Comparative studies on sporulation-promotive actions on cyclic AMP, theophylline and caffeine in Saccharomyces cerevisiae.

Cyclic AMP, theophylline and caffeine promoted sporulation when added to a presporulation medium containing glucose. Caffeine promoted sporulation even when added to a presporulation medium containing acetate as the carbon source, but cyclic AMP and theophylline did not. Caffeine did not increase the intracellular cyclic AMP level, while theophylline did significantly when added to a presporulation medium containing glucose. Caffeine inhibited the vegatative DNA synthesis with little effect on RNA and protein synthesis, resulting in the increase in cell volume, dry weight, and RNA and protein contents, but cyclic AMP and theophylline did not show such effects.     

Effect of cyclic AMP,theophylline and caffeine on the glucose repression of sporulation inSaccharomyces cerevisiae

Summary Repression of the sporulation ability ofSaccharomyces cerevisiae by glucose present in the presporulation medium was studied. Glucose lowered sporulation ability when added to the presporulation medium containing yeast extract but did not do so when added to the presporulation medium without glucose. The glucose-repressed sporulation ability was recovered by the addition of cyclic AMP, and theophylline or caffeine to the presporulation culture. Theophylline promoted the action of cyclic AMP, but caffeine did not. The effect of caffeine to reverse glucose repression was greater than that of cyclic AMP and theophylline.     

PROMOTION OF CELL ARREST IN G2 DURING CELL MATURATION IN ROOTS OF PISUM SATIVUM BY EXOGENOUSLY APPLIED CYCLIC AMP

Cyclic AMP and dibutyryl cyclic AMP promoted cell arrest in G2 in roots during tissue maturation in seedlings of Pisum sativum when applied in lanolin after cotyledon excision. 5' AMP did not promote arrest in G2 in a similar manner. Results demonstrate that cyclic AMP did not inhibit cell proliferation in root meristems when applied exogenously.     

Effect of cyclic 3',5'-adenosine monophosphate on the sporulation of Saccharomyces cerevisiae

Cyclic 3',5'-adenosine monophosphate and sodium dibutyryl cyclic3',5'-adenosine monophosphate had no effect on sporulation ofSaccharomyces cerevisiae, when added to a sporulation mediumnot enriched with glucose. They did, however, reverse the repressionof sporulation by glucose, when added to the sporulation mediumtogether with glucose. 5'-AMP, 5'-ADP and 5'-ATP did not reversethe repression of sporulation by glucose. (Received February 24, 1972; )     

Effects of isoproterenol,theophylline and carbachol on cyclic nucleotide levels and relaxation of bovine tracheal smooth muscle

The effect of theophylline and isoproterenol on bovine tracheal smooth muscle tension and cyclic AMP levels was investigated. Concentrations of isoproterenol (4 × 10^?6 M) and theophylline (10 mM) that relaxed carbachol-contracted tracheal muscle by 85–95% did not significantly elevate control levels of cyclic AMP. In the absence of carbachol, several-fold increases in cyclic AMP were caused by isoproterenol although no elevations by theophylline were measurable. However, when isoproterenol and theophylline were administered together, theophylline potentiated the rise in cyclic AMP caused by isoproterenol. Phosphodiesterase studies in tracheal muscle showed the presence of a high and a low K_m enzyme which were inhibited by theophylline. Cyclic GMP levels were elevated in muscles contracted by carbachol as well as in carbachol-contracted muscles that had been relaxed by theophylline. In non-tension studies, in which the tracheal muscle was not under isometric tension, carbachol or theophylline alone increased cyclic GMP and together they synergistically elevated cyclic GMP. Atropine blocked the elevation caused by carbachol but not that caused by theophylline. In contrast to theophylline, isoproterenol did not elevate cyclic GMP, and in carbachol-contracted muscles that had been relaxed by isoproterenol, cyclic GMP levels were no different from control. Also, in non-tension studies, isoproterenol decreased basal cyclic GMP and antagonized the increase in cyclic GMP due to carbachol.The results indicate that whole-tissue levels of cyclic AMP and cyclic GMP do not correlate with the state of tracheal smooth muscle tension. Cyclic GMP levels do not clearly correlate with either contraction or relaxation. The inhibition by carbachol of increases in cyclic AMP due to isoproterenol and the inhibition by isoproterenol of increases in cyclic GMP due to carbachol provide evidence for a reciprocal cholinergic-adrenergic antagonism of cyclic AMP and cyclic GMP levels. The antagonism did not appear to be due to either cyclic nucleotide affecting the elevation of the other since the levels of both cyclic nucleotides were depressed.     

A possible role of cyclic AMP on tracheary element formation in cultured carrot-root slices

When the root-phloem slices ofDaucus carota cv. Hokkaidô-gosun were cultured on a Murashige and Skoog's medium containing 2,4-dichlorophenoxyacetic acid (2,4-D medium) and cyclic AMP or its analogues, tracheary elements were formed in the dark, while they were not formed on the medium containing only 2,4-D in the dark. The number of tracheary elements induced by cyclic AMP was far less than that induced by cytokinin or 8-bromo-cyclic AMP. But when theophylline, an inhibitor of cyclic AMP phosphodiesterase, was used in combination with cyclic AMP in the culture, the number of tracheary elements was significantly increased. A remarkable increase in cytokinin activity was found in the hydrolyzate of soluble RNA extracted from the slices cultured on the 2,4-D medium containing 8-bromo-cyclic AMP, but only negligible cytokinin activity was detected in the hydrolyzate of soluble RNA extracted from the slices cultured on the 2,4-D medium without 8-bromo-cyclic AMP. Since cytokinin production occurred in the slices cultured in the light, it was supposed that light irradiation might induce cyclic AMP production. The mechanism of cytokinin production leading to tracheary element formation mediated by cyclic AMP level is discussed.     

Effect of glucose on adenosine 3', 5'-monophosphate levels in rat pancreatic islets.

Time course of the changes in insulin release and cyclic AMP levels in isolated rat islets incubated in media containing 5 or 16.7 mM of glucose were followed. The higher glucose concentration caused a slight but significant increase of cyclic AMP levels after 10 min incubation, but not 5 min incubation, whereas the stimulation of insulin release by 16.7 mM of glucose was apparent in both incubation times. Theophylline increased cyclic AMP levels markedly but did not stimulate insulin release when the glucose concentration was 5 mM. A slight augmentation by theophylline of insulin release was observed in the incubation medium containing 16.7 mM glucose. All these findings suggest that the elevation of cyclic AMP in islets may not play a role for the initiation of the insulin release induced by glucose, though it may act to modulate the glucose effect.     

Auxin-induced growth of tuber tissue of Jerusalem artichoke VIII. Role of cyclic AMP in the action of auxin, cytokinin and gibberellic acid

Cyclic AMP showed no growth-promoting effect when given aloneto unaged slices excised from Jerusalem artichoke tubers. Butit synergistically enhanced cell expansion when given togetherwith an auxin, 2,4-dichlorophenoxyacetic acid. Growth responsesof aged slices to cyclic AMP were much smaller than those ofunaged slices. Cyclic AMP was substantially effective when sliceswere aged in the presence of cyclic AMP, then were transferredto a growth solution containing auxin. Interactions betweencyclic AMP and gibberellic acid or kinetin were additive inpromoting auxininduced cell expansion. Both caffeine and theophylline,inhibitors of phosphodiesterase, enhanced the stimulating effectof applied cyclic AMP on auxin-induced cell expansion. But theydid not enhance the promoting effect of gibberellic acid orkinetin on auxininduced cell expansion. These results suggestthat cyclic AMP did not act as a second messenger for any auxin,gibberellic acid and cytokinin in the promotion of cell expansionin this tissue. (Received September 27, 1972; )     

Cyclic AMP and growth of Ehrlich ascites tumor cells. Lack of cyclic AMP elevation in nutritionally deprived cells and mechanism of retardation of growth by dibutryl cyclic AMP.

Cyclic AMP levels in Ehrlich ascites tumor cells changed little after deprivation of cells of essential nutrients, serum, glucose and amino acids, deprival of each of which leads to marked inhibition of growth and protein synthesis. Cyclic AMP levels also changed little after the addition of these nutrients to deprived cells. Thus cyclic AMP is not likely to be the intracellular mediator for growth regulation by these three nutrients. Elevation of cyclic AMP levels for short periods by exposure of cells to choleratoxin or theophylline produced only slight changes in parameters of protein synthesis (polyribosome pattern and rate of [3H]leucine incorporation). An exposure for 1 day to dibutyryl cyclic AMP did not inhibit cell growth. However, prolonged exposure to dibutyryl cyclic AMP inhibited the multiplication of Ehrlich ascites cells both in suspension and in stationary cultures. No morphological effects were evident in the former; in the latter, cells attached firmly to the substratum and formed elongated cytoplasmic processes. Inhibition of cell multiplication by dibutyryl cyclic AMP was related to cell density and to serum concentration. Cells in dibutyryl cyclic AMP-containing media plated at low cell densities multiplied as rapidly as control cells. The final densities cells reached were determined by the serum concentration; in dibutyryl cyclic AMP-containing media these densities were about one-half those of respective control cells. Limitation of cell multiplication by dibutyryl cyclic AMP was reversed by the addition of serum, by resuspending cells at lower densities, or by resuspending cells in media without dibutyryl cyclic AMP. These findings suggested that dibutyryl cyclic AMP may affect the utilization of serum factors by cells. Dibutyryl cyclic AMP did not inactivate serum factors and did not change the rate at which cells depleted the growth medium of serum factors. Dibutyryl cyclic AMP may limit cell multiplication by increasing the cellular requirement for serum factors.     

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.     

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.     

Erythrophagocytosis by macrophages: suppression of heme oxygenase by cyclic AMP.

On incubation of peritoneal macrophages with antibody-coated radiolabeled erythrocytes, a reproducible fraction of the erythrocytes was phagocytized and heme oxygenase was induced. Addition of cyclic AMP, dbcyclic AMP, or theophylline to the incubation medium suppressed the substrate-mediated induction of heme oxygenase in a dose-related manner but did not impair the rate or extent of erythrophagocytosis. A similar effect was produced by epinephrine, norepinephrine, isoproterenol, and prostaglandins, which generate endogenous cyclic AMP by stimulating the adenyl cyclase system. Propanolol completely blocked the suppressive effect of epinephrine, while phentolamine was ineffective. In contrast to the cyclic adenosine nucleotide, cyclic GMP probably slightly enhanced the substrate-mediated induction of heme oxygenase and partly reversed the suppressive effect of cyclic AMP. Cyclic adenosine nucleotides, prostaglandin, and theophylline significantly reduced the incorporation of labeled uridine or leucine into RNA and protein of erythrophagocytic macrophages, but failed to impair the uptake of these precursors by the phagocytizing cells. These compounds also reduced the conversion of [1-¹⁴C] glucose to ¹⁴CO₂ by the incubated macrophages, whereas ¹⁴CO₂ formation was enhanced by epinephrine. None of these effects was reversible by addition of insulin or by glucose supplementation, which is in sharp contrast to the suppressive effect of glucocorticoids on heme oxygenase induction.     

Inhibitory effect of glucose and adenosine 3',5'-monophosphate on the synthesis of inducible N-acetylglucosamine catabolic enzymes in yeast

Glucose can block the utilization of N-acetylglucosamine in Saccharomyces cerevisiae, a facultative aerobe, but not in Candida albicans, an obligatory aerobe. Furthermore, glucose represses the synthesis of the enzymes of the N-acetylglucosamine catabolic pathway in S. cerevisiae, but not in C. albicans. The results suggest that catabolite repression is present in S. cerevisiae, but not in C. albicans. Cyclic AMP added to S. cerevisiae cells maintained in a glucose medium cannot bring about their release from catabolite repression. On the contrary, the synthesis of inducible enzymes of N-acetylglucosamine pathway was inhibited by cyclic AMP in both the yeasts. This seems to indicate that cyclic AMP can penetrate into the yeast cells. Furthermore, cyclic AMP inhibits protein synthesis, suggesting that protein synthesis in yeast is under cyclic AMP control.     

Evidence that a glucose-mediated rise in cyclic AMP triggers germination ofPilobolus longipes spores

Spores ofPilobolus longipes incubated in phosphate buffer were activated within 5–10 min following the addition of either glucose or 6-deoxyglucose. Cyclic AMP content increased in response to glucose or 6-deoxyglucose, and the increase consistently preceded spore activation. Dibutyryl cyclic AMP also caused activation. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) did not cause activation, but, when added to spores with a suboptimal level of 6-deoxyglucose, it amplified the signal to produce a large increase in activation. IBMX increased intracellular cyclic AMP levels when it was applied with 6-deoxyglucose, but had no effect when it was applied alone. Phosphodiesterase activities in cell extracts from dormant and activated spores were not significantly different. These results indicate that the rise in cyclic AMP that follows exposure to glucose may play an important role in triggering spore germination.     

Cyclic AMP and genetic sterility of a male restricted (H re /+) mutant of Rattus

Restricted (H ^re /+) male rats marked by a coat color pattern have normal testes at birth. By 9 days postpartum, testes of the mutant animals are smaller than normal and by approximately 90 days of age the animals are sterile. The genetically sterile testes are totally devoid of spermatogonial cells, spermatocytes, spermatids, and spermatozoa, with only Sertoli cells remaining in the seminiferous tubules. Cyclic AMP concentrations in the whole testes (and the seminiferous tubules) of the mutant males are approximately 10–35% greater than in testes of control males when tested at intervals from 5 to 120 days of age. The possible role of excess cyclic AMP in reducing the rate of mitotic division of spermatogonial cells while enhancing differentiation of spermatogonial cells into spermatozoa is discussed. Such a change in the respective rates of mitotic and meiotic divisions would ultimately deplete the mutant testes of all spermatogonial cells.     

The role of adenosine 3′:5′-cyclic monophosphate in the regulation of insulin release by isolated rat islets of Langerhans

1. Concentrations of cyclic AMP (adenosine 3':5'-cyclic monophosphate) and rates of insulin release were measured in islets of Langerhans isolated from rat pancreas and incubated for various times in the presence of glucose, 3-isobutyl-1-methylxanthine, caffeine, theophylline, adrenaline and diazoxide. 2. Caffeine and theophylline produced small but significant increases in both cyclic AMP and release of insulin when they were incubated in the presence of 10mm-glucose. 3. 3-Isobutyl-1-methylxanthine produced a marked increase in the intracellular concentration of cyclic AMP in the presence of 5mm- and 10mm-glucose. However, insulin release was stimulated only in the presence of 10mm-glucose. 4. In response to rising concentrations of extracellular glucose (5-20mm) there was no detectable increase in the intracellular concentration of cyclic AMP even though there was a marked increase in the rate of insulin release. 5. In response to 10mm-glucose insulin release occurred in two phases and 3-isobutyl-1-methylxanthine potentiated the effect of glucose on both phases. The intracellular concentration of cyclic AMP remained constant with glucose and rose within 10min to its maximum value with 3-isobutyl-1-methylxanthine. 6. Adrenaline and diazoxide inhibited insulin release and lowered the intracellular concentration of cyclic AMP when islets were incubated with glucose or 3-isobutyl-1-methylxanthine. 7. It is suggested that glucose does not stimulate insulin release by increasing the concentration of cyclic AMP in islet cells. However, the concentration of cyclic AMP in islet cells may modulate the effect of glucose on the release process.