Stimulation by Dibutyryl Cyclic AMP of Serotonin Synthesis and Tryptophan Transport in Brain Slices

N⁶,O²-′Dibutyryl cyclic AMP (dibutyryl cyclic AMP), a derivative of 3′,5′-adenosine monophosphate (cyclic AMP) resistant to phosphodiesterase inactivation, has been reported to stimulate serotonin and melatonin synthesis in the pineal gland in vitro^1–3. In brain adenyl cyclase and phosphodiesterase, which catalyse the formation and the inactivation of cyclic AMP, are found chiefly in the synaptosomal fraction of the tissue homogenates⁴, where vesicles containing monoamine are also present⁵. These factors prompted us to study the effects of cyclic AMP and its dibutyryl derivative on the synthesis of brain monoamines.     

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.     

Difference in the Cyclic Adenosine 3′,5′-Monophosphate Levels in Normal and Transformed Cells

CYCLIC 3′5′-adenosine monophosphate (cyclic AMP) regulates many physiological phenomena^1,2. Cellular morphology changes when the dibutyryl derivative of cyclic AMP is added in vitro to the nutrient media of cultured mammalian cells^3–6. Dibutyryl cyclic AMP has also been shown to restore controlled growth to transformed cells³, change the cell's surface architecture^3,7 and induce axon formation⁸ with an accompanied increase in acetylcholinesterase activity⁹ in neuroblastoma cells growing in culture. These effects suggest that the cyclic AMP moiety may have some basic regulatory action on cell growth and cell specialization.     

Similar and opposite effects of dibutyryl cyclic AMP and insulin on glucose metabolism in adipose tissue

The effects of N⁶-2′-O-dibutyryl cyclic AMP on glucose metabolism and lipolysis in fragments of rat epididymal adipose tissue were studied. Measurements were made of glucose uptake, conversion of glucose carbon to CO₂ and tissue fatty acids and glyceride-glycerol, lactate production, and glycerol release. Low concentrations of dibutyryl cyclic AMP (0.1–0.5 mM) increased all parameters of glucose metabolism and inhibited glycerol release in tissue from both normally fed and fasted rats. Higher concentrations of dibutyryl cyclic AMP (3–5 mM) diminished glucose utilization and greatly accelerated lipolysis. Insulin, 50 μunits/ml, accelerated glucose metabolism in the presence of either low or high concentrations of dibutyryl cyclic AMP though the effect of insulin was greatly reduced by 3 mM dibutyryl cyclic AMP. Tissue exposed to concentrations of dibutyryl cyclic AMP which inhibited glucose metabolism (5 mM), then rinsed and reincubated without dibutyryl cyclic AMP, displayed increased glucose utilization. The results of these experiments emphasize the need for caution in interpretation of the effects of dibutyryl cyclic AMP on adipose tissue metabolism and the need for further research to elucidate the role of cyclic AMP in the regulation of glucose metabolism.     

Cyclic AMP and platelet prostaglandin synthesis

The present study has investigated the influence of agents which elevate intracellular levels of endogenous platelet adenosine 3′5′-cyclic monophosphate (cyclic AMP), and the effect of the exogenous cyclic AMP analog, dibutyryl cyclic AMP, on the conversion of ¹⁴C-arachidonic acid by washed platelets. Prostaglandin E₁ (PGE₁), PGE₁ with theophylline, or dibutyryl cyclic AMP incubated with washed platelets prevented arachidonic acid induced platelet aggregation, but had no effect on the conversion of arachidonic acid to 12L-hydroxy-5,8,10, 14-eicosatetraenoic acid (HETE), 12L-hydroxy-5,8,10 heptadecatrienoic acid (HHT), or thromboxane B₂. Ultrastructural studies of the platelet response revealed that agents acting directly or indirectly to increase the level of cyclic AMP inhibited the action of arachidonic acid on washed platelets and prevented internal platelet contraction as well as aggregation. The influence of PGE₁ with theophylline, and dibutyryl cyclic AMP on the thrombin induced release of ¹⁴C-arachidonic acid from platelet membrane phospholipids was also investigated. These agents were found to be potent inhibitors of the thrombin stimulated release of arachidonic acid from platelet phospholipids, due most likely to an inhibition of platelet phospholipase A activity. The results show that dibutyryl cyclic AMP and agents which elevate intracellular cyclic AMP levels act to inhibit platelet activation at two steps 1) internal contraction and 2) release of arachidonic acid from platelet phospholipids.     

N<Superscript>6</Superscript>,O<Superscript>2</Superscript>′-Dibutyryl Adenosine 3′,5′-Monophosphate induces Pigment Production in Melanoma Cells

IN VITRO studies have suggested that adenosine 3′,′-monophosphate (cyclic AMP) regulates cell morphology. During treatment with the dibutyryl analogue of cyclic AMP, N⁶,O²′-dibutyryl cyclic AMP, transformed fibroblasts acquire several morphological characteristics of untransformed fibroblasts^1,3. Cell processes are extended, the cells occupy a greater surface area and in some cases there is a parallel alignment of cells. Chinese hamster ovary cells are affected in the same way. In neuroblastoma cells⁵, dibutyryl cyclic AMP induces neurite extension and increases the activity of acetylcholinesterase, an indicator of biochemical differentiation⁶. Cyclic AMP is known to control the dispersion of melanin^7,8 and the differentiation of melanoblasts into melanocytes. We have now found that during treatment with dibutyryl cyclic AMP, melanoma cells spread out, appear larger and produce considerably more pigment than untreated cells.     

Cyclic AMP-induced tyrosinase synthesis in Neurospora crassa

Cyclic AMP induces the synthesis of tyrosinase in $\text{Neurospora crassa}$. Adenine, adenosine, 3′-AMP, 5′-AMP, and 2′,3′-cyclic AMP have no inductive effect while 8-bromocyclic AMP and dibutyryl cyclic AMP are good inducers. Caffeine and theophylline, inhibitors of cyclic AMP phosphodiesterase, also induce tyrosinase. A possible relationship between cyclic AMP induction and previously reported induction by cycloheximide is suggested.     

Two independent mechanisms of induction of 2′:3′-cyclic-nucleotide 3′-phosphohydrolase in glioma cells by cyclic AMP and high cell density

Specific activity of the myelin enzyme, 2′:3′-cyclic-nucleotide 3′-phosphohydrolase (EC 3.1.4.37), increases 2- to 10-fold when sparsely inoculated cultures of C₆ rat glioma cells are allowed to grow to high cell density. Cyclic-nucleotide phosphohydrolase specific activity is also induced in C₆ cells and in oligodendrocytes by dibutyryl cyclic AMP or by agents that elevate intracellular cyclic AMP. In this report, we have compared the density-dependent induction of cyclic-nucleotide phosphohydrolase activity with the cyclic AMP-dependent induction. Dibutyryl cyclic AMP induced cyclic-nucleotide phosphohydrolase specific activity in both sparse and dense cultures which had very different density-dependent cyclic-nucleotide phosphohydrolase activities. Induction of both cyclic-nucleotide phosphohydrolase specific activity and intracellular cyclic AMP content by norepinephrine also occurred to a similar degree in sparse and dense cultures. Similar results were obtained for several clones of C₆ cells, and for a clone of oligodendrocyte x C₆ cell hybrids. Induction of cyclic-nucleotide phosphohydrolase by norepinephrine or dibutyryl cyclic AMP was not due to a change in cell density or rate of cell proliferation, nor did cell density have any appreciable effect on cyclic AMP content of the cells. These results show that regulation of cyclic-nucleotide phosphohydrolase activity in C₆ cells involves two distinct mechanisms.     

Morphologic Differentiation of Mouse Neuroblastoma Cells induced <Emphasis Type="Italic">in vitro</Emphasis> by Dibutyryl Adenosine 3′:5′-Cyclic Monophosphate

The mechanism of mammalian neural differentiation is still obscure; but the availability of mouse neuroblastoma cells in vitro provides an opportunity to study some possible inducers of differentiation and this may help to elucidate the events involved at the molecular level. We have reported¹ that X-irradiation of mouse neuroblastoma cells in vitro induces the formation of axons. The differentiated cells seem to undergo maturation: the soma and nucleus increase in size and the cytoplasm becomes granular. Here we report that N⁶O² dibutyryl adenosine 3′:5′-cyclic monophosphate (dibutyryl cyclic AMP) induces axon formation in mouse neuroblastoma cells in vitro.     

Uptake and metabolism of 3′,5′-cyclic adenosine monophosphate and N,O′-dibutyryl 3′,5′-cyclic adenosine monophosphate in isolated bovine thyroid cells

1.

1. Accumulation of intracellular radioactivity was measured during incubation of isolated bovine thyroid cells with cyclic [³²P]AMP, cyclic [8-³H]AMP and dibutyryl cyclic [8-³H]AMP. With cyclic [³²P]AMP, ³²P cell/medium ratios ranged from 0 to to 0.04 compared to a maximum ³H cell/medium ratio of 0.29 with cyclic [³H]AMP and 0.16 with dibutyryl cyclic [³H]AMP. The excess of intracellular cyclic [³H] over cyclic [³²P]AMP radioactivity was due to extracellular formation of more penetrable dephosphorylated cyclic AMP metabolites which probably served as precursor of intra-cellular cyclic AMP.     

Hormonal regulation of amino acid accumulation in human fetal liver explants Effects of dibutyryl cyclic AMP,glucagon and insulin

α-Aminoisobutyrate accumulation by human fetal liver explants in organ culture is stimulated by dibutyryl cyclic AMP ($\text{N}{}^6\text{,}{}^2\text{′O-}\text{dibutyryl}$ adenosine 3′–5′: cyclic monophosphate), glucagon or insulin. Theophylline increased the effect of submaximal concentrations of dibutyryl cyclic AMP or glucagon. Maximal concentrations of glucagon and dibutyryl cyclic AMP yielded the same results as either agent alone. A period of about 4–6 h was required to observe the stimulatory effect of dibutyryl cyclic AMP or insulin, which could be completely prevented by simultaneous incubation with cycloheximide. Maximal effects of either dibutyryl cyclic AMP or glucagon plus insulin produced additive results. These data support the hypothesis that insulin acts via a mechanism independent of the glucagon—cyclic AMP pathway in liver tissue.In addition, the pharmacologic receptor for glucagon was detected in liver explants from a 30-mm (crown - rump) specimen (6 weeks gestation). The liver had the competence to respond to dibutyryl cyclic AMP by the 36-mm stage. Tissue from a 36-mm specimen did not respond to insulin, but a clear response was elicited from a specimen at the 48-mm stage. These data demonstrate the ability of human fetal liver to respond to hormones at a very early stage in gestation.     

Regulation of phospholipid synthesis inMycobacterium smegmatis by cyclic adenosine monophosphate

Forskolin, an adenylate cyclase activator and a cyclic AMP analogue, dibutyryl cyclic AMP have been used to examine the relationship between intracellular levels of cyclic AMP and lipid synthesis inMycobacterium smegmatis. Total phospholipid content was found to be increased in forskolin grown cells as a result of increased cyclic AMP levels caused by activation of adenylate cyclase. Increased phospholipid content was supported by increased [¹⁴C] acetate incorporation as well as increased activity of glycerol-3-phosphate acyltransferase. Pretreatment of cells with dibutyryl cyclic AMP had similar effects on lipid synthesis. Taking all these observations together it is suggested that lipid synthesis is being controlled by cyclic AMP in mycobacteria.     

Effect of dibutyryl cyclic AMP on the restoration of contact inhibition in tumor cells and its relationship to cell density and the cell cycle

KB cell cultures exposed to 10⁻⁴ M dibutyryl cyclic AMP were significantly inhibited and exhibited contact inhibition of growth at cell densities of 8 × 10⁴/cm² irrespective of the initial plating density. Control cultures reached densities of 2.5 × 10⁵/cm². Inhibition of growth did not occur in KB cells when the density was below 1 × 10⁴ cells/cm². When dibutyryl cyclic AMP was removed from KB cells in the contact-inhibited state, growth resumed with DNA synthesis beginning in about 6 h. Labeled metaphases increased rapidly after 22 h without the appearance of an early rise in unlabeled metaphases. This suggests that the inhibitory effect of dibutyryl cyclic AMP is on the G1 phase of the cell cycle.     

Effect of Noradrenaline and Prostaglandin E<Subscript>1</Subscript> on Adenosine 3′,5′;-Monophosphate Formation in Isolated Pericardial Fat Cells of Man

NORADRENALINE increases the intracellular concentration of adenosine 3′,5′-monophosphate (cyclic AMP)^1,2 which, in turn, enhances glycogenosis³ and lipolysis^4,5 in adipose tissue by increasing Phosphorylase and lipase activities. Prostaglandin E₁ (PGE₁) antagonizes the induced increases in Phosphorylase activity^6,7 and glycerol release in human adipose tissues^8,9 and isolated adipocytes⁷. The finding that the stimulatory effects of the cyclic AMP analogue N⁶—O² dibutyryl cyclic AMP, which mimics the hormonal effect of noradrenaline in human fat cells, are not blocked by PGE₁⁷ suggests that noradrenaline and PGE₁ alter fat cell metabolism by acting on the adenyl cyclase system¹⁰. Whether noradrenaline and PGE₁ alter concentrations of cyclic AMP in human fat cells, however, has not been reported.     

The effect of cyclic AMP on glycoprotein secretion in isolated rat hepatocytes

The effects of dibutyryl cyclic AMP on glycoprotein biosynthesis, intracellular mobilization, and secretion in isolated rat hepatocytes are described. Dibutyryl cyclic AMP (2.5 mm) initially suppresses [³H]glucosamine or [³H]fucose incorporation into cellular macromolecular material; however, after $\text{3}\text{1}\text{2}$ h, the incorporation of these radiolabeled carbohydrates into macromolecular material was stimulated relative to control cells. The stimulation in accumulation of cellular glycoprotein occurred in membrane-associated fractions, with most of this accumulation occurring in the Golgi elements. The glycoprotein produced in the presence of dibutyryl cyclic AMP was quantitatively precipitated by antibodies directed against rat serum, suggesting that the accumulated cellular material is normally destined for secretion from the cell. Dibutyryl cyclic AMP also produced a drastic inhibition of glycoprotein secretion which persisted during the cellular accumulation of glycosylated material. Exposure of the hepatocytes to colchicine (10 μm) produced a similar increase in accumulation of [³H]glucosamine-containing immunoprecipitable material in the cellular fraction and a similar inhibition in secretion. The initial dibutyryl cyclic AMP-mediated suppression of synthesis of intracellular glycosylated material occurred entirely in non-membrane-associated intracellular fractions. Also, the initial accumulation of [³H]glucosamine-containing immunoprecipitable material was not suppressed during the first $\text{3}\text{1}\text{2}$ h after exposure to dibutyryl cyclic AMP, suggesting the initial suppression represents a metabolic process unrelated to secretion. The incorporation of [³H]leucine into macromolecular material was inhibited in both cellular and secreted fractions after exposure to dibutyryl cyclic AMP; however, the accumulation into the extracellular environment was inhibited to a greater extent. The patterns of [³H]glucosamine-containing lipid biosynthesis were unaffected by dibutyryl cyclic AMP.     

Hormone-induced changes in pyruvate kinase activity in isolated hepatocytes II. Relation to the hormonal regulation of gluconeogenesis gluconeogenesis

1. 1. Incubation of isolated hepatocytes with glucagon (10⁻⁶ M) or dibutyryl cyclic AMP (0.1 mM) causes a decrease in pyruvate kinase activity of 50%, measured at suboptimal substrate (phosphoenolpyruvate) concentrations and 1 mM Mg_free²⁺. The magnitude of the decrease in activity is not influenced by the applied extracellular concentrations of lactate (1 and 5 mM), glucose (5 and 30 mM) or fructose (10 and 25 mM). With all three substrates comparable inhibition percentages are induced by glucagon or dibutyryl cyclic AMP.

2. 2. The extent of inhibition of pyruvate kinase induced by incubation of hepatocytes with glucagon or dibutytyl cyclic AMP is not influenced by the extracellular Ca²⁺ concentration nor by the presence of 2 mM EGTA. The reactivation of pyruvate kinase seems to be inhibited by a high concentration of extracellular Ca²⁺ (2.6 mM) as compared to a low concentration of extracellular Ca²⁺ (0.26 mM).

3. 3. Incubation of hepatocytes in a Na⁺-free, high K⁺-concentration medium does not influence the magnitude of the pyruvate kinase inhibition induced by dibutyryl cyclic AMP. However, the reactivation reaction is stimulated under these incubation conditions.

4. 4. Incubation of hepatocytes with dibutyryl cyclic GMP (0.1 mM) leads to a 25% decrease in pyruvate kinase activity. The magnitude of the inhibition by dibutyryl cyclic (GMP) is not influenced by the presence of pyruvate (1 mM) or glucose (5 mM and 30 mM).

5. 5. The relative insensitivity of the pyruvate kinase inhibition induced by glucagon, dibutyryl cyclic AMP and dibutyryl cyclic GMP to the extracellular environment leads to the conclusion that the hormonal regulation of pyruvate kinase is not the only site of hormonal regulation of glycolysis and gluconeogenesis. It is concluded that hormonal regulation of pyruvate kinase activity is exerted by changes in the degree of (de)phosphorylation of the enzyme reflecting acute hormonal control as well as by changes in the concentration of the allosteric activator fructose 1,6-diphosphate. The latter depends at least in part on the hormonal control of the phosphofructokinase-fructose-1,6-phosphatase cycle.

On the role of cellular calcium in the response of the parotid to dibutyryl and monobutyryl cyclic AMP.

The role of intracellular Ca in the exocytosis of α-amylase stimulated by derivatives of cyclic AMP was investigated. Partial depletion of cellular Ca stores was accomplished by prolonged (100–120 min) incubation in media containing no added Ca and 5.0 mM ethyleneglycolbis (aminoethylether)-N,N′-tetraacetic acid (EGTA). Release of α-amylase in response to the N⁶, O²′-dibutyryl or N⁶-monobutyryl derivatives of cyclic adenosine-3′,5′-monophosphate (cyclic AMP) was significantly inhibited by this procedure. When [K⁺]° was increased from 5.0 mM to 25.0 mM, Ca-depletion was accelerated, as was the inhibition of the response to the monobutyryl derivative. The Ca-depletion regimen did not affect the cellular content of other cations, suggesting that the effects were specific for Ca. The effects of the cyclic AMP derivatives on release of Ca was also investigated. Both monobutyryl and dibutyryl cyclic AMP significantly enhanced the rate of release of ⁴⁵Ca from pre-loaded parotid slices. These observations lend support to the hypothesis previously set forth suggesting that in the parotid, cyclic AMP acts to release Ca from intracellular stores. It is this rise in cytosolic Ca which may catalyze the events ultimately leading to exocytosis.