free energy--RT ln alpha, and the intrinsic binding constant K and cooperativity parameter alpha were computed. Double-label experiments showed that cGMP is strictly competitive with cAMP, and its binding parameters K and alpha are not very different from that for cAMP. Since two binding sites exist for each of the cyclic nucleotides in dimeric CRP and no change in the quaternary structure of the protein is observed on binding the ligands, it is proposed that the cooperativity originates in ligand/ligand interactions. When bound to double-stranded deoxyribonucleic acid (dsDNA), CRP binds cAMP more efficiently, and the cooperativity is positive even in conditions of low ionic strength where it is negative for the free protein. By contrast, cGMP binding properties remained unperturbed in dsDNA-bound CRP. Neither the intrinsic binding constant K nor the cooperativity parameter alpha was found to be very sensitive to changes of pH between 6.0 and 8.0 at 0.2 M ionic strength and 20 degrees C. For these conditions, the intrinsic free energy and entropy of binding of cAMP are delta H degree = -1.7 kcal . mol-1 and delta S degree = 15.6 eu, respectively.     

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.     

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.     

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.