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.     

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 Na Fstimulated 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.     

Cyclic nucleotide levels in brains of control and hypertensive rats.

Adenosine 3′, 5′-monophosphate (cyclic AMP) and guanosine 3′,5- monophosphate (cyclic GMP) levels were measured in seven brain areas of spontaneously hypertensive rats (SHR) and two groups of control rats. In cerebral cortex, hypothalamus, pons-medulla oblongata and cerebellum cyclic AMP levels were higher in SHR than in Wistar-Kyoto controls. Cyclic GMP levels were higher in SHR than in Wistar-Kyoto rats in all brain areas except for the striatum and hippocampus where the levels were lower. There were also some differences in cyclic nucleotide levels between Wistar-Kyoto and Wistar-Charles River controls.     

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.     

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.     

Cyclic nucleotide signaling changes associated with normal aging and age-related diseases of the brain

Deficits in brain function that are associated with aging and age-related diseases benefit very little from currently available therapies, suggesting a better understanding of the underlying molecular mechanisms is needed to develop improved drugs. Here, we review the literature to test the hypothesis that a break down in cyclic nucleotide signaling at the level of synthesis, execution, and/or degradation may contribute to these deficits. A number of findings have been reported in both the human and animal model literature that point to brain region-specific changes in Galphas (a.k.a. Gαs or Gsα), adenylyl cyclase, 3′,5′-adenosine monophosphate (cAMP) levels, protein kinase A (PKA), cAMP response element binding protein (CREB), exchange protein activated by cAMP (Epac), hyperpolarization-activated cyclic nucleotide-gated ion channels (HCNs), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), soluble and particulate guanylyl cyclase, 3′,5′-guanosine monophosphate (cGMP), protein kinase G (PKG) and phosphodiesterases (PDEs). Among the most reproducible findings are 1) elevated circulating ANP and BNP levels being associated with cognitive dysfunction or dementia independent of cardiovascular effects, 2) reduced basal and/or NMDA-stimulated cGMP levels in brain with aging or Alzheimer's disease (AD), 3) reduced adenylyl cyclase activity in hippocampus and specific cortical regions with aging or AD, 4) reduced expression/activity of PKA in temporal cortex and hippocampus with AD, 5) reduced phosphorylation of CREB in hippocampus with aging or AD, 6) reduced expression/activity of the PDE4 family in brain with aging, 7) reduced expression of PDE10A in the striatum with Huntington's disease (HD) or Parkinson's disease, and 8) beneficial effects of select PDE inhibitors, particularly PDE10 inhibitors in HD models and PDE4 and PDE5 inhibitors in aging and AD models. Although these findings generally point to a reduction in cyclic nucleotide signaling being associated with aging and age-related diseases, there are exceptions. In particular, there is evidence for increased cAMP signaling specifically in aged prefrontal cortex, AD cerebral vessels, and PD hippocampus. Thus, if cyclic nucleotide signaling is going to be targeted effectively for therapeutic gain, it will have to be manipulated in a brain region-specific manner.     

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.     

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.     

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.     

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.     

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.     

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.     

Rolipram,a selective c-AMP phosphodiesterase inhibitor suppresses oro-facial dyskinetic movements in rats

Since striatal dopamine D2 receptor supersensitivity in the etiology of tardive dyskinesia has been suggested and dopamine D2 receptors are known to inhibit adenylate cyclase activity resulting in a decrease of cyclic adenosine 3′,5′-monophosphate (cAMP) levels, we hypothesized that an increase in cAMP levels ameliorates the condition. In the present study, 21-day haloperidol treatment (1.5 mg/kg I.P.) in rats resulted in an increase in striatal [³H]-spiperone (D2) binding whereas [³H] SCH23390 (D1) binding was unaltered. This haloperidol treatment also induced a significantly increase in the frequency of involuntary chewing movements and tongue protrusions, which are considered as a model of tardive dyskinesia. These dyskinetic movements were suppressed by administration of rolipram (0.5 and 1.0 mg/kg I.P.), an inhibitor of the cAMP phosphodiesterase type IV. The present results suggest that selective cAMP phosphodiesterase type IV inhibitors could be putative therapeutic drugs for tardive dyskinesia.     

Properties and cellular distribution of cyclic AMP-dependent protein kinase from thymus

A protein kinase that catalyzes the phosphorylation of histone was partially purified from rat thymus, and the rate of histone phosphorylation was stimulated three- to fourfold by 1 × 10^?6 M adenosine 3′,5′-monophosphate (cyclic AMP). Thymic protein kinase was more active than the enzyme from spleen. Histone fractions f1, f2a, f2b, and f3 were all capable of serving as phosphate acceptors for the thymic protein kinase, and the rate of phosphorylation of each fraction was stimulated by cyclic AMP. The ability of various 3′,5′-mononucleotides to stimulate protein kinase activity was compared. Inosine 3′,5′-monophosphate (cyclic IMP) was the most effective substitute for cyclic AMP. The cellular distribution of cyclic AMP-dependent protein kinase and adenylate cyclase activities in the thymus was determined. Cyclic AMP-dependent protein kinase activity is present in both small thymocytes and residual thymic tissue. The specific activity of protein kinase from residual tissue, both for basal and cyclic AMP-stimulated enzyme, was greater than that of enzyme from small thymocytes. In contrast to this, adenylate cyclase activity is predominately localized in the thymocytes.     

In vivo regulation of hepatic protein kinase by adenosine 3',5'-monophosphate mediated glucagon stimulation

$\text{In vivo}$ administration of glucagon caused an increase in the dissociation of protein kinase subunits which was accompanied by elevated adenosine 3′,5′-monophosphate concentrations in the rat liver. Concomitantly, there was a decrease in non saturated adenosine 3′,5′-monophosphate binding sites. A reduction in protein kinase activity measured in the presence of the cyclic nucleotide was apparent at 5 minutes of glucagon administration while enzyme activity assayed in the absence of adenosine 3′,5′-monophosphate was already increased after one minute. Glucose, given through an intragastric tube, caused no changes in the effect of glucagon on hepatic protein kinase.     

Protamine kinase independent of adenosine 3',5'-monophosphate from rat brain cytosol

A protein kinase was obtained from rat brain cytosol which phosphorylated preferentially protamine and to some extent histone. This enzyme was independent of adenosine 3′,5′-monophosphate (cyclic AMP) and was not identical with the catalytic unit of cyclic AMP-dependent protein kinase. The enzyme and cyclic AMP-dependent protein kinase from this tissue were distinguishable from each other in their kinetic and catalytic properties, and phosphorylated different seryl and threonyl residues of protamine and histone.     

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.     

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.     

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.     

Effect of phosphaden and unithiol on the cyclase system of the small intestine mucosa in rabbits in experimental salmonellosis

The study was made on 59 chinchilla rabbits. S. typhimurium 1847 live culture was introduced into the lumen of an isolated loop of the thin intestine. The activity of adenylate cyclase (AC), guanylate cyclase (GC), the levels of cyclic adenosine 3,5-monophosphate (cAMP), cyclic guanosine 3,5-monophosphate (cGMP), the activity of cAMP- and cGMP-phosphodiesterases were determined in the mucous membrane of the ligated part of the intestine. Considerable fluid accumulation in the loop, activation of AC and cGMP-phosphodiesterase, a rise in the level of cAMP and a drop in the level of cGMP in the mucosa of the ligated part of the intestine were registered. In one group of the animals phosphadene and in the other group unitiol were introduced into the infected intestinal loop; as a result, a decrease in the accumulation of fluid in the loop, on the average, by 40% and a tendency to an increase in the level of cAMP and a drop in the level of cGMP in the mucous membrane of the ligated part of the intestine were observed. Changes in the level of cGMP play, seemingly, a more important role in the development of diarrhea in salmonellosis.