Diurnal rhythms in cyclic nucleotide metabolism in Helix nervous system.

Concentrations of cAMP (cyclic adenosine 3',5'-monophosphate) and cGMP (cyclic guanosine 3',5'-monophosphate), in ganglia from the garden snail Helix pomatia, vary considerably over the course of the day. There is a maximum in the concentration of both cyclic nucleotides between 08:00 and 12:00 (lights on 06:00 to 18:00), with the cAMP maximum occurring slightly later than that in cGMP. In addition there can be several smaller maxima in cAMP and cGMP levels; the timing of these can be markedly different from experiment to experiment, with cAMP and cGMP sometimes in and sometimes out of phase with each other. This pattern is observed in Helix which had been activated from the dormant state 4-6 days earlier, but is not present in dormant or in long-active animals. The cyclic nucleotide rhythm can be seen in ganglia maintained in organ culture, and persists for at least 24 hours after removal of the tissue from the animal. There appears to be little change in the level of basal or NaF-stimulated adenylate cyclase activity in Helix ganglia over the course of the day. On the other hand, both cAMP and cGMP phosphodiesterase activities exhibit rhythms which are consistent with the rhythms in cAMP and cGMP concentrations.     

The effect of locomotor activity on cerebellar levels of cGMP.

The effect of locomotor activity on brain regional levels of cyclic guanosine 3′, 5′-monophosphate (cGMP) and cyclic adenosine 3′, 5′-monophosphate (cAMP) was examined in rats trained to run in an activity wheel. Following 5 minutes of running, there was a two-fold elevation over control levels of cerebellar cGMP. Significant elevations were seen in eight other regions. No changes were observed in cAMP. Plasma levels of hormones indicative of stress were not significantly different between groups. We suggest that locomotor activity may contribute to elevations in cGMP in cerebellum and other brain regions in rats exposed to a variety of conditions.     

Tissue Distribution,Developmental Changes and Some Catalytic Properties of Guanosine 3′,5′-Monophosphate-dependent Protein Kinase of Bombyx mori

The levels of guanosine 3′,5′-monophosphate (cGMP)-dependent protein kinase in the larval and pupal tissues of Bombyx mori were estimated. This activity was highest in the fat body of the female pupa. The enzyme showed a significant variation in activity during development of adult in female. Male silkworm gave less significant results. The cGMP-dependent kinase partially purified from the pupa could be activated by a high concentration of adenosine 3′,5′-monophosphate (cAMP) as reported for cGMP-dependent protein kinases from other sources. The nature of the enzyme thus activated and that of the enzyme activated by a low concentration of cGMP were found to be similar in several aspects. This indicates that the intrinsic activity of protein kinase from the silkworm pupa is independent of the kind of cyclic nucleotide as an activator.     

Allosteric regulation of phosphodiesterase from Portulaca callus by cGMP and papaverin

Three fractions of phosphodiesterase activity capable of hydrolysing cyclic 3′,5′-AMP and cyclic 3′,5′-GMP were purified from Portulaca callus. Hydrolysing bis-(p-nitrophenyl)-phosphate, two fractions showed linear Lineweaver-Burk plots. One fraction showed positive cooperativity. This fraction can be activated competitively by blue dextran, indicating a possible allosteric regulation by nucleotides, demonstrated by changing from being positively cooperative, to following Michaelis-Menten kinetics by cGMP and papaverin. cGMP triggers an enzyme highly active against 3′,5′cAMP and 3′5′cGMP, and papaverin triggers high activity against 2′,3′cAMP, demonstrated by two separate enzyme fractions.     

Microinjection of cyclic nucleotides provides evidence for a diffusional mechanism of intraneuronal control

Cyclic 3′,5′-AMP and cyclic 3′,5′-GMP injected into large neurons of the snail Helix lucorum altered neuron activity. The effect of cAMP is usually depolarizing and that of cGMP hyperpolarizing. The results are specific for 3′,5′-cyclic nucleotides. The experiments support the hypothesis that reaction-diffusion processes involving cyclic nucleotides from the basis of an intraneuronal system of information processing.     

A melanin-dispersing effect of cyclic adenosine monophosphate on Fundulus melanophores

Treatment of Fundulus melanophores with adenosine 3′,5′-monophosphate (cyclic AMP) is followed by reversible melanin dispersion in these cells. Adenosine 3′-monophosphate and adenosine 5′-monophosphate both have a similar, but weaker dispersing action. In addition, adenosine 5′-monophosphate also has a melanin aggregating effect. These results are interpreted to mean that nerve transmitters may act by controlling the level of cyclic AMP within the Fundulus melanophore.     

Resting and concanavalin-A stimulated levels of cyclic nucleotides in splenic cells of aging mice with spontaneous cancers.

The resting levels of cyclic 3′, 5′ -adenosine monophosphate (cAMP) and cyclic 3′, 5′ -guanosine monophosphate (cGMP) in splenic lymphoid cells of 25 aged (C57BL/10 × C3H)F₁ hybrid mice with spontaneous tumors, including 5 with hepatoma, 10 with lung tumor, 2 with lymphoma, and 8 with several varieties of tumor, as well as in 18 young and 13 tumor-free aging mice, were measured. The alterations in cyclic nucleotide levels in spleen cells characteristic of normal aging in tumor-free animals may be additionally influenced by the occurrence of spontaneous neoplasia. Furthermore, the levels may vary with different types of late-life tumors. For example, levels of cAMP in resting spleen cells of old mice with hepatomas were not different than in age-matched controls, whereas spleen, cells from old mice with lung tumors showed exceedingly high levels of resting cAMP. Upon $\text{in vitro}$ stimulation by Con-A, the splenic lymphoid cells from mice bearing spontaneous late-life lung and liver tumors displayed different kinetic patterns of percent changes in cAMP, cGMP and cAMP/cGMP ratios when compared to either young or age-matched tumor-free controls. Thus, both resting and Con-A stimulated levels of cAMP and cGMP and their ratios in splenic lymphoid cells may be affected by spontaneous cancer elsewhere in the body, including cancer of non-lymphoid type and origin. These findings plus the known functional decline in immune response capacity and the increase in spontaneous tumor incidence with age may suggest the existence of a complex relationship among cyclic nucleotide levels, immunity, aging, and cancer.     

In vitro response of goldfish (Carassius auratus L.) dermal melanophores to cyclic 3',5'-nucleotides, nucleoside 5'-phosphates and methylxanthines

cAMP, dbcAMP, cCMP, cGMP, theophylline and caffeine caused reversible melanosome dispersion within 5 minutes at 10 mM in the dermal melanophores of the black goldfish, Carassius auratus L. cTMP, cUMP, 5′-AMP, 5′-CMP, 5′-GMP, 5′-TMP, and 5′-UMP did not produce melanosome dispersion or aggregation in this melanophore system. cAMP was the most effective nucleotide in the induction of melanosome dispersion; at 10 mM, cGMP and at 5 mM, dbcAMP were the least effective of those nucleotides inducing melanosome dispersion. At the 10 mM level dbcAMP required 30 minutes to evoke the same degree of melanosome dispersion as 5 minutes cAMP treatment. Theophylline was more effective than caffeine in eliciting melanosome dispersion. At 1 mM, theophylline and caffeine first induced melanosome dispersion which was followed by aggregation in the course of the 30 minute test period. These reactions suggest both a high melanophore phosphodiesterase activity and competitive inhibition of phosphodiesterase by theophylline and caffeine. Induction of melanosome dispersion by several cyclic 3′,5′-nucleotides suggest multi-nucleotide control of melanosome dispersion. These findings also support a proposed mechanism of prostaglandin induced melanosome dispersion as well as the “second messenger” hypothesis.     

Studies on cyclic adenosine 3' ,5'-monophosphate levels, Adenylate cyclase and phosphodiesterase activities in the dimorphic fungus Mucor rouxii.

The germination of spores of Mucor rouxii into hyphae was inhibited by 2 mm dibutyryl cyclic adenosine 3′,5′-monophosphate or 7 mm cyclic adenosine 3′,5′-monophosphate; under these conditions spores developed into budding spherical cells instead of filaments, provided that glucose was present in the culture medium. Removal of the cyclic nucleotides resulted in the conversion of yeast cells into hyphae. Dibutyryl cyclic adenosine 3′,5′-monophosphate (2 mm) also inhibited the transformation of yeast to mycelia after exposure of yeast culture to air.Since in all living systems so far studied adenylate cyclase and cyclic adenosine 3′,5′-monophosphate phosphodiesterase are involved in maintaining the intracellular cyclic adenosine monophosphate level, the activity of both enzymes and the intracellular concentration of cyclic adenosine monophosphate were investigated in yeast and mycelium extracts. Cyclic adenosine monophosphate phosphodiesterase and adenylate cyclase activities could be demonstrated in extracts of M. rouxii. The specific activity of adenylate cyclase did not vary appreciably with the fungus morphology. On the contrary, cyclic adenosine monophosphate phosphodiesterase activity was four- to sixfold higher in mycelial extracts than in yeast extracts and reflected quite accurately the observed changes in intracellular cyclic adenosine monophosphate levels; these were three to four times higher in yeast cells than in mycelium.     

cAMP AND cAMP-STIMULATED PROTEIN PHOSPHORYLATION IN ZOOSPORES OF BROWN ALGAE1

Adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP) were detected at concentrations of 8–11 and 10–20 pmol · mg^?1 protein, respectively, in zoospores of a brown alga, Undaria pinnatifida (Harvey) Suringer. Cellular levels of these cyclic nucleotides did not substantially change during dark to light transition. cAMP-stimulated protein phosphorylation was found in soluble cell-free extracts of zoospores of Undaria pinnatifida and Laminaria angustata Kjellman.     

Cyclic guanosine 3',5'-monophosphate and phosphodiesterase activity in mitogen-stimulated human lymphocytes.

The cyclic adenosine 3′,5′-monophosphate (cyclic AMP) phosphodiesterase from human leukemic lymphocytes differes from the normal cell enzyme in having a much higher activity and a loss of inhibition by cyclic guanosine 3′,5′-monophosphate (cyclic GMP). In an effort to determine the mechanism of these alterations, we have studied this enzyme in a model system, lectin-stimulated normal human lymphocytes. Following stimulation of cells with concanavalin A (con A) the enzyme activity gradually becomes altered, until it fully resembles the phosphodiesterase found in leukemic lymphocytes. The changes in the enzyme parallel cell proliferation as measured by increases in thymidine incorporation into DNA. The addition of a guanylate cyclase inhibitor preparation from the bitter melon prevents both the changes in the phosphodiesterase and the thymidine incorporation into DNA. This blockage can be partially reversed by addition of 8-bromo cyclic guanosine 3′,5′-monophosphate (8-bromo cyclic GMP) to the con A-stimulated normal lymphocytes. These results indicate a possible role of cyclic GMP in a growth related alteration of cyclic AMP phosphodiesterase.     

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The bioreduction capacity of Cr(VI) by Shewanella is mainly governed by its bidirectional extracellular electron transfer (EET). However, the low bidirectional EET efficiency restricts its wider applications in remediation of the environments contaminated by Cr(VI). Cyclic adenosine 3′,5′-monophosphate (cAMP) commonly exists in Shewanella strains and cAMP–cyclic adenosine 3′,5′-monophosphate receptor protein (CRP) system regulates multiple bidirectional EET-related pathways. This inspires us to strengthen the bidirectional EET through elevating the intracellular cAMP level in Shewanella strains. In this study, an exogenous gene encoding adenylate cyclase from the soil bacterium Beggiatoa sp. PS is functionally expressed in Shewanella oneidensis MR-1 (the strain MR-1/pbPAC) and a MR-1 mutant lacking all endogenous adenylate cyclase encoding genes (the strain Δca/pbPAC). The engineered strains exhibit the enhanced bidirectional EET capacities in microbial electrochemical systems compared with their counterparts. Meanwhile, a three times more rapid reduction rate of Cr(VI) is achieved by the strain MR-1/pbPAC than the control in batch experiments. Furthermore, a higher Cr(VI) reduction efficiency is also achieved by the strain MR-1/pbPAC in the Cr(VI)-reducing biocathode experiments. Such a bidirectional enhancement is attributed to the improved production of cAMP–CRP complex, which upregulates the expression levels of the genes encoding the c-type cytochromes and flavins synthetic pathways. Specially, this strategy could be used as a broad-spectrum approach for the other Shewanella strains. Our results demonstrate that elevating the intracellular cAMP levels could be an efficient strategy to enhance the bidirectional EET of Shewanella strains and improve their pollutant transformation capacity.     

In Central Obesity,Weight Loss Restores Platelet Sensitivity to Nitric Oxide and Prostacyclin

Central obesity shows impaired platelet responses to the antiaggregating effects of nitric oxide (NO), prostacyclin, and their effectors—guanosine 3′,5′‐cyclic monophosphate (cGMP) and adenosine 3′,5′‐cyclic monophosphate (cAMP). The influence of weight loss on these alterations is not known. To evaluate whether a diet‐induced body‐weight reduction restores platelet sensitivity to the physiological antiaggregating agents and reduces platelet activation in subjects affected by central obesity, we studied 20 centrally obese subjects before and after a 6‐month diet intervention aiming at reducing body weight by 10%, by measuring (i) insulin sensitivity (homeostasis model assessment of insulin resistance (HOMA_IR)); (ii) plasma lipids; (iii) circulating markers of inflammation of adipose tissue and endothelial dysfunction, and of platelet activation (i.e., soluble CD‐40 ligand (sCD‐40L) and soluble P‐selectin (sP‐selectin)); (iv) ability of the NO donor sodium nitroprusside (SNP), the prostacyclin analog Iloprost and the cyclic nucleotide analogs 8‐bromoguanosine 3′,5′‐cyclic monophosphate (8‐Br‐cGMP) and 8‐bromoadenosine 3′,5′‐cyclic monophosphate (8‐Br‐cAMP) to reduce platelet aggregation in response to adenosine‐5‐diphosphate (ADP); and (v) ability of SNP and Iloprost to increase cGMP and cAMP. The 10 subjects who reached the body‐weight target showed significant reductions of insulin resistance, adipose tissue, endothelial dysfunction, and platelet activation, and a significant increase of the ability of SNP, Iloprost, 8‐Br‐cGMP, and 8‐Br‐cAMP to reduce ADP‐induced platelet aggregation and of the ability of SNP and Iloprost to increase cyclic nucleotide concentrations. No change was observed in the 10 subjects who did not reach the body‐weight target. Changes of platelet function correlated with changes of HOMA_IR. Thus, in central obesity, diet‐induced weight loss reduces platelet activation and restores the sensitivity to the physiological antiaggregating agents, with a correlation with improvements in insulin sensitivity.     

Preparative solvent partition chromatography of butyrylated cyclic AMP analogues

A simple and effective separation of cyclic adenosine-3′,5′-monophosphate (cAMP) and its butyryl-substituted analogues using partition chromatography on columns of Sephadex gel in isopropanol/0.5 m ammonium acetate (4:1) is described. The technique is suitable for preparative separations as demonstrated by revised uv spectral data obtained on butyrylated cAMP's purified by this technique. In addition, it has analytical utility in that it allows complete separation of N⁶-monobutyryl cAMP from O^2′-monobutyryl cAMP, thereby permitting simultaneous and independent assessment of the rate of acyl substituent hydrolysis from the disubstituted derivative (N⁶,O^2′-dibutyryl cAMP), and this is demonstrated under several conditions.     

Dibutyryl cyclic adenosine monophosphate enhancement of α-methyl-d-glucoside accumulation by kidney cortex slices

Cyclic adenosine 3′,5′-monophosphate and N⁶-2′-O-dibutyryl cyclic adenosine 3′,5′-monophosphate increase the accumulation of α-methyl-d-glucoside by cortical slices from rat, rabbit, dog and human kidney. The characteristics of the effect have been studied in rat tissue. At least 90 min of exposure of the tissue to cyclic nucleotide prior to onset of glucoside accumulation is required as well as presence of the cyclic nucleotide during the accumulation phase. Inhibition of protein synthesis does not abolish the effect of N⁶-2′-O-dibutyryl cyclic adenosine 3′,5′-monophosphate. The cyclic nucleotide causes an increase in the initial entry rate of α-methyl-d-glucoside into cells and an increase in the intracellular steady state concentration. The cyclic nucleotide does not affect the apparent K_m of the glucoside entry process but increases the maximum velocity of accumulation.     

Interaction of calcium and cyclic nucleotides in thyroliberin-stimulated prolactin release

The object of the present study was to determine the relative importance of Ca++ and cyclic nucleotides as “second messengers” in thyroliberin (TRH)-mediated prolactin (PRL) release in the GH₃ and GH₄ rat pituitary tumor cell lines. PRL, cyclic adenosine 3': 5'-monophosphate (cAMP), and cyclic guanosine 3': 5'-monophosphate (cGMP) were measured by radioimmunoassay (RIA) following TRH stimulation. TRH increased PRL release and cAMP levels in GH₃ and GH₄ cells, but cGMP increases were variable. Treatment with 1 mM theophylline increased PRL release and raised cAMP and cGMP. Addition of TRH to theophylline-pretreated cells produced further significant increases in PRL release without any additional increases in cAMP and cGMP. Co++, a Ca++ antagonist, abolished TRH-induced PRL release in a dose-dependent manner. The Co++ inhibition was partially reversed by Ca++ in GH₃ or GH₄ cells. Furthermore, the Ca++ ionophore A23187 stimulated PRL release. We conclude that Ca++ is the primary “second messenger” for TRH-mediated PRL release from GH₃ or GH₄ cells.     

Effects of multiple phorbol myristate acetate treatments on cyclic nucleotide levels in mouse epidermis.

The basal levels of 3′,5′ adenosine monophosphate and 3′,5′ guanosine monophosphate were measured in mouse epidermis after initiation with 7,12 dimethylbenzanthracene and 1,2,10 or 20 skin treatments with the tumor promoter phorbol myristate acetate. Slight but significant decreases in cAMP and dramatic (5–10 fold) increases in cGMP were found after multiple treatments with the promoter. The cyclic nucleotide levels found in isolated solid tumors closely paralleled these changes.     

Cyclic nucleotide phosphodiesterases in somatic and germ cells of mouse seminiferous tubules

The distribution of phosphodiesterase forms in somatic and germ cells, and their variations during testicular development and germ cell differentiation have been investigated. Seminiferous tubules from immature mice and Sertoli cells in culture possessed two enzyme activities which were comparable to forms described for different tissues and species: (a) a calcium-calmodulin-dependent enzyme with high affinity for guanosine 3',5'-(cyclic)-monophosphate (cGMP), and (b) a calcium-calmodulin-independent enzyme with high affinity for adenosine 3',5'-(cyclic)-monophosphate (cAMP) the activity of which increased in cultured Sertoli cells after treatment with FSH or dibutyryl cAMP. Seminiferous tubules from adult animals and germ cells at the meiotic and post-meiotic stage of differentiation possessed two enzyme forms that could be distinguished from those present in somatic cells of the seminiferous tubules: (a) a calcium-calmodulin-dependent form with high affinity for both cAMP and cGMP, similar to forms described in other tissues from different species, and (b) a calcium-calmodulin-independent phosphodiesterase with high affinity for cAMP and present only in post-meiotic cells, previously identified also in germ cells of the rat.     

Variation of the levels of cyclic AMP and cyclic GMP during development of the insect Ceratitis capitata.

Changes in the levels of adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP) during development were studied in the Dipterous Ceratitis capitata. The developmental patterns were different to each other. Cyclic AMP showed a sharp maximum in the larval stage to decrease afterwards during adult development. Changes of cyclic GMP exhibited an opposite pattern, although its levels were always higher than those of cyclic AMP.     

The effect of N6-2'-O-dibutyryl-adenosine 3',5'-monophosphate on rat liver calciferol 25-hydroxylase.

Liver calciferol 25-hydroxylase activity of vitamin-D deficient rats was enhanced 24 hours following the intravenous injection of N⁶-2′-O-dibutyryl adenosine 3′,5′-monophosphate. Sodium butyrate administered in the same way had no effect on this enzyme system. Administration of actinomycin D with N⁶-2′-O-dibutyryl adenosine 3′,5′-monophosphate abolished the stimulatory effect of the cyclic nucleotide. Direct addition to the incubation medium of adenosine 3′,5′-cyclic monophosphate or of its dibutyryl derivative did not influence the hepatic conversion of cholecalciferol to 25-hydroxycholecalciferol. These results suggest a possible role for the cyclic nucleotide in the regulation of this enzyme system.