Adenosine 3′,5′-Cyclic Monophosphate-Deficient Mutants of Vibrio cholerae

Two adenosine 3',5'-cyclic monophosphate (AMP)-deficient mutants of Vibrio cholerae (biotype El Tor) were successfully isolated by nitrosoguanidine treatment followed by pencillin screening for pleiotropic sugar-negative clones. Exogenous cyclic AMP is required for the fermentation of sucrose, trehalose, fructose, maltose, and mannose but not of glucose, as well as for the formation of normal flagella and specific somatic antigens. A striking characteristic of the mutants is their growth behavior at higher temperatures. They cannot grow on TCBS selective plates at 37 C or higher unless they are provided with a supply of exogenous cyclic AMP, although they are capable of producing colonies on the same medium, even without cyclic AMP, at temperatures lower than 30 C. Since the mutants are converted to spheroplasts, spindle forms, and spiral filaments in cyclic AMP-free media at 37 C, and this phenomenon is stopped by the addition of cyclic AMP or a combination of 20% sucrose and 0.2% magnesium chloride, it is assumed that cyclic AMP is essential for the synthesis of the cell wall of V. cholerae at higher temperatures.     

Effects of Adenosine 3′,5′-Monophosphate and Adenosine 5′-Monophosphate on Glycogen Degradation and Synthesis in Dictyostelium discoideum

Data are presented demonstrating that the presence in vivo of adenosine 3',5'-monophosphate (3',5'-AMP) causes a rapid depletion of glycogen storage material in the cellular slime mold. The effect of adenosine 5'-monophosphate (5'-AMP) is twofold, stimulating both glycogen degradation and synthesis. In pseudoplasmodia, cell-free extracts appear to contain at least two species of glycogen phosphorylase, one of which is severely inhibited by glucose-1-phosphate and another which is only partially inhibited by this hexose-phosphate. In some cases, 5'-AMP partially overcomes the inhibition by glucose-1-phosphate. Data presented here also indicate the existence of two forms of glycogen synthetase, the total activity of which does not change during 10 hr of differentiation from aggregation to culmination. During this period there is a quantitative conversion of glucose-6-phosphate-independent enzyme activity to glucose-6-phosphate-dependent activity. It is suggested that one effect of 3',5'-AMP is closely related to enzymatic processes involved in the rapid conversion of glycogen to cell wall material and other end products accumulating during sorocarp construction.     

Cyclic 3′,5′-Adenosine Monophosphate and N-Acetyl-glucosamine-6-Phosphate as Regulatory Signals in Catabolite Repression of the lac Operon in Escherichia coli

When an Escherichia coli mutant lacking the enzyme N-acetyl-glucosamine-6-phosphate (AcGN6P) deacetylase is grown in a succinate-mineral salts medium and exposed to an exogenous source of N-acetylglucosamine, approximately 20 to 30 pmoles of AcGN6P per mug of cell dry weight will accumulate in these cells. This accumulation occurs within 2 to 4 min after the addition of N-acetylglucosamine and is coincident with the production of a severe permanent catabolite repression of beta-galactosidase synthesis. This repression does not occur if adenosine 3',5'-cyclic phosphate (cyclic AMP) is added to the cells before AcGN6P accumulates. An immediate derepression occurs when cyclic AMP is added to cells that have already accumulated a large AcGN6P pool. These findings are consistent with the view that low-molecular-weight carbohydrate metabolites and cyclic AMP play key roles in the catabolite repression phenomenon, and that metabolites such as AcGN6P may participate in the represion mechanism by influencing either the formation or degradation of cyclic AMP in E. coli.     

Relationship of Adenosine 3′,5′-Monophosphate to Growth and Metabolism of Tetrahymena pyriformis

The concentration of adenosine 3',5'-monophosphate (cyclic AMP) and the activity of adenylate cyclase were determined for the first time in conjuncation with cyclic 3',5'-nucleotide phosphodiesterase (phosphodiesterase) during the growth cycle of Tetrahymena pyriformis. High levels of cyclic AMP observed during early exponential and late stationary phases were associated with elevated adenylate cyclase and decreased phosphodiesterase activities. Adenylate cyclase and cyclic AMP were decreased and phosphodiesterase was increased in cells grown in glucose-supplemented medium. In contrast to findings in mammalian liver, cyclic AMP was decreased during active gluconeogenesis in Tetrahymena. This suggests a different modulation of carbohydrate metabolism in the two species. The results illustrate that both the content of cyclic AMP and its action as a regulatory agent in Tetrahymena are uniquely suited to the metabolism of this organism.     

Release of the β-Galactosidase-Synthesizing System from Ultraviolet Catabolite Repression by Cyclic 3′,5′-Adenosine Monophosphate, Dark Repair, Photoreactivation, and Cold Treatment

Recovery from the inhibitory effect of ultraviolet irradiation on the induced synthesis of beta-galactosidase was studied in Escherichia coli B/r. When irradiated cells (520 ergs/mm(2) at 254 nm) were induced and incubated in minimal medium supplemented with Casamino Acids (conditions of catabolite repression), the ability to form enzyme was greatly reduced for about 100 min and then recovery began. The inhibition observed immediately after ultraviolet irradiation was partially reversed by cyclic 3',5'-adenosine monophosphate (cyclic AMP) or by photoreactivation treatment. Inhibition was reduced if the cells were given cold treatment (5 C) before or during irradiation; the kinetics of induced enzyme formation in each case were similar to those of irradiated cells receiving cyclic AMP. These kinetics suggest that the cold treatments, like cyclic AMP, cause the release of the beta-galactosidase-synthesizing system from catabolite repression. When irradiated cells were incubated for various times before cyclic AMP or photoreactivation treatment, some reversal of the inhibition of induced enzyme formation was obtained, but by 100 min the treatments were ineffective. Because 100 min was also the time at which dark recovery of enzyme formation began, the recovery process was interpreted to be the result of completion of DNA repair, which, in turn, released the beta-galactosidase-synthesizing system from catabolite repression.     

Inhibition of hepatoma cell growth by analogs of adenosine and cyclic AMP and the influence of enzymes in mammalian sera

The following evidence suggests that inhibition of hepatoma cell (HTC) growth by cyclic nucleotides is an adenosine-like effect that is greatly modified by the type and treatment of serum used in the culture medium and is probably not mediated by cyclic AMP-dependent protein kinase: 1) Heating serum reduces its phosphodiesterase content, thereby slowing metabolism of cyclic AMP and reducing the inhibition of HTC cell growth by cyclic AMP; 2) Using medium that contains phosphodiesterase but lacks adenosine deaminase causes adenosine to accumulate from cyclic AMP and increases the toxicity of cyclic AMP; 3) Uridine or cytidine reverses the growth inhibition caused by adenosine, 5'-AMP or cyclic AMP; 4) adenosine, 5'-AMP and N6-(delta 2-isopentenyl) adenosine are more toxic for HTC cells than is cyclic AMP, and N6,O2-dibutyryl cyclic AMP is not toxic; and 5) N6,O2'-dibutyryl cyclic AMP inhibits growth of Reuber H35 cells, but uridine prevents this inhibition of growth. We conclude that most, if not all, of the inhibitory effects of cyclic AMP and N6,O2'-dibutyryl cyclic AMP on HTc and Reuber H35 hepatoma cell growth are due to the generation of toxic metabolites.     

Requirement of Cyclic Adenosine 3′,5′-Monophosphate for the Thermosensitive Effects of Rts1 in a Cyclic Adenosine 3′,5′-Monophosphate-Less Mutant of Escherichia coli

Previous publications showed that a covalently closed circular (CCC) Rts1 plasmid deoxyribonucleic acid (DNA) that confers kanamycin resistance upon the host bacteria inhibits host growth at 42 degrees C but not at 32 degrees C. At 42 degrees C, the CCC Rts1 DNA is not formed, and cells without plasmids emerge. To investigate the possible role of cyclic adenosine 3',5'-monophosphate (cAMP) in the action of Rts1 on host bacteria, Rts1 was placed in an Escherichia coli mutant (CA7902) that lacks adenylate cyclase or in E. coli PP47 (a mutant lacking cAMP receptor protein). Rts1 did not exert the thermosensitive effect on these cells, and CCC Rts1 DNA was formed even at 42 degrees C. Upon addition of cAMP to E. coli CA7902(Rts1), cell growth and formation of CCC Rts1 DNA were inhibited at 42 degrees C. The addition of cAMP to E. coli PP47(Rts1) did not cause inhibitory effects on either cell growth or CCC Rts1 DNA formation at 42 degrees C. The inhibitory effect of cAMP on E. coli CA7902(Rts1) is specific to this cyclic nucleotide, and other cyclic nucleotides such as cyclic guanosine 3',5'-monophosphate did not have the effect. For this inhibitory effect, cells have to be preincubated with cAMP; the presence of cAMP at the time of CCC Rts1 DNA formation is not enough for the inhibitory effect. If the cells are preincubated with cAMP, one can remove cAMP during the [(3)H]thymidine pulse and still observe its inhibitory effect on the formation of CCC Rts1 DNA. The presence of chloramphenicol during this preincubation period abolished the inhibitory effect of cAMP. These observations suggest that cAMP is necessary to induce synthesis of a protein that inhibits CCC Rts1 DNA formation and cell growth at 42 degrees C.     

Activation of protein kinase and glycogen phosphorylase in isolated rat liver cells by glucagon and catecholamines.

In liver cells isolated from fed female rats, glucagon (290nM) increased adenosine 3':5'-monophosphate (cyclic AMP) content and decreased cyclic AMP binding 30 s after addition of hormones. Both returned to control values after 10 min. Glucagon also stimulated cyclic AMP-independent protein kinase activity at 30 s and decreased protein kinase activity assayed in the presence of 2 muM cyclic AMP at 1 min. Glucagon increased the levels of glycogen phosphorylase a, but there was no change in total glycogen phosphorylase activity. Glucagon increased glycogen phosphorylase a at concentrations considerably less than those required to affect cyclic AMP and protein kinase. The phosphodiesterase inhibitor, 1-methyl-3-isobutyl xanthine, potentiated the action of glucagon on all variables, but did not increase the maximuM activation of glycogen phosphorylase. Epinephrine (1muM) decreased cyclic AMP binding and increased glycogen phosphorylase a after a 1-min incubation with cells. Although 0.1 muM epinephrine stimulated phosphorylase a, a concentration of 10 muM was required to increase protein kinase activity. 1-Methyl-3-isobutyl xanthine (0.1 mM) potentiated the action of epinephrine on cyclic AMP and protein kinase. (-)-Propranolol (10muM) completely abolished the changes in cyclic AMP binding and protein kinase due to epinephrine (1muM) in the presence of 0.1mM 1-methyl-3-isobutyl xanthine, yet inhibited the increase in phosphorylase a by only 14 per cent. Phenylephrine (0.1muM) increased glycogen phosphorylase a, although concentrations as great as 10 muM failed to affect cyclic AMP binding or protein kinase in the absence of phosphodiesterase inhibitor. Isoproterenol (0.1muM) stimulated phosphorylase and decreased cyclic AMP binding, but only a concentration of 10muM increased protein kinase. 1-Methyl-3-isobutyl xanthine potentiated the action of isoproterenol on cyclic AMP binding and protein kinase, and propranolol reduced the augmentation of glucose release and glycogen phosphorylase activity due to isoproterenol. These data indicate that both alpha- and beta-adrenergic agents are capable of stimulating glycogenolysis and glycogen phosphorylase a in isolated rat liver cells. Low concentrations of glucagon and beta-adrenergic agonists stimulate glycogen phosphorylase without any detectable increase in cyclic AMP or protein kinase activity. The effects of alpha-adrenergic agents appear to be completely independent of changes in cyclic AMP protein kinase activity.     

Metabolism of Adenosine 3′,5′-Cyclic Monophosphate and Induction of Fruiting Bodies in Coprinus macrorhizus

The adenyl cyclase and phosphodiesterase metabolizing adenosine 3',5'-cyclic monophosphate (cyclic AMP) were detected in mycelia of strains of Coprinus macrorhizus which form fruiting bodies, but not in those of strains which do not form fruiting bodies. The adenyl cyclase synthesized cyclic AMP from adenosine triphosphate. The phosphodiesterase degr[UNK]ded cyclic AMP to adenosine-5'-monophosphate and was inhibited by adenosine-3'-monophosphate, theophylline, and caffeine. The strains which form fruiting bodies incorporated and metabolized cyclic AMP, but strains which do not form fruiting bodies did not. The possible participation of cyclic AMP in the induction of fruiting bodies is discussed.     

Role of Cyclic Adenosine 3′,5′-Monophosphate in the In Vivo Expression of the Galactose Operon of Escherichia coli

Studies of levels of galactokinase in Escherichia coli with mutations affecting synthesis of, or response to, cyclic adenosine 3',5'-monophosphate show that this nucleotide does not play a major role in expression of the galactose operon, causing at most a twofold stimulation. The discrepancy between our in vivo results and the marked stimulation by cyclic adenosine 3',5'-monophosphate in in vitro systems indicates that current cell-free systems lack a factor which allows efficient expression of the galactose operon even in the absence of cyclic adenosine 3',5'-monophosphate or of the binding protein for this nucleotide.     

Effects of adenosine 5'-monophosphate and adenosine 3',5'-cyclic monophosphate on l cells.

The adenine nucleotides, 5'-AMP and 3',5'-cyclic AMP block L cells in the S-phase of the cell cycle. The intracellular level of cyclic AMP is reduced after incubation of cells with 5'-AMP, and rates of uridine transport are increased after incubation with either 5'-AMP or cyclic AMP. On the contrary, cyclic AMP levels are increased and uridine transport decreased in cells treated with an inhibitor of the cyclic AMP phosphodiesterase. This inhibitor partially reverses the growth-inhibitory effect of cyclic AMP, indicating that a breakdown product is the effective inhibitor of growth. The inhibition of cell growth induced by the adenine nucleotides is prevented by uridine, suggesting that the block in S is due to a lack of availability of pyrimidines.     

Role of AMP on the activation of glycogen synthase and phosphorylase by adenosine, fructose, and glutamine in rat hepatocytes

The mechanism for glycogen synthesis stimulation produced by adenosine, fructose, and glutamine has been investigated. We have analyzed the relationship between adenine nucleotides and glycogen metabolism rate-limiting enzymes upon hepatocyte incubation with these three compounds. In isolated hepatocytes, inhibition of AMP deaminase with erythro-9-(2-hydroxyl-3nonyl)adenine further increases the accumulation of AMP and the activation of glycogen synthase and phosphorylase by fructose. This ketose does not increase cyclic AMP or the activity of cyclic AMP-dependent protein kinase. Adenosine raises AMP and ATP concentration. This nucleotide also activates glycogen synthase and phosphorylase by covalent modification. The correlation coefficient between AMP and glycogen synthase activity is 0.974. Nitrobenzylthioinosine, a transport inhibitor of adenosine, blocks (by 50%) the effect of the nucleoside on AMP formation and glycogen synthase but not on phosphorylase. 2-Chloroadenosine and N6-phenylisopropyladenosine, nonmetabolizable analogues of adenosine, activate phosphorylase (6-fold) without increasing the concentration of adenine nucleotides or the activity of glycogen synthase. Cyclic AMP is not increased by adenosine in hepatocytes from starved rats but is in cells from fed animals. [Ethylenebis (oxyethylenenitrilo)]tetraacetic acid (EGTA) blocks by 60% the activation of phosphorylase by adenosine but not that of glycogen synthase. Glutamine also increases AMP concentration and glycogen synthase and phosphorylase activities, and these effects are blocked by 6-mercaptopurine, a purine synthesis inhibitor. Neither adenosine nor glutamine increases glucose 6-phosphate. It is proposed that the observed efficient glycogen synthesis from fructose, adenosine, and glutamine is due to the generation of AMP that activates glycogen synthase probably through increases in synthase phosphatase activity. It is also concluded that the activation of phosphorylase by the above-mentioned compounds can be triggered by metabolic changes.     

Induction of Nonpigmented Variants of Erwinia herbicola by Incubation at Supraoptimal Temperatures

As with other inducible enzymes, the induced synthesis of l-arabinose isomerase (l-arabinose ketol isomerase, EC 5.3.1.4) in Salmonella typhimurium is subject to catabolite repression. Of the three catabolite repressors tested, glucose produces maximum repression. Analogues of catabolite repressors like 2-deoxy-d-glucose and d-fucose also inhibit the synthesis of the enzyme. The catabolite repression is completely reversed in the presence of 1.5 x 10(-3)m cyclic 3',5'-adenosine monophosphate (AMP). The maximum repression is produced in glucose-grown cells in glucose-containing induction medium. Cyclic 3',5-AMP reverses this repression provided that the cells are treated with ethylenediaminetetraacetic acid (EDTA). In normal cells, cyclic 3',5'-AMP has no effect on the induction but in EDTA-treated cells the cyclic nucleotide enhances synthesis of the enzyme. The inhibition produced by d-fucose cannot be reversed by cyclic 3',5'-AMP. d-Fucose competes with the inducer l-arabinose in some step(s) involved in the process of induction.     

Regulation of carbohydrate permeases and adenylate cyclase in Escherichia coli. Studies with mutant strains in which enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system is thermolabile.

Carbohydrate uptake and cyclic adenosine 3':5'-monophosphate (cyclic AMP) synthesis were studied employing mutant strains of Escherichia coli in which Enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system was heat-labile. Partial loss of Enzyme I activity, which resulted from incubation of cells at the nonpermissive temperature, depressed the rate and extent of methyl alpha-glucoside uptake. Temperature inactivation of Enzyme I also rendered cyclic AMP synthesis and the uptake of several carbohydrates (glycerol, maltose, melibiose, and lactose) hypersensitive to inhibition by methyl alpha-glucoside. Protein synthesis did not appear to be required for these effects. The parental strains and "revertant" strains in which Enzyme I was less sensitive to temperature did not exhibit heat-enhanced regulation. Inhibition was abolished by the crr mutation. The results suggest that Enzyme I functions as a catalytic component of the regulatory system. Simple positive selection procedures are described for the isolation of bacterial mutants which are deficient for either Enzyme I or the heat-stable protein of the phosphotransferase system.     

Bordetella pertussis-induced Alteration of the Normal Hyperglycemic Response of Mice to 3′,5′-Adenosine Phosphate

Blockade of the adrenergic receptors or the injection of Bordetella pertussis vaccine prevented the normal hyperglycemic response of CFW mice to exogenous cyclic 3',5'-adenosine phosphate (3',5'-AMP). The cyclic nucleotide was also ineffective in offsetting the histamine hypersensitivity of the beta-adrenergically blocked and the pertussis-sensitized groups. These observations suggest that the autonomic malfunction occurs at a point(s) subsequent to release of 3',5'-AMP. Neither the hyperglycemic effect of epinephrine or 3',5'-AMP, or both, nor the histamine sensitivity of pertussis-pretreated animals was influenced by the subcutaneous administration of theophylline.     

Cyclic Adenosine 3′,5′-Monophosphate and Morphogenesis in Mucor racemosus

Yeastlike cells of Mucor racemosus grown under 100% CO(2) underwent morphogenesis to hyphae after exposure to air. The addition of dibutyryl cyclic adenosine monophosphate (dbcAMP) to yeastlike cultures inhibited this morphogenesis in media containing 2% glucose. The maintenance of uniformly spherical, budding cells required 1 mM dbcAMP in a defined medium containing Casamino Acids, and 3 mM dbcAMP in a medium containing yeast extract and peptone. At these concentrations, dbcAMP also induced yeastlike development in young aerobic hyphae grown in media containing 2% glucose. Removal of dbcAMP resulted in hyphal development. The endogenous cyclic AMP (cAMP) content of yeastlike cultures was measured after a shift from CO(2) to air. A fourfold decrease in intracellular cAMP preceded the appearance of hyphal germ tubes. These results indicate that cAMP plays a role in the control of morphogenesis in Mucor racemosus.     

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.