Calcium-dependent accumulation induced by carbamylcholine of guanosine 3′,5′-monophosphate in rabbit choroid plexus

An active guanylate cyclase system was detected in isolated choroid plexus of rabbits by sodium azide (6 × 10^?5 mol/l) which increased cGMP levels tenfold within 15 min. Inhibition of cGMP phosphodiesterase by sodium azide was excluded. cGMP accumulation was also raised dose-dependently by carbamylcholine, a cholinergic agonist. Pretreatment of chroid plexus with atropine (10^?7 mol/l) reduced the effect of carbamylcholine (5 × 10^?5 mol/l) by 80%. Both carbamylcholine and sodium azide induced accumulation of cGMP also in the incubation medium, indicating rapid extrusion of the nucleotide from choroid plexus cells. The effect of carbamylcholine could be mimicked by the calcium ionophore A 23187. Incubation in calcium-free medium abolished cGMP accumulation by carbamylcholine and A 23187 but not by sodium azide, indicating a different mechanism of action. Sodium azide, carbamylcholine and A 23187 had no effect on cyclic AMP levels. Withdrawal of calcium led to an enhanced efflux of both cAMP and cGMP. Since a cholinergic innervation of stroma and epithelial cells has been described, we hypothesize that cGMP and calcium may be involved in cholinergic transmission regulating blood flow or transport processes of the choroid plexus.     

Effects of glucagon, 3′,5′-AMP and 3′,5′-GMP on ion fluxes and transmembrane potential in perfused livers of normal and adrenalectomized rats

The effects of glucagon, 3′,5′-AMP, 3′,5′-GMP and dexamethasone on ion fluxes and transmembrane-potential changes were compared in perfused livers from normal and adrenalectomized rats. Glucagon and cyclic nucleotide administration resulted in a similar redistribution of Na⁺ and K⁺ and membrane hyperpolarization in both groups. Dexamethasone at a dose which restores the gluconeogenic response after adrenalectomy, had no effect on either the ion movements or membrane potential and did not alter the responses to cyclic nucleotides or glucagon in either normal or adrenalectomized rat livers. These results suggest that the permissive effect of glucacorticoids on gluconeogenesis might be related to an event following ion movement.     

Effect of cyclic 3′:5′-AMP derivatives,prostaglandins and related agents on human chorionic gonadotropin secretion in human malignant trophoblast in culture

Summary The secretion of human chorionic gonadotropin (hCG) is stimulated by addition of N⁶, O^2′-dibutyryl cyclic 3′:5′-AMP (dbcAMP) or theophylline to normal term placenta and human malignant trophoblast cells in vitro. To understand better the specificity of this process. malignant trophoblast cultures were incubated with 3′:5′-cyclic AMP (cAMP) derivatives, prostaglandins and other agents for 1 to 3 days, and the secretion of radioimmuno-assayable hCG was measured. Whereas dbcAMP was the most potent agent in stimulating secretio of hCG, the N⁶- and O^2′-monobutyryl derivatives of cAMP and phosphodiesterase inhibitors (theophylline, papaverine, 3-isobutyl-1-methylxanthine) also increased the secretion of the hormone. A slight increase in hCG secretion was observed following addition of adenine. By contrast, butyrate, cAMP, cyclic 3′:5′-GMP (cGMP), dbcBMP, 5′-AMP, adenosine, L-epinephrine and prostaglandins E₁, E₂, F_1α and F_2α were ineffective. Particulate fractions from sonicates of malignant trophoblast cultures contained adenylate cyclase activity which was stimulated more than 10-fold by NaF, but not by either catecholamines or prostaglandins. The relatively specific stimulation of hCG secretion suggested that a regulatory process involving cAMP may have physiological significance in the trophoblast. This investigation was supported by Grant Nos. CA14232 and CA16539 awarded by the National Cancer Institute, DHEW.     

Influence of theophylline on concentrations of cyclic 3′,5′-adenosine monophosphate and cyclic 3′,5′-guanosine monophosphate of rat brain

The influence of theophylline (2.5–100 mg/kg p.o.) on cyclic 3,5-adenosine monophosphate (cAMP) and cyclic 3,5-guanosine monophosphate (cGMP) in brain of Sprague-Dawley rats (0.5–3.0 hr after administration of theophylline) was investigated. It was found that theophylline increases cAMP and cGMP levels when administered in a dose of 25 mg/kg or higher. A significant decrease of cGMP level was observed after administration of 10 mg/kg. The results of this study suggest that the influence of theophylline on cyclic nucleotide levels of rat brain is the result of two factors: (a) inhibitory properties of theophylline on cAMP and cGMP phosphodiesterases and (b) competition of theophylline with adenosine.     

Mutants of Serratia marcescens lacking cyclic nucleotide phosphodiesterase activity and requiring cyclic 3′,5′-AMP for the utilization of various carbohydrates

Adenine requiring mutants of Serratia marcescens SM-6-F'lac ⁺ have been found to grow well in minimal-glucose medium solely supplemented with cAMP. From one of these ade strains double mutants (called ade cpd) were isolated which could no longer utilize cAMP but which still grew on 5′AMP. Dialyzed cell extracts (soluble fraction) of the double mutants, assayed for cAMP phosphodiesterase, were unable to hydrolyze cAMP whereas cell extracts of the parental strains yielded 5′AMP at a rate of 1.6–2.0 μmoles min⁻¹ mg⁻¹ protein. The loss of the phosphodiesterase activity in S. marcescens cpd W1181 did not cause an accumulation of large amounts of cAMP as was found for the diesterase-negative mutant AB257^pc-1 of Escherichia coli. The induced synthesis of β-galactosidase in mutant cpd W 1181 showed about the same sensitivity to transient and permanent catabolite (glucose) repression as the corresponding cpd ⁺ strain. Starting from S. marcescens cpd W1181 three independent double mutants (called cpd cya) were isolated which required exogenous cAMP for utilizing various carbohydrates as carbon source, for motility and for the formation of extracellular lipase and the red pigment prodigiosine. The intracellular concentration of cAMP in these mutants, grown in nutrient broth, was 40–60% of that of the parental strain which is about 4×10⁻⁴ M. However, the adenylate cyclase in cell extracts of the mutants W1237 and W1270 was like that of the corresponding cya ⁺ strain (about 2×10⁻² μmoles min⁻¹ mg⁻¹ protein).     

Role of cyclic adenosine-3′,5′-monophosphate in olfactory reception

The action of cyclic adenosine-3,5-monophosphate (3,5-AMP) and of substances modifying the rate of its breakdown (inhibitors and activators of phosphodiesterase) on the olfactory epithelium was investigated in frogs. The slow electrical response of the olfactory epithelium to stimulation by solutions of various substances was recorded. Cyclic 3,5-AMP and its dibutyryl derivative were found to excite the olfactory receptors effectively. Responses to these substances developed after an appreciably longer delay than responses to stimulation by solutions of odiferous substances. It is postulated that the depolarizing action of 3,5-AMP and dibutyryl 3,5-AMP is manifested only after they have penetrated inside the receptor cell through its membrane. Both 5-AMP and cyclic 2,3-AMP were ineffective. In the next series of experiments the integral receptor potential was recorded in response to short stimulation by the vapor of an odiferous substance. The duration of this potential was increased after treatment of the olfactory epithelium with phosphodiesterase inhibitors: methylxanthines or papaverine. Conversely, the negative wave of the integral receptor potential was shortened under the influence of the phosphodiesterase activator imidazole. Cyclic 3,5-AMP is considered to play the role of mediator in the mechanism of excitation of the olfactory receptor; during interaction between an odiferous substance and the receptor, adenyl cyclase is activated and the concentration of 3,5-AMP increases; this, in turn, causes depolarization of the receptor cell membrane.Institute of Chemical Physics, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 5, No. 4, pp. 415–422, July–August, 1973.     

Ontogeny of adenosine 3′,5′-monophosphate metabolism in rabbit cerebral cortex

The effects of norepinephrine (NE), histamine (HIST), glutamate, and adenosine, singly and in combinations, on the accumulation of adenosine 3,5-monophosphate (cyclic AMP) in slices of rabbit cerebral cortex were examined using tissue from animals 4 days before to 38 days after birth. A response to NE became visible 2 days before birth and increased to - maximum at 7 days after birth before declining toward the small adult value during the second post-natal week. During this period NE was at least twice as efficacious as isoproterenol, and both - and -adrenergic antagonists had prominent inhibitory effects. Responses to HIST were already apparent 4 days before birth and increased in an irregular fashion thereafter, sometimes exceeding the adult value during the second post-natal week. The response to adenosine was not visible until birth and gradually increased toward the adult value during the entire period examined. Synergistic responses to various combinations of the three agents were first detected at 2 to 4 days before birth. The degree of synergism was larger during the neonatal period than that found in adult tissue; no synergism between HIST and adenosine persisted in the adult. During the first post-natal week, L-glutamate produced very large increases of cyclic AMP accumulation in the presence of either adenosine or histamine plus theophylline; smaller but substantial responses occurred in combination with NE. Responses to glutamate declined progressively after about the tenth post-natal day.     

Rapid change in cyclic 3′,5′-AMP concentration triggered by a light-off or light-on signal in Anabaena cylindrica

In a nitrogen-fixing cyanobacterium, Anabaena cylindrica, an immediate light-to-dark transition caused an increase in cAMP concentration within 1 min. Conversely, a dark-to-light transition caused a rapid fall in cellular cAMP concentration. The concentration of cyclic 35-GMP(cGMP) was several times lower than that of cAMP and was not largely affected by a light-off or light-on signal.     

Studies of effects of cyclic adenosine 3′,5′-monophosphate in regulation of human hemopoiesis in vitro

Summary Prostaglandin E₁ (PGE₁), high concentrations of dibutyryl cyclic AMP (dbcAMP), and theophylline were strikingly inhibitory both to tritiated thymidine ([³H]TdR) incorporation into bone marrow deoxyribonucleic acid (DNA) in vitro and to granulocytic colony growth. Autoradiography revealed that lower concentrations of dbcAMP were stimulatory to red blood cell precursors. This study was supported in part by United States Public Health Service Grant AM15163, by Health Research Council of the City of New York Career Scientist Award I-683, and by a Veterans Administration Medical Investigatorship to V. H.     

Histochemical method for localization of cyclic 3′, 5′-nucleotide phosphodiesterase

Summary A histochemical method has been described for demonstration of cyclic 3, 5-nucleotide phosphodiesterase in tissues. 5-AMP is formed due to splitting of substrate cyclic 3, 5-AMP by cyclic 3, 5-AMPase. The 5-AMP is split into adenosine and phosphate by the 5-nucleotidase from added snake venom. Endogenous tissue 5-nucleotidase would contribute to this activity. The phosphate was in turn visualized by conversion to the lead salt in the presence of lead acetate and finally into brownish-black lead sulphide by treatment with yellow ammonia sulphide. Control studies with and without substrate and snake venom, as well as inhibition by theophylline, indicate the test to be specific for cyclic 3, 5-AMPase.In the eye the conjunctiva, ciliary process, choroid and retina all showed strongly positive activity. In the kidney the proximal and distal tubules both ascending and descending and the loop of Henle show strongly positive activity — the rest of the elements being negative. The cardiac and skeletal muscle exhibited very little positive activity. The liver showed only mildly positive activity. The villi of the small intestine showed strongly positive activity at the apical part of the cells. Neurons showed very little positive activity in either the cerebral cortex or the cerebellum. On the other hand, the molecular layer in the cerebellum and the plexiform layer of the cerebral cortex showed strongly positive activity. The significance of these findings are briefly discussed. T. R. Shanthaveerappa — in previous publications.     

Involvement of cyclic adenosine-3′, 5′-monophosphate in chloronema differentiation in protonema cultures of Funaria hygrometrica

The role of purine and pyrimidine ribosides, nucleotides and substituted xanthines in the differentiation of chloronema filaments in suspension cultures of protonema of the moss Funaria hygrometrica Hedw. has been examined. Cyclic adenosine-3,5-monophosphate (cAMP) and mono-and dibutyryl cAMP evoked the maximum response in wild-type protonema. ADP and ATP also enhanced chloronema differentiation but were less active than cAMP; pyrimidine derivatives were completely inactive. Inhibitors of cyclic-nucleotide phosphodiesterase aminophylline, theophylline and ICI 58, 301 (3-acetamido-6-methyl-8-n-propyl-s-triazolo-(4,3a)-pyrazine)-mimicked the effect of cAMP. A leaky, chloronema-repressed mutant was isolated and in this mutant cAMP was much more active than cyclic guanosine monophosphate and ADP in enhancing chloronema differentiation. These results strongly indicate that cAMP is involved in chloronema differentiation in Funaria, and a hypothesis on growth regulation in protonema cell cultures is proposed.Abbreviations cAMP, cyclic AMP cyclic adenosine-3, 5-monophosphate - cCMP, cGMP, cIMP cyclic cytosine-, guanosine-and inosine-3, 5-monophosphates, respectively - IAA indole-3-acetic acid - ICI 58,301 3-acetamido-6-methyl-8-n-propyl-s-triazolo-(4,3a)-pyrazine     

Naturally produced isocoumarins: inhibitors of calmodulinsensitive cyclic guanosine 3′,5′-monophosphate phosphodiesterase

Summary Three isocoumarins have been isolated from a strain ofStreptoverticillium sp. and all inhibit the calmodulin-sensitive cyclic guanosine 3,5-monophosphate phosphodiesterase (EC 3.1.4.17, Boehringer Mannheim). Two of the compounds, 6,8-dihydroxy-7-methoxy-3-methyl isocoumarin and 6,7,8-trihydroxy-3-methyl isocoumarin have previously been isolated fromStreptomyces. The third fermentation product, 6,8-dihydroxy-3-methyl isocoumarin, was also found as a metabolite ofCeratocystis minor, a fungal species associated with the blue stain disease of pine [2,3].     

Does cyclic-3′,5′-AMP induce glutamine synthetase in embryonic neural retina?

The inducibility of retinal glutamine synthetase (GS) by dibutyryl cyclic-3′,5′-AMP (DB-cAMP) was re-examined in view of conflicting reports. Various lots of DB-cAMP were compared for a) ability to induce GS in cultures of embryonic chick neural retina, and b) their composition as visualized by paper chromatography. Chromatographically purified DB-cAMP did not induce retinal GS, nor did cAMP, DB-cGMP, epinephrine, or norepinephrine; none of these enhanced the induction of GS by hydrocortisone. Some of the agents occasionally caused small increases in GS activity; however, these were invariably below the GS levels induced consistently by hydrocortisone. A single lot of DB-cAMP was found which significantly raised GS activity in the retina; it contained a contaminant which when isolated was found to be responsible for this effect.     

Relative sensitivity of undifferentiated and cyclic adenosine 3′, 5′-monophosphate-induced differentiated neuroblastoma cells to cyclosporin A: potential role of β-amyloid and ubiquitin in neurotoxicity

Cyclosporin A is routinely used in transplant therapy following allogeneic or xenogeneic tissue transplantation to prevent rejection. This immunosuppressive drug is also neurotoxic; however, its mechanisms of action for neurotoxicity are poorly understood. Undifferentiated and cyclic adenosine 3',5'-monophosphate (cAMP)-induced differentiated neuroblastoma (NB) cells were used as an experimental model to study the toxicity of cyclosporin A. Results showed that cyclosporin A promoted the outgrowth of neurites and inhibited the growth of undifferentiated NB cells. When cyclosporin A was added simultaneously with RO20-1724, an inhibitor of cyclic nucleotide phosphodiesterase, or with prostaglandin E1, a stimulator of adenylate cyclase, it markedly enhanced the growth inhibitory and differentiation effects of these cAMP-stimulating agents. In addition, cyclosporin A added to cAMP-induced differentiated NB cells caused dose-dependent degeneration of these cells as evidenced by the vacuolization of cytoplasm and the fragmentation of nuclear and cytoplasmic materials; however, neurites remained intact. Cyclosporin A alone did not alter the intensity of cell immunostaining for ubiquitin or beta-amyloid peptide (amino acids 1-14) (Abeta1-14); however, it enhanced the intensity of staining for both ubiquitin and Abeta in cells that were treated with cAMP-stimulating agents. The intensity of staining of amyloid precursor protein (amino acids 44-63) (APP44-66) did not change in any treated group, suggesting that the increase in Abeta staining is due to increased processing of APP to Abeta. We propose that one of the mechanisms of cyclosporin A-induced neurotoxicity involves increased levels of Abeta and ubiquitin.     

Effect of adenosine 3′,5′-(cyclic)-monophosphate on the synthesis of progestational steroids by rabbit ovarian tissue in vitro

1. Adenosine 3',5'-(cyclic)-monophosphate (3',5'-AMP) stimulates the synthesis of progestational steroids by rabbit ovarian tissue in vitro. 2. Other adenosine phosphates fail to increase steroidogenesis. 3. The ratio of 20alpha-hydroxypregn-4-en-3-one to progesterone, the maximal response of the tissue, and the responses of separated corpora lutea and interstitial tissue produced by luteinizing hormone are closely paralleled by 3',5'-AMP. 4. In tissues maximally stimulated by luteinizing hormone, 3',5'-AMP fails to produce an additional response. 5. The addition of theophylline, an inhibitor of phosphodiesterase, potentiates the effects of 3',5'-AMP and also luteinizing hormone. 6. The results obtained suggest that 3',5'-AMP is a mediator of the action of luteinizing hormone on progestational steroid synthesis by rabbit ovarian tissue.     

Induction of blastocladiella emersonii germination by cyclic adenosine-3′, 5′-monophosphate

The role of adenosine 3′,5′-monophosphate (cyclic AMP) in the control of Blastocladiella emersonii germination was studied. This differentiative transition may be induced by replacing K⁺, a classical inducer, by cyclic AMP or by competitive inhibitors of cyclic AMP phosphodiesterase activity. When zoospores are treated simultaneously with two inducers at non-effective concentrations, a synergistic effect is observed between cyclic AMP and either KCl or adenine. The calcium ionophore A23187 per se is not able to elicit germination, but the association of A23187 and sub-optimal concentrations of cyclic AMP is effective. These results suggest that germination may depend on a correlation between the intracellular mobilization of calcium and cyclic AMP levels.     

Adsorption of 5′-AMP and catalytic synthesis of 5′-ADP onto phosphate surfaces: Correlation to solid matrix structures

A non-enzymatic formation of 5-ADP starting from phosphorylation of 5-AMP in the presence of either calcium phosphate or calcium pyrophosphate precipitates is reported. This reaction is taken as a model for the study of heterogeneous catalysis of transphosphorylation in prebiotic conditions. Experiments were performed in completely aqueous media and in media containing dimethyl sulfoxide (Me₂S0), to simulate periods of dehydration in primitive aquatic environments. It has been observed that the nucleotide is adsorbed onto both calcium phosphate and calcium pyrophosphate in accordance with Langmuir isotherms. Adsorptive capacity and affinity of the precipitates for nucleotide are changed by the presence of Me₂SO, suggesting that the interaction between biomonomers and surfaces can be modulated by the degree of hydration of the anionic components of these compounds. In completely aqueous environments, formation of 5-ADP from 5-AMP adsorbed on precipitates of calcium phosphate and calcium pyrophosphate is very small. However, in the presence of 60% Me₂SO this synthesis increases by factors of 3 and 6 for surfaces of calcium phosphate and calcium pyrophosphate, respectively, and follows first-order kinetics. Determinations of free energy changes show that phosphorylation of 5-AMP adsorbed to these precipitates is thermodynamically favorable. Depending on the precipitation time of the samples and the composition of the medium, structural analysis of these precipitates by electron and X-ray diffraction shows changes in their cristallinity grade. It is proposed that these changes are responsible for the modulation of the quantity of adsorbed nucleotides to the surface of solid matrices as well as the catalytic activity of the precipitates.Abbreviations 5-AMP 5-adenosine monophosphate - 5-ADP 5-adenosine diphosphate - BTP l,3-bis[tris(hydroxymethyl)-methylamino]propane - CTEM conventional transmission electron microscopy - Tris tris(hydroxymethyl)aminomethane - Pi (H₂PO ₄ ^– /HPO ₄ ^2– ) orthophosphate - Pi.Ca calcium phosphate - PPi (H₃P₂O ₇ ^– /H₂P₂O ₇ ^2– ) pyrophosphate - PPi.Ca calcium pyrophosphate - EGTA [ethylenebis(oxyethylene)nitrilo]tetraacetic acid This work has been submitted to the Department of Biochemistry, Institute of Biomedical Sciences, UFRJ, by A.C.T. in partial fulfillment of requirements for the MS degree.     

In vivo binding of 2,3,6,2′,3′,6′-hexachlorobiphenyl and 2,4,5,2′,4′,5′-hexachlorobiphenyl to mouse liver macromolecules

The role of metabolic activation in the binding of polychlorinated biphenyls (PCBs) to cellular macromolecules was investigated in vivo by comparing the relative binding of 2,4,5,2′,4′,5′-[U-¹⁴C]hexachlorobiphenyl (2,4,5), a slowly metabolized PCB, with that of 2,3,6,2′,3′,6′-[U-¹⁴C]hexachlorobiphenyl (2,3,6), a rapidly metabolized PCB, and the appropriate controls. Each hexachlorobiphenyl was administered to mice, orally for 5 days (7.28 mg/kg/day). Following the dosing schedule, animals were killed at 1, 5 and 8 days. The concentration of each PCB was determined in liver, muscle and kidney and in purified macromolecules isolated from those tissues. The concentration of 2,4,5 was consistently higher than the concentration of 2,3,6 in all tissues studied. However, the amount of 2,3,6 bound to the purified macromolecules was consistently at least one order of magnitude greater than that of 2,4,5. The greatest binding was observed in RNA followed by protein and DNA, respectively. The purity of the macromolecules and the presence of PCB-derived radioactivity at the monomer level were confirmed. This is the first report of ¹⁴C-labeled PCB being bound to purified RNA, DNA, and proteins isolated from the tissues of animals treated in vivo. The binding is thought to be covalent and to be the result of metabolic activation.     

New Approach to the Synthesis of 2′,3′-dideoxyadenosine and 2′,3′-Dideoxyinosine

Abstract

A new approach to the synthesis of 2′,3′-dideoxyadenosine and 2′,3′-dideoxyinosine based on deoxygenation of 2′,3′-di-O-mesylnucleosides was developed.