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.     

Elevation of guanosine 3′,5′-monophosphate level by adenosine in cerebellar slices of guinea pig

Effect of adenosine on the level of guanosine 3′,5′-monophosphate in guinea pig cerebellar slices was investigated. Adenosine increased the concentration of guanosine 3′,5′-monophosphate in the slices 3–4-fold. Upon removal of adenosine from the medium, the concentration of guanosine 3′,5′-monophosphate returned to the initial level. AMP, ADP or ATP also increased the guanosine 3′,5′-monophosphate level to the same extent as adenosine, while adenine or other nucleotides were not effective. In the absence of Ca²⁺ in the incubation medium, adenosine did not increase the concentration of guanosine 3′,5′-monophosphate in cerebellar slices although level of adenosine 3′,5′-monophosphate was elevated by adenosine.Anticholinergic agents, adrenergic blocking agents or antihistaminics did not prevent the increase of guanosine 3′,5′-monophosphate by adenosine indicating that the effect of adenosine was not mediated by the release of neurotransmitters.The combination of adenosine with depolarizing agents showed an additive effect on the level of guanosine 3′,5′-monophosphate indicating that adenosine increased the level of guanosine 3′,5′-monophosphate by a different mechanism from the depolarization.     

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.     

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.     

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.     

MEDIATION BY β-ADRENERGIC RECEPTORS OF EFFECT OF NOREPINEPHRINE ON PINEAL SYNTHESIS OF [14C]SEROTONIN AND [14C]MELATONIN

Rat pineal organs maintained in organ culture converted [¹⁴C]tryptophan to [¹⁴C]serotonin and [¹⁴C]melatonin. The synthesis of both indoles was stimulated by the presence of norepinephrine or dibutyryl adenosine 3′,5′-monophosphate. This effect of norepinephrine could be blocked by the α-adrenergic blocking drug, propranolol, but was not modified by the a-adrenergic blocking agent, phenoxybenzamine. Neither blocking agent modified the pineal response to dibutyryl adenosine 3′,5′-monophosphate. Unlike dibutyryl adenosine 3′,5′-monophosphate, the naturally occurring adenosine phosphates did not stimulate synthesis of [¹⁴C]melatonin in vitro.     

N6,O2′-dibutyryl adenosine 3′,5′-monophosphate-stimulated release of proteoglycans from cultured immature rabbit ear cartilage

The stimulatory effects of N⁶,O^2′-dibutyryl adenosine 3′,5′-monophosphate on proteoglycans released from immature rabbit ear cartilage were studied in vitro. Cartilage incubated in medium containing dibutyryl cyclic AMP resulted in a significant increase of proteoglycans released in concentrations above 0.5 mM. Theophylline (1 mM) which did not significantly stimulate proteoglycans released alone, was found to potentiate the action of this nucleotide. ATP, 5′-AMP and butyric acid in the presence of theophylline, did not stimulate proteoglycans released. The addition of protein or RNA synthesis inhibitors depressed proteoglycans released by dibutyryl cyclic AMP and theophylline.Gel chromatographic and chemical investigations of the proteoglycans released into the culture media in the presence of dibutyryl cyclic AMP indicated a reduction in the proportion of protein associated with these complexes. This result, together with enzyme inhibitor studies, leads us to speculate that the observed action of dibutyryl cyclic AMP on rabbit ear cartilages may be mediated by the neural proteases.     

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.     

Changes in adenosine 3',5'-monophosphate induced by parathyroid hormone and theophylline in the developing rat renal cortex

To test the biochemical responsiveness of developing rat renal cortex to parathyroid hormone (PTH), intracellular concentrations of adenosine 3′,5′-monophosphate (cyclic AMP) were measured. Renal cortical slices from 10-day-, 20-day-, and 12-week-old animals contained higher concentrations of cyclic AMP when incubated in the presence of theophylline than in its absence. In the absence of theophylline, tissue from all three age groups responded to PTH with dose-dependent increases in cyclic AMP. In the presence of theophylline the response of tissue from 10-day-old animals was greater than that of 12-week-old animals.It is suggested that the differential effect of theophylline with respect to age may be the result of higher turnover rates of cyclic AMP in the young animals.     

The effect of theophylline on adenosine 3′, 5′-monophosphate-dependent protein kinase and ACTH1–24 stimulated steroidogenesis in bovine adrenal cortical cells

Theophylline (theo), a known phosphodiesterase (PDE) inhibitor, was tested for its effects on ACTH_1–24 regulated steroidogenesis in isolated bovine adrenal cortical cells. Theo produced a dose related inhibition of ACTH_1–24 stimulated cortisol synthesis with half maximal inhibition occuring at 7 mM. Theo enhanced ACTH_1–24 stimulated cellular adenosine 3′, 5′-monophosphate (cAMP) levels above that produced by ACTH_1–24 alone confirming its inhibition of cAMP PDE. When tested on cAMP binding protein and cAMP-dependent protein kinase activities in cytosol prepared from bovine adrenal cortex, theo displaced ³H-cAMP binding to cAMP binding protein and inhibited cAMP-stimulated protein kinase activity. The half maximal inhibition of cAMP binding and protein kinase activity was observed at 10 and 5 mM, respectively. Inhibition of cAMP-dependent protein kinase by theo provides a possible explanation of its inhibitory effects on adrenal steroidogenesis and further implicates cAMP-dependent protein kinase in mediating ACTH stimulated steroidogenesis. Furthermore these studies suggest a novel mechanism of action for theo in addition to its known action on cAMP PDE.     

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.     

Adenosine as a putative transmitter in the cerebral cortex. Studies with potentiators and antagonists.

Adenosine has a potent depressant action on cerebral cortical neurons, including identified corticospinal cells. Adenosine 2′-, 3′- and 5′-phosphates, including adenosine 5′-imidodiphosphate, had comparable depressant actions and 2-chloroadenosine was an even more potent depressant. Inhibitors of adenosine uptake, hexobendine and papaverine, potentiated the actions of adenosine and adenosine 5′-monophosphate. Theophylline and caffeine antagonized the depressant actions of adenosine and adenosine 5′-monophosphate. The results are compatible with the hypothesis that adenosine depresses neurons by activating an extracellular receptor and that this effect can be blocked by theophylline and caffeine.     

Glycogen Phosphorylase and Synthase Activities of Rumen Ciliates of the Genus Entodinium

Glycogen phosphorylase and synthase activities were detected in the sonic lysate of rumen ciliates of the genus Entodinium. The ciliate phosphorylase had the following properties. The pH optimum was narrow and centered at pH 5.9. The activity was maximum at 30°C; above 40°C a rapid inactivation occurred. The K_m value for glucose-1-phosphate (G-1-P) and for glycogen was 15 mM and 0.069% (w/v), respectively. NaF and ethylenediamine tetraacetic acid had no stimulative effect on the enzyme activity, though adenosine 3′,5′-monophosphate and theophylline activated it. NaHSO₃ inhibited the enzyme activity at a concentration of 1 mM. The inhibition of glucose was noncompetitive for G-1-P. Glycolytic intermediates and nucleotides had a minor effect on phosphorylase activity. Glycogen synthase existed in two forms, glucose-6-phosphate dependent and independent forms: the proportion of the latter form increased with the decrease of reserve polysaccharide levels in the ciliates. Correlations between glycolytic enzyme activities included phosphorylase and synthase activities and reserve polysaccharide contents in the ciliates were determined, and a possible regulatory mechanism of polysaccharide synthesis and degradation was discussed.     

THE EFFECT OF ELECTRICAL STIMULATION UPON THE ACCUMULATION OF ADENOSINE 3'',5''-PHOSPHATE IN ISOLATED CEREBRAL TISSUE

The application of electrical pulses to slices of guinea pig cerebral cortex led to an increase in the levels of adenosine 3′,5′-phosphate (cyclic 3′,5′-AMP) of more than 11-fold within 10 min. This effect of electrical pulses was severely reduced in the presence of theophylline. Cyclic 3′,5′-AMP accumulation in slices was increased in the presence of norepinephrine and histamine about 1·5-fold and six-fold, respectively; the effect of electrical pulses was augmented in the presence of maximal amounts of either amine. For these and other reasons, the accumulation of cyclic 3′,5′-AMP induced by electrical stimulation cannot be ascribed to the release and action of either histamine or norepinephrine.     

POSTSYNAPTIC ADRENERGIC-CYCLIC AMP CONTROL OF THE SEROTONIN CONTENT OF CULTURED RAT PINEAL GLANDS

Abstract— This investigation was designed to determine whether the amount of serotonin (5-HT) in cultured pineal glands can be altered by norepinephrine (NE). Treatment with l -NE (10^?5-10^?7m ) for 4-6 h caused a gradual decrease in the concentration of 5-HT to a value that was less than 30% of that in the untreated control gland. This effect was observed using chronically denervated pineal glands. d -Norepinephrine (10^?6-10^?7m ) and dopamine (10^?4m ) were ineffective in lowering 5-HT. The effect of l -NE was completely blocked by a β-adrenergic receptor blocker, propranolol and was only slightly decreased by α-adrenergic receptor blockers. These observations indicate that l -NE acts post-synaptically via a highly specific β-adrenergic mechanism. The effect of l -norepinephrine was mimicked by theophylline and N⁶, 2′0-dibutyryl adenosine 3′,5′-monophosphate, an indication that adenosine 3′,5′-monophosphate is involved in the effect of l -NE on 5-HT. Treatment with cycloheximide, which by itself caused a decrease in pineal 5-HT, also blocked any further decrease caused by treatment with l -NE, an indication that protein synthesis is necessary for maintenance of baseline levels of serotonin and for the effect of l -NE to be observed. The total amount of l -[³H]NE and degradation products of L-[³H]NE in the gland after 6 h of treatment with l -[³H]NE was less than 3 pmol. This amount of l -NE and degradation products of l -NE could not account for the decrease of 100-200 pmol of 5-HT on the basis of a mole for mole replacement of 5-HT by l -NE. These findings are consistent with the hypothesis that non-neuronal pineal 5-HT is physiologically regulated by the release of l -NE from the sympathetic nerve network.     

Adducts of Mannose 6-Phosphate with 5-Iodo-2′-Deoxyuridine and 2-5A as Potential Antiviral Agents

Abstract

To examine the possibility that the mannose 6-phosphate receptor system might be capitalized upon to facilitate uptake of nucleotides or nucleotides into cell, adducts of mannose 6-phosphate with 5-iodo-2′-deoxyuridine 5′-monophosphate and with adenosine 5′-monophosphate, p5′A2′p5′A and p5′A2′p5′A2′p5′A were prepared and evaluated for their antiviral activities. The adducts with 2′,5′-oligoadenylates possessed no significant antiviral activity. The adduct with 5-iodo-2′-deoxyuridine 5′-monophosphate showed activity that could be fully explained by extracellular cleavage to free 5-iodo-2′-deoxyuridine.     

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.     

Effect of cyclic AMP and cytochalasin B on tissue cultured melanophores of Xenopus laevis

The effects of cytochalasin B or low concentrations of adenosine 3′,5′-monophosphate (cyclic AMP) were tested on melanophores in hanging drop preparations of neural fold explants from Xenopus laevis embryos in Barths' solution. After one week in culture, the melanophores were punctate in this medium. Cyclic AMP at 5 mM consistently caused reversible morphological transformation of these cells to the stellate state, whether they were situated within an epithelial outgrowth or isolated on the surface of the coverglass. Only the isolated melanophores consistently responded to 1 mM cyclic AMP. Cytochalasin B at 1–10 μg/ml caused aggregation of melanin granules in stellate cells, but left long, narrow cell branches containing some melanosomes. Its effect was at least partially reversible and appeared to be dose dependent. At 1% concentration, dimethyl sulfoxide caused melanin dispersion.     

Comparison of cytokinin activities of the base,ribonucleoside and 5′- and cyclic-3′,5′-monophosphate ribonucleotides of N6-isopentenyl-, N6-benzyl or 8-bromo-adenine

The cytokinin activities of adenosine 3′,5′-monophosphate, N⁶,O^2″-dibutyryladenosine 3′,5−'monophosphate, 8-bromoadenosine 3′,5′-monophosphate, N⁶-(Δ²-isopentenyl)adenosine 3′,5′-monophosphate, and N⁶-benzyladenosine 3′,5′-monophosphate were determined in the tobacco bioassay and compared with the activities of the corresponding non-cyclic nucleotides, nucleosides and bases of the N⁶-isopentenyl-substituted, N⁶-benzyl-substituted, 8-bromo-substituted, and unsubstituted adenine series. In each of these series the cytokinin activities in decreasing order were: bases ⪢ nucleosides ⪖ nucleotides > cyclic nucleotides. All members of the N⁶-isopentenyl- substituted and N⁶-benzyl-substituted series were highly active cytokinins, reaching maximum activity at concentrations of 1 μM or less, whereas, as expected, all members of the unmodified adenine series were inactive in the tested concentration ranges of up to 180 and 200 μM for adenosine and adenine, and 40 μM for the adenine nucleotides. Members of the 8-bromo-substituted adenine series were much weaker cytokinins than the N⁶-substituted adenine derivatives but showed activity in the same sequence starting at a concentration of about 5 μM. Thus, in the cases of 8-bromoadenosine 3′,5′-monophosphate and N⁶,O^2′-dibutyryl-adenosine 3′,5′-monophosphate, both of which have been reported to promote cell division and growth of plant tissues, the cytokinin activity is related to the 8-bromo substituent and to the N⁶-butyryl substituent, respectively, rather than to the 3′,5′-cyclic monophosphate moiety.     

The effect of calcium concentration on ACTH stimulation of steroidogenesis in mouse adrenal tumor cells

Calcium is required for ACTH stimulated steroidogenesis in adrenal tumor cells in tissue culture. In the absence of calcium, the dose of ACTH required to induce half maximum steroidogenesis was increased 30 fold. In contrast to intact adrenal glands or isolated adrenal cells, high doses of ACTH (50 mU/ml) maximally stimulated steroidogenesis in the absence of calcium. Growth for up to six days in medium with low calcium did not affect basal or ACTH induced steroidogenesis. The addition of calcium to cells incubated with ACTH produced a maximum steroidogenic response in 15 minutes. In contrast to intact adrenal glands, calcium is not required for adenosine-3′,5′-cyclic monophosphate (cyclic AMP) stimulated steroidogenesis in adrenal tumor cells. These experiments support the concept that calcium is important at the level of ACTH-membrane receptor site interaction or activation of adenyl cyclase in adrenal tumor cells.