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Endogenous cyclic AMP in thyroid cell culture. Effect of thyroid-stimulating hormone and dibutyryl cyclic AMP.

We have studied the variations of endogenous cyclic AMP levels in thyroid cells cultured over a period of 7 days in several conditions: in the presence of thyroid-stimulating hormone or dibutyryl cyclic AMP which both promote the aggregation of isolated cells into follicles, and in their absence when cells develop as a typical monolayer. In follicle-forming cells, the cyclic AMP level was found to rise during the first day of culture, then to fall rapidly. In monolayer-forming cells, the cyclic AMP content slightly increases attaining the same level as found in other cells at the fourth day, which remains stable till the seventh day. We have investigated the response of these cells cultured in the presence of dibutyryl cyclic AMP retain the capability of increasing their cyclic AMP concentration whereas monolayer-forming cells do not preserve this quality of thyroid cells.

Animals

Comparative activation by AMP and cyclic-AMP of rat erythrocyte and reticulocyte glycolysis.

Incubation of raty erythrocytes and reticulocytes in Tris-Ringer's medium with 5 mM cyclic-AMP or AMP increased lactate formation and glucose utilization. The glycolysis-stimulating effect of cyclic-AMP is very similar to that of AMP and, in both cases, it seems to be higher in reticulocytes than in erythrocytes. 0.5 mM norepinephrine produced a much higher lactate formation in reticulocytes than in erythrocytes, suggesting a greater adenylate cyclase activity in younger cells. 300 micrometer cyclic-AMP and AMP reverse inhibition produced by ATP (up to 1.5 micrometer) on phosphofructokinase from rat reticulocyte haemolysates. Both nucleotides are positive allosteric effectors of the enzyme as shown by displacement of F6P-saturation curve to hyperbolic kinetics. Similar results were previously obtained with rat erythrocytes. This deinhibitory effect is suggested to be responsible of the above glycolysis-stimulating effect.

Adenosine Monophosphate

Kinetic studies of adenylyl cyclase of fat cell membranes. II. Comparison of activities measured in the presence of Mn++-AMP-P(NH)P and Mg++-AMP-P(NH)P. Effects of insulin, fluoride, isoproterenol, and GMP-P(NH)P.

The kinetics of fat cell adenylyl cyclase were studied, with AMP-P(NH)P and Mn++ or Mg++ as the divalent cation. In general, the reaction times were not linear. In the presence of fluoride or GMP-P(NH)P, the time curves were concave upwards; in other cases (i.e., basal activity, insulin, or isoproterenol), transient rates tended to decrease with time during the assay. Kinetic data were analyzed according to a previously described procedures (Torres et al., 1978b) which isolates two kinetic components: initial and final. With AMP-P(NH)P, kinetic activities were about ten times lower than those for ATP. With Mn++, activities were at least two-times higher than for Mg++. Spontaneous inactivation of adenylyl cyclase was higher in assays containing Mg++ than in those supplemented with Mn++. In the latter case, insulin was able to increase the inactivation rate. Fluoride and isoproterenol both activated adenylyl cyclase in both the initial and final kinetic components; under most of the conditions explored, their effects on the final component appeared to be more dramatic. Assays with GMP-P(NH)P showed inhibited activity in the initial component and increased activity in the final one. When the results obtained with AMP-P(NH)P are compared with those of ATP (Torres et al., 1978b. J. Membrane Biol. 43:000), the following differences were found: (i) in the presence of insulin and Mn++, cyclase inactivation was higher with AMP-P(NH)P than with ATP; (ii) fluoride stimulation of the final component was more marked with ATP than with AMP-P(NH)P; (iii) cyclase stimulation by isoproterenol was slightly higher with the nucleotide analog; and (iv) GMP-P(NH)P stimulation of the final component resulted in higher activity with ATP than with AMP-P(NH)P.

Adenylyl Cyclases

[Conformation of nucleotides, oligonucleotides and their analogues in aqueous solution. II. Syn-anti-equilibrium in solutions of adenosine, 5'-AMP, 3'-AMP, 5'-CMP and 3'-CMP].

A method for determining the equilibrium constant of sin-anti-states in the aqueous solution of purine and pyrimidine nucleotides and nucleosides has been proposed. This method is based on the measurement of the spin-lattice relaxation rate of H(1') atom of purine nucleotides before and after exchange of H(8) deuterium in the case of purine nucleotides or H(1') and H(5) atoms of pyrimidine nucleotides. The results obtained were interpreted by the two-state dynamic model. The method applied for investigation of the conformation situation in solutions of Ado, 5'-AMP, 3'-AMP, 5'-CMP and 3'-CMP. 5'-AMP and 5'-CMP were shown to exist predominantly in the anti-state (90%). In the case of 3'-nucleotides the enhancement of the relativity weight of sin-populations to 0.2 and 0.85 for 3'-CMP and 3'-AMP, respectively, was found. In the solution Ado both anti- and sin-state were equiprobable. Experimentally measured relaxation rates of H(8) of adenine or H (6) of cytosine nucleotides were used for determination of the time average orientation of nucleic bases towards the ribose ring in the anti-state. For all compounds investigated with the exception of 3'-AMP the "normal" anti-conformation was confirmed. The 3'-AMP was found to be characterized by high anti-conformation. The probable causes of the conformational situation organization and correlation between the N/S and sin/anti rations are discussed.

Adenosine Monophosphate

The dependence of Escherichia coli asparaginase II formation on cyclic AMP and cyclic AMP receptor protein.

The amount of asparaginase II in an Escherichia coli wild-type strain (cya+, crp+) markedly increased upon a shift from aerobic to anaerobic growth. However, no such increase occurred in a mutant (cya) lacking cyclic AMP synthesis unless supplemented with exogenous cyclic AMP. Since a mutant (crp) deficient in cyclic AMP receptor protein also did not support the anaerobic formation of this enzyme, it is concluded that the formation of E. coli asparaginase II depends on both cyclic AMP and cyclic AMP receptor protein.

Aerobiosis

Induction of germination in Blastocladiella emersonii by cyclic AMP and inhibitors of cyclic AMP phosphodiesterase.

Since K+-induced germination of Blastocladiella emersonii is accompanied by a rapid decrease of a specific cyclic AMP phosphodiesterase (cAMP PDE) activity and a transient cyclic AMP accumulation, the effects of this compound as well as of some inhibitors of cAMP PDE on the induction of germination were tested. Adenine and caffeine, competitive inhibitors of zoospore cAMP PDE, were able to elicit germination in substitution for K+. Cyclic AMP is a poor inducer, but a synergistic effect was evident when non-effective concentrations of K+ and cyclic AMP were added together to the medium. At the same concentration, cyclic GMP had no effect as compared with cyclic AMP. Lanthanum, a specific antagonist of calcium in several biological systems, completely blocked the germination induced by potassium.

3',5'-Cyclic-AMP Phosphodiesterases

CYCLIC AMP/*SECRETl cyclic AMP into push-pull perfusates in freely moving rats.

Cyclic AMP was found in perfusates of push-pull cannula's, chronically placed in the lateral ventricle of freely moving rats. After addition of norepinephrine, dopamine or adenosine to the perfusates the output of cyclic AMP was enhanced, whilst serotonin and histamine were found to be ineffective. The effects of noradrenaline and dopamine on cyclic AMP were found to be mediated by different receptors: haloperidol antagonized only the dopamine response. The effect of norepinephrine was mimicked by isoprenaline, a beta-adrenergic agonist, but was not blocked by propranolol. L-DOPA, given intraperitoneally, increased cyclic AMP levels in the perfusate. We conclude that the release of cyclic AMP into the extracellular medium is a normally occurring phenomenon in brain tissue.

Adenosine

Cyclic AMP, membrane transport and cell division. I. Effects of various chemicals on cyclic AMP levels and rate of transport of neucleosides, hypoxanthine and deoxyglucose in several lines of cultured cells.

Nutrient transport rates and cyclic AMP levels have been implicated in the regulation of cell proliferation. In the present study, however, changes in intracellular cyclic AMP level in several lines of cultured cells (normal 3T3 and SV40 and polyomavirus-transformed 3T3 cells; 3T6, C6 GLIOMA, MOUSE L, and Novikoff rat hepatoma cells) by treatment with papaverine, prostaglandine E1 or isoproterenol did not correlate with the inhibition of the uridine, hypoxanthine or deoxyglucose transport rates by these chemicals. Transport inhibitions by above chemicals or Persantin or Cytochalasin B occurred in most cell lines in the absence of any measurable change in intracellular cyclic AMP concentration. Furthermore, treatment of several cell lines with 1 mM dibutyryl cyclic AMP had no immediate effect on the transport of uridine, thymidine or deoxyglucose, although the transport capacity of the cells for uridine and thymidine, but not that for deoxyglucose, decreased progressively with time of treatment. We also observed that the uridine transport system of all cell lines derived from 3T3 cells and the hypoxanthine transport system of L cells exhibited high degrees of resistance to inhibition by the various chemicals. On the other hand, deoxyglucose transport was inhibited to about the same extent by these chemicals in all the cell lines investigated.

Animals

Stimulation of calcium uptake into aortic microsomes by cyclic AMP and cyclic AMP-dependent protein kinase.

Enhancement of calcium uptake into rabbit aortic microsomes was seen at a cyclic AMP concentration of 10(-6) M in the presence of cyclic AMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37). Protein kinase alone also increased calcium uptake, but cyclic AMP alone was without effect. The results suggest that stimulation of calcium sequestration may be the mechanism of cyclic AMP involvement in vascular smooth muscle relaxation.

Animals

Cyclic AMP accumulation in psoriatic skin: differential responses to histamine, AMP, and einephrine by the uninvolved and involved epidermis.

Using the uninvolved and involved skin from psoriatic patients, we investigated the effects of histamine and AMP (or adenosine) in vitro on the intracellular cyclic AMP levels. Both agents activated adenylate cyclase of the uninvolved and involved resulting in the accumulation of cyclic AMP. Without a cyclic nucleotide phosphodiesterase (PDE) inhibitor, these responses were biphasic and the maximal accumulation was observed in 5 min. With the PDE inhibitor both responses were markedly potentiated and high levels of cyclic AMP were observed for more than 20 min. The response to histamine by the involved skin was much greater than that by the uninvolved. The degree of the response to adenosine was approximately equal. In accordance with our previous work, the response to epinephrine by the involved skin was much less than that by the uninvolved. Thus adenylate cyclases of involved skin from psoriatic patients exhibit a markedly diminished response to epinephrine while at the same time exhibiting a markedly enhanced response to histamine. This precludes the possibility that the unresponsiveness to epinephrine can be due to a generalized inability of the epidermal psoriatic plaque cell to make a functioning cell membrane.

Adenosine Monophosphate

Adenosine, AMP, cyclic AMP, theophylline and the action and production of erythropoietin.

Erythropoiesis, as measured by the uptake of 59Fe into plethoric mice, is stimulated by adenosine, AMP, cyclic AMP, and dibutyryl cyclic AMP, but not by cytidine, its nucleotides or cyclic GMP. This stimulation is erythropoietin dependent, because it is prevented by anti-erythropoietin. Theophylline neither stimulates erythropoiesis nor potentiates the action of erythropoietin on bone marrow cells in plethoric mice. Theophylline does potentiate the production of erythropoietin in rats following a frief hypoxic exposure but does not cause a similar increase in mice.

Adenine Nucleotides

[Further developments of the AMP-system. A report on the 2nd AMP-symposium and training seminar (author's transl)].

This is a report of a symposium and training seminar of the "Arbeitsgemeinschaft für Methodik und Dokumentation in der Psychiatrie" (AMP). Video-recordings of two psychiatric interviews were shown during the seminar and rated by the participants using the AMP-3-Scale (Psychopathology). Aim of the seminar was a rater training and the preparation of an expert standard rating. These videotapes with the corresponding standard ratings will enable the different clinics using the AMP-System to compare their rating behaviour. The process of achieving an expert standard rating is described. The training seminar was followed by a symposium during which a series of lectures was presented on "Standardized Assessment in Psychiatry", The different working groups of the Association reported about their work and future prospects of the Association were discussed.

Congresses as Topic

Effects of cyclic AMP and dibutyryl cyclic AMP on amino acid transport in the isolated rat ovary.

Prepubertal rat ovaries were incubated in medium containing the non-utilizable amino acids alpha-aminoisobutyric acid (AIB-14C) or 1-aminocyclo-pentane-carboxylic acid (cycloleucine-14C). The rate of uptake of the two amino acids was studied in the isolated ovaries after different incubation periods. Addition of 5mM cyclic AMP (cAMP) caused a slight stimulation of the AIB-transport but in higher concentrations (10-25 mM) an inhibition was noted. With dibutyrl cyclic AMP (dbcAMP) a dose-dependent increase was seen with 0.5-5 mM concentrations with no further effect of higher concentrations. Time course studies were performed with both AIB and cycloleucine in presence of 10 mM dbcAMP and increased uptake values were noted at each time studied (30-240 min). The phosphodiesterase inhibitor aminophyline in lower concentrations did not influence AIB-transport but 5-10 mM caused increased uptake values in the ovaries. The stimulatory action of dbcAMP on amino acid transport was augmented by a low concentration of aminophylline (0.5 mM). Experiments were in addition carried out in the presence of puromycin and under these circumstances it was still possible to enhance amino acid transport by addition of dbcAMP. The results are discussed in relation to earlier reported effects of gonadotropins on ovarian amino acid transport.

Amino Acids

Enzymatic radioiodination of succinyl cyclic AMP tyrosine methyl ester by lactoperoxidase and radioimmunoassay for cyclic AMP.

A highly specific and simple radioimmunoassay for cyclic AMP with a sensitivity of 0.04 picomoles/tube has been developed according to the method of Steiner et al., using 125I-succinyl cyclic AMP tyrosine methyl ester as a tracer. The tracer with higher immunoactivities could be simply and constantly prepared by an enzymatic iodination procedure utilizing lactoperoxidase, radioactive iodide and hydrogen peroxide generated by glucose-glucose oxidase system, rather than by chloramine-T procedure.

Adipose Tissue

[Interrater reliability of diagnosis, AMP syndromes and AMP symptoms (author's transl)].

Psychic disorders can be classified into three levels: symptom, syndrome, and diagnosis. For each of these levels of reference, interrater reliability has been calculated. For this purpose, 48 patients (25 depressives and 23 schizophrenics) were interviewed by two raters each and the diagnoses were registered on the AMP system (forms 3 and 4). Additionally, each rather made an ICD diagnosis. With ICD numbers of three digits interrater reliability amounted to Kappa = 0.84; with ICD numbers of four digits it amounted to Kappa = 0.65. According to the degree of accuracy, numerical agreement with the AMP syndromes lies between Kappa = 0.61 and 0.85. Single symptoms had the lowest reliability (median: Kappa 0.45 and 0.53). Reasons for these differences and possibilities for improvement are discussed.

Adolescent

Cyclic AMP in action of antidiuretic hormone: effects of exogenous cyclic AMP and its new analogue.

Exogenous cyclic 3',5'-adenosine monophosphate (cAMP) stimulates the effect of the antidiuretic hormone, vasopressin (VP), only in pharmacologic quantities and results have often been inconsistent. The present study examined the ability of a new analogue, 8-[p-Cl-phenylthio]cyclic 3',5'-adenosine monophosphate (C1-PheS-cAMP) to mimic the effect of VP, both biochemically (protein kinase activation) and functionally (hydrosomatic response of perfused collecting tubules) in mammalian kidney tissue. C1PheS-cAMP was found to be about 100 times as effective as cAMP both biochemically and functionally. The increased effectiveness of C1PheS-cAMP is probably is probably due to a greater permeability across the cell membrane and to the resistance of C1PheS-cAMP to enzymatic degradation, Cyclic AMP phosphodiesterase inhibition was observed with C1PheS-cAMP, but its contribution to overall effect was minor. C1PheS-cAMP was found to be more effective than exogenous vasopressin, an effect probably due primarily to its resistance to catabolism. The results provide further new evidence that cAMP and protein kinase are involved in the cellular action of vasopressin. C1PheS-cAMP proved to be a useful tool in the study of hormone action, especially in steps subsequent to cAMP generation.

3',5'-Cyclic-AMP Phosphodiesterases

Influence of dibutyryl cyclic AMP on thymidine uptake by herpes simplex virus infected cells and the intracellular level of cyclic AMP.

Dibutyryl cyclic AMP inhibits the increase of dThd and BrdUrd transport normally observed after infection with Herpesvirus hominis, type I and II. Incorporation is also reduced. Inhibition of uptake is non-competitive as analysed by the Lineweaver-Burk plot. Addition of this drug to infected cells also reduces the activity of the thymidine kinase (EC 2.7.1.75). Transport of dUrd, dCyd and dAdo is not reduced. 4--8 h after infection with thymidine kinase (+) herpes strains the level of cAMP increases. On infection with a thymidine kinase (-) virus, only a small elevation of cAMP can be shown. It was also found that early addition of actinomycin D or of cycloheximide prevents the increase of the cAMP level. This increase seems to depend on the activity of the herpes genome, because ultraviolet irradiation of infective particles destroys this ability.

Biological Transport

Cyclic AMP-dependent protein kinase mediates a cyclic AMP-stimulated decrease in ornithine and S-adenosylmethionine decarboxylase activities.

Incubation of S49 lymphoma cells with N6,O2'-dibutyryl cyclic AMP (Bt2cAMP) decreases the activities of ornithine decarboxylase (L-ornithine carboxy-lyase; EC 4.1.1.17) and S-adenosylmethionine decarboxylase (S-adenosyl-L-methionine carboxy-lyase; EC 4.1.1.50), the two principal enzymes in the pathway of polyamine synthesis. This decrease is dose-dependent, commences after a 3-hr delay, virtually abolishes the assayable activities of the two enzymes, and is not associated with a soluble inhibitor of the enzyme activities. Studies in mutant S49 clones that have altered protein kinase indicate that cAMP-dependent protein kinase mediates the decreases in enzyme activities. The dose-response pattern for the cAMP-stimulated decrease in enzyme activities parallels the pattern for the cAMP-stimulated, cell cycle-specific (G1) growth arrest of S49 cells. The activity of ornithine decarboxylase decreases faster than Bt2cAMP arrests wild-type S49 cells and, similarly, release of cells from the cAMP-stimulated arrest in G1 increases the activity of ornithine decarboxylase faster than cells exit from G1. These findings contrast with reports that cAMP induces ornithine decarboxylase in other cell types and further suggest that passage of cells through cell cycle is required for maintaining the activities of ornithine and S-adenosylmethionine decarboxylases.

Adenosylmethionine Decarboxylase