Exogenous Cyclic AMP Research Articles

Increased cyclic nucleotide phosphodiesterase activity in leukaemic lymphocytes

THE intracellular concentration of cyclic AMP influences cellular proliferation and maturation in both normal and malignant tissue. For example, the concentration of cyclic AMP—changes during the cell cycle being lowest during mitosis1—is inversely related to the rate of cell growth2, and is elevated in contact-inhibited cell populations3,4. In studies of malignant tissue, the concentration of cyclic AMP is lower in cells transformed in vitro by oncogenic viruses than in untransformed cells3–5, and is lower in certain tumours grown in vivo than in the corresponding normal tissue6,7. Furthermore, in malignant cells, exogenous cyclic AMP or agents that increase the intracellular concentration of this cyclic nucleotide decrease the rate of growth8 and induce morphological and biochemical differentiation9,10.

Effect of Exogenous Cyclic AMP and other Adenine Nucleotides on Neutrophil Chemotaxis and Motility

Cyclic AMP placed in the bottom of the modified Boyden chemotaxis chamber is weakly chemotactic for rabbit peritoneal neutrophils. Dibutyryl-cyclic AMP under similar conditions gives a biphasic response, being weakly chemotactic at low concentrations and inhibiting cell movement at higher concentrations. When mixed with the cells in the upper compartment of the chemotaxis chamber, cyclic AMP and dibutyryl-cyclic AMP enhance neutrophil spontaneous motility; on the other hand, dibutyryl-cyclic AMP mixed with the cells in the upper compartment consistently inhibits neutrophil chemotactic responsiveness, while cyclic AMP has a variable effect on chemotaxis. The effect of ADP and ATP was to inhibit chemotaxis and spontaneous motility. AMP is totally without effect.

Selective inhibition of urea transport by oxidizing agents

Urea and water transport across the toad bladder epithelial cell appears to take place through independent vasopressin-stimulated pathways. Agents such as chromate, for example, when added to the luminal bathing medium, inhibit urea transport without inhibiting osmotic water flow, providing evidence for such independent pathways. In the present study, selective inhibition of urea transport is shown for permanganate and methylene blue, which like chromate, are oxidizing agents. Permanganate inhibits urea transport irreversibly, while methylene blue acts reversibly. Not all oxidizing agents are inhibitory; perchlorate, peroxide and ferricyanide have no effect on urea transport or water flow. Permanganate and chromate both act at a point beyond the generation of cyclic AMP, since they continue to inhibit urea transport in bladders treated with exogenous cyclic AMP, 8-bromoadenosine 3', 5'-cyclic monophosphate, and a combination of cyclic AMP and theophylline. These findings suggest that selective inhibition of urea transport can be brought about by oxidation of one or more components in its transport pathway, and that, in the case of chromate and permanganate, these components may be in the luminal membrane itself.

The effect of catecholamines and prostaglandins upon human and rat erythrocytes

Both human and rat erythrocytes respond to low doses (10 −11-10 −9 M) of L-isoproterenol and Lepinephrin with an increased degree of hypotonic hemolysis and a decreased rate of filtration through standardized paper filters. The receptors in both cell types have many of the characteristics of β-receptors for catecholamines. However, hormone-receptor interaction in the human cell does not lead to an increase in intracellular cyclic AMP concentration, but in the rat cell, hormone-receptor interaction does lead to a significant increase in cylic AMP content. Thus, catecholamine-β-receptor interaction, at least in the human red cell, leads to a change in red cell properties which are not mediated by adenylate cyclase activation. Likewise, prostaglandin E 2, at 10 −12-10 −10 M, causes an increased degree of hypotonic hemolysis and a decreased rate of filtration through standardized paper filters, but it also does not increase the cyclic AMP content of the human erythrocyte but does increase that of the rat erythrocyte. Nevertheless, exogenous cyclic AMP, when added at a concentration of 10 −8 M to washed human erythrocytes, increases the degree of hypotonic hemolysis. Conversely, prostaglandin E 1, at 10 −12-10 −10 M, causes a decreased degree of hypotonic hemolysis and an increased rate of filtration through a standard filter. Both prostaglandin E 2 and the catecholamines decrease the size of a rapidly exchangeable calcium pool, and prostaglandin E 1 increases it.

Synergistic Effects of Adenosine and Compounds Related to Adenosine 3′: 5′-cyclic Monophosphate on Dedifferentiating Iris Epithelial Cells in Culture

Addition of adenosine or 5′ AMP to the primary culture of newt iris epithelial cells produces an extensive array of fine cytoplasmic processes in a fraction of the cell population. Dibutyryl cyclic AMP, monobutyryl cyclic AMP, cyclic AMP and theophylline have the same effect, but the minimum effective concentrations of the latter four compounds are higher than those of the former two. When the primary cultures are treated simultaneously with two effective compounds at various concentrations a synergis-tic effect is observed between dibutyryl cyclic AMP and theophylline; cyclic AMP and theophylline; adenosine and theophylline; adenosine and cyclic AMP. The results support the interpretation that an increase in cellular level of cyclic AMP, which is produced by exogenous adenosine or 5′ AMP as well as by exogenous cyclic AMP, its derivatives or theophylline, is responsible for the formation of radial cytoplasmic processes. The morphogenetic response does not depend on the presence of serum in the culture medium. The possibility is discussed that the cellular level of cyclic AMP is involved as one link of the sequential events which control the dedifferentiation of iris epithelial cells in cell-type conversion.

Factors released from sea urchin eggs affect cyclic nucleotide metabolism in sperm

CYCLIC nucleotide phosphodiesterase inhibitors and exogenous cyclic AMP or cyclic GMP can increase or maintain mammalian sperm motility, respiration rate and fructolytic rate1–7. Sperm from various species have been shown to contain high activities of cyclic AMP-dependent protein kinase8–10, guanylate cyclase11,12, cyclic nucleotide phosphodiesterase11, phospho-protein phosphatase13, and adenylate cyclase11,14. These and other observations have led to speculation that cyclic nucleotides regulate or modulate sperm motility and metabolism15, and that this modulation may occur during the processes of capacitation or fertilisation15–17. The latter hypothesis requires that factors from the female or from the ovum itself alter sperm cyclic nucleotide levels. Until now there has been no demonstration of a naturally occurring factor of animal origin that can substantially change cyclic nucleotide levels in sperm15. The purpose of this study was to determine whether or not there are factors released from eggs that can alter sperm cyclic nucleotide levels. Gametes from sea urchins were used because both eggs and sperm can be obtained in large quantities, and because factors released from sea urchin eggs have been known for some years to alter sea urchin sperm motility18–20 and respiration20,21.

Metabolism of cyclic adenosine 3',5'-monophosphate and induction of tryptophanase in Escherichia coli

The relationship between cyclic adenosine 3',5'-monophosphate (cyclic AMP) metabolism and the induction of tryptophanase and beta-galactosidase was studied in several strains of Escherichia coli grown with succinate, acetate, glycerol, or glucose as the carbon source. No consistent relationship between the intracellular concentration of cyclic AMP in the several strains cultured and the various carbon sources was discerned. In E. coli K-12-1 the induction of tryptophanase was found to vary in the order: succinate greater than acetate greater than glycerol greater than glucose, and that of beta-galactosidase was found in the order: glycerol greater than acetate greater than succinate greater than glucose. Rate of accumulation of cyclic AMP in the culture filtrate was in the order: succinate greater than acetate greater than glycerol greater than glucose. The addition of glycerol to E. coli K-12-1 grown in acetate caused inhibition of tryptophanase and slight inhibition of accumulation of extracellular cyclic AMP. These same conditions caused beta-galactosidase induction to be stimulated. The addition of exogenous cyclic AMP to cultures grown with four different carbon sources had an effect characteristic for each of the two enzymes studied as well as each individual carbon source. The results suggest that there are control elements distinct from cyclic AMP and its receptor protein which respond to the catabolic situation of the cell.

Hormonal control of cyclic 3':5'-AMP levels and gluconeogenesis in isolated hepatocytes from fed rats

Glucagon can stimulate gluconeogenesis from 2 mM lactate nearly 4-fold in isolated liver cells from fed rats; exogenous cyclic adenosine 3':5'-monophosphate (cyclic AMP) is equally effective, but epinephrine can stimulate only 1.5-fold. Half-maximal effects are obtained with glucagon at 0.3 nM, cyclic AMP at 30 muM and epinephrine at 0.2 muM. Insulin reduces by 50% the stimulation by suboptimal concentrations of glucagon (0.5 nM). A half-maximal effect is obtained with 0.3 nM insulin (45 microunits/ml). Glucagon in the presence of theophylline (1 mM) causes a rapid rise and subsequent fall in intracellular cyclic AMP with a peak between 3 and 6 min. Some of the fall can be accounted for by loss of nucleotide into the medium. This efflux is suppressed by probenecid, suggesting the presence of a membrane transport mechanism for the cyclic nucleotide. Glucagon can raise intracellular cyclic AMP about 30-fold; a half-maximal effect is obtained with 1.5 nM hormone. Epinephrine (plus theophylline, 1 mM) can raise intracellular cyclic AMP about 2-fold; the peak elevation is reached in less than 1 min and declines during the next 15 min to near the basal level. Insulin (10 nM) does not lower the basal level of cyclic AMP within the hepatocyte, but suppresses by about 50% the rise in intracellular and total cyclic AMP caused by exposure to an intermediate concentration of glucagon. No inhibition of adenylate cyclase by insulin can be shown. Basal gluconeogenesis is not significantly depressed by calcium deficiency but stimulation by glucagon is reduced by 50%. Calcium deficiency does not reduce accumulation of cyclic AMP in response to glucagon but diminishes stimulation of gluconeogenesis by exogenous cyclic AMP. Glucagon has a rapid stimulatory effect on the flux of 45Ca2+ from medium to tissue.

Effects of TSH on Iodide Transport by MouseThyroid Lobesin Vitro

Thyroidal iodide concentration by in vitro mouse thyroid lobes was studied by determining the equilibrium tissue/medium iodide, concentration ratio (T/M[I-]), THYROIDAL I- influx, and thyroidal I-efflux in the presence and absence of ClO4-. Chronic thyroid stimulation by low dietary iodine increased the T/M[I-], and the increase was linearly related to I- influx. There was no difference in efflux rate when animals fed low and high iodine diets were compared, although the efflux with C104- added to block influx was increased by low iodine diet. Addition of TSH in vitro caused a delayed fall in T/M[I-] with a similar TSH concentration-dependence as colloid droplet formation. The TSH effect was mimicked by exogenous cyclic AMP but could be dissociated from stimulation of hormone release by colchicine. Thyroidal I- efflux was increased up to 20-fold by C104-. In the presence of C104- short-term and equilibrium-labeled I- exited at the same rate, but in the absence of C104- short-term-labeled I- efflux was consistently higher. The TSH-induced fall in T/M I- could be accounted for by increased iodide efflux. As TSH also increased efflux when influx was blocked by C104-, the TSH effect would seem due to an increased intrinsic follicular leakiness.

Control of mitogen-induced lymphocyte activation: II. Analysis of cell populations and metabolic events involved in cyclic AMP-mediated recovery of DNA synthesis suppressed by mitogens

Exposure of human peripheral blood lymphocytes to increased doses of PHA (0–2000 μg/ml PHA) leads to a decrease in thymidine incorporation. The addition of exogenous cyclic AMP (10 −3–10 −4 M) to lymphocyte cultures exposed to such high levels of PHA results in a recovery of the ability to incorporate both thymidine and uridine. Using this system as a model for the study of cyclic nucleotide metabolism, experiments have demonstrated a differential sensitivity to changes in cyclic nucleotide levels among lymphocyte subpopulations exposed to PHA, concanavalin A, or pokeweed mitogen. Further evidence for differential sensitivity is also obtained using lymphocyte populations separated on the basis of rosette formation. Measurement of intracellular cAMP and cGMP levels in lymphocyte cultures exposed to PHA, concanavalin A, or pokeweed mitogen suggests that while cGMP is the mitogenic signal, cAMP may control deactivation, thereby exerting an overide effect on cGMP mediated events. In addition, metabolic studies suggest that exogenous cAMP may influence PHA-induced transformation up to 67 hr after culture initiation and that the effects of Ca 2+ ions and cyclic nucleotides are interrelated in the activation of lymphocytes by PHA.

Utilization of gluconate by Escherichia coli. A role of adenosine 3′:5′-cyclic monophosphate in the induction of gluconate catabolism

1. Cultures of Escherichia coli growing on gluconate use both gluconate and glucose when glucose is added. 2. Glycerol-grown cells adapt to gluconate utilization even in media containing glucose as well as gluconate. 3. The rates of gluconate utilization by cells growing on a mixture of glucose and gluconate, and the specific activities of the gluconate uptake system and of gluconate kinase, are greater if adenosine 3':5'-cyclic monophosphate (cyclic AMP) is present in the medium than in its absence. 4. Growth on media containing gluconate and cyclic AMP is accompanied by the formation of methyl glyoxal and pyruvate, and progressive inhibition of growth. 5. A mutant devoid of adenylate cyclase activity (cya) grew well on glucose in the absence of exogenous cyclic AMP but grew only poorly on gluconate; neither the gluconate uptake system nor gluconate kinase was adequately induced. The addition of cyclic AMP promoted growth on gluconate and facilitated the induction of proteins required for gluconate catabolism. 6. Phage Pl-mediated transduction of cya+ into the cya-mutant also restored the wild-type phenotype in its ability to adapt to gluconate utilization.

Modulation of Stimulated Acid Secretion by Dibutyryl Cyclic Amp in the Dog Stomach

An in vivo canine gastric chamber preparation was used to determine effects of exogenous cyclic AMP and dibutyryl cyclic AMP on histamine-driven acid secretion and the role of blood flow in these responses. Histamine was infused intravenously at a submaximal rate 1.2 mu/kg-min) followed by the intraarterial infusion of either cyclic AMP (0.15 mg/kg-min) or dibutyryl cyclic AMP (0.15 mg/kg-min. Acid secretion and mucosal blood flow (aminopyrine clearance) were measured at 15-minute intervals, and electrical potential difference was measured continuously. Histamine alone decreased the potential difference from 82 mV to 48 mV within 60 minutes, increased aminopyrine clearance from 1.0 ml/min to 2.4 ml/min and increased acid output to 220 muEq/15 min. All values returned towards control following termination of histamine infusion. Dibutyryl cyclic AMP significantly increased histamine stimulated acid secretion to 350 muEq/15 min, but did not alter mucosal blood flow or electrical potential. Cyclic AMP did not change secretory, circulatory or electrical response to histamine. Therefore, dibutyryl cyclic AMP appears to increase histamine-stimulated acid secretion, and the effect is not mediated by increased mucosal blood flow.

Protective effect of thymidine against cytostatic action of cyclic-AMP in mammalian cell cultures

Under the influence of 1 mM cyclic-adenosine-3′,5′-monophosphate (cyclic-AMP) the degree of survival and rate of reproduction of Chinese hamster cells in culture were reduced to 27 and 42% of the control level, respectively. Addition of 0.02 mM thymidine along with the cyclic-AMP almost completely abolished the cytostatic effect of the latter. Thymidine also prevented the cytostatic effect of noncyclic 5′-AMP, but did not affect death of the cells due to the action of dibutyryl cyclic-AMP and theophylline. Thymidine did not prevent the inhibitory action of cyclic-AMP on a mutant line of mouse cells deficient in thymidine kinase. It is concluded that, in the concentrations used, the cytostatic action of exogenous cyclic-AMP on mammalian cells is the result of its splitting to 5′-AMP in the culture medium, and that it acts by blocking one of the stages of TMP biosynthesis.

Stimulation of cartilage amino acid uptake by growth hormone-dependent factors in serum: Mediation by adenosine 3′:5′-monophosphate

The effects of growth hormone-dependent serum factors on amino acid transport and on cartilage cyclic AMP levels in embryonic chicken cartilage were studied in vitro. Cartilages incubated in medium containing rat serum showed a significantly greater uptake of α-amino [1-14C] isobutyrate or [1-14C] cycloeeucine than control cartilages incubated in medium alone. Normal rat serum (5%) added to the incubation medium also caused an increase in cartilage cyclic AMP content (from as little as 23% to as much as 109%). The factors in serum which increase cartilage cyclic AMP and amino acid uptake are growth hormone dependent, since neither growth hormone itself nor serum from hypophysectomized rats affects either parameter. Growth hormone treatment of hypophysectomized rats restores these serum factors. Studies comparing the ability of sera with varying amounts of growth hormone-dependent factors to stimulate α-aminoisobutyrate transport and to increase cartilage cyclic AMP show a striking linear correlation between the two effects (r = 0.977). Theophylline and prostaglandin E1, which raise cartilage cyclic AMP also increase α-aminoisobutyrate transport. Exogenous cyclic AMP, N6-monobutyrll cyclic AMP and N6, O2′-dibutyryl cyclic AMP increase cartilage α-aminoisobutyrate transport. The data are compatible with the thesis that growth hormone-dependent serum factors increase cartilage amino acid transport by elevating cartilage cyclic AMP.

Rabbit testicular capsular contractions— Prostaglandins, cyclic adenosine 3′,5′-monophosphate and epinephrine

Log dose-response curves for the effect of prostaglandin E 1, E 2, F 1α, and F 2α on rabbit testicular capsules in vitro showed that F-type prostaglandins were stimulatory throughout the concentration range tested. On the other hand, E-type prostaglandins were stimulatory in low concentrations, but became less effective at higher concentrations, and exogenous cyclic AMP potentiated the testicular capsule's inhibitory response to PGE 1. Dibutyryl cyclic AMP inhibited tonus, whereas theophylline and isoproterenol inhibited both tonus and contractility in vivo and in vitro. Epinephrine stimulated contractions equally in vivo and in vitro; however, pretreatment of the preparations with ergotamine tartrate eliminated the stimulatory response to epinephrine. Pretreatment with propranolol eliminated the inhibitory response to isoproterenol. Ergotamine tartrate inhibited spontaneous contractions in vivo, but propranolol had no effect on capsular motility in vivo or in vitro. These data suggest that inhibition of capsular motility by higher concentrations of prostaglandin E 1 and E 2 may be mediated by cyclic AMP and confirm the presence of α-and β-adrenergic receptors in rabbit testicular capsules. Moreover, the data affirm the importance of adrenergic stimulation in regulating spontaneous contractions of testicular preparations in vivo.

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.

Regulation of pancreatic acinar function: effects of cyclic AMP, dibutyryl cyclic AMP, and theophylline in vitro.

The role of cyclic AMP in the regulation of pancreatic acinar function has been assessed by measuring the effects of exogenous cyclic AMP, dibutyryl cyclic AMP, and theophylline on protein synthesis and amylase secretion. The rate at which slices of rat pancreas incorporated leucine into protein did not change as a consequence of treatment with either cyclic AMP or dibutyryl cyclic AMP, nor did the slices alter their rate of amylase secretion. Moreover, theophylline did not enhance the ability of submaximal doses of pancreozymin to stimulate amylase secretion or to suppress protein synthesis. These results fail to demonstrate that cyclic AMP regulates either the synthesis or secretion of pancreatic digestive enzymes but they do not rule out the possibility.

Role of cyclic adenosine 3':5'-monophosphate in the action of 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT)on hepatic and renal metabolism.

The possibility whether alterations in the cyclic AMP-adenylate cyclase-phosphodiesterase system play a role in the action of 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) on hepatic and renal carbohydrate metabolism was investigated. Administration of exogenous cyclic AMP (10mg/100g) was found to mimic the action of DDT which enhanced the activities of pyruvate carboxylase, phosphoenolpyruvate carboxylase, fructose 1,6-diphosphatase and glucose 6-phosphatase in both liver and kidney cortex, elevated the concentration of blood glucose and urea and decreased the amount of hepatic glycogen. Treatment with theophylline augmented the effects of a submaximal dose of this halogenated hydrocarbon on serum urea and glucose as well as the key gluconeogenic enzymes in liver and kidney cortex. Addition of DDT in vitro to liver and kidney homogenates resulted in a significant enhancement of adenylate cyclase activity. Hepatic and renal slices from rats already treated with DDT displayed an increased ability to convert [(3)H]adenosine into cyclic [(3)H]AMP. Whereas kidney-cortex slices excised from rats given caffeine and DDT produced an even greater amount of cyclic [(3)H]AMP, imidazole, propranolol and hydrazine prevented the insecticide-stimulated rise in cyclic nucleotide production. In contrast, prostaglandin E(1) failed to exert any significant effect on DDT-induced increases in cyclic [(3)H]AMP synthesis from radioactive adenosine. The present study and our previous findings (Kacew & Singhal, 1973e) support the concept that the DDT-induced alterations in carbohydrate metabolism of liver and kidney cortex may be related to an initial stimulation of the cyclic AMP-adenylate cyclase system in these tissues.

Stimulation of cytochrome synthesis in Escherichia coli by cyclic AMP

A cyclic AMP-requiring mutant of Escherichia coli K12 which grows slowly on glucose was found to contain reduced levels of cytochrome b 1 and cytochrome oxidase o. The addition of exogenous cyclic AMP stimulated the synthesis of these cytochrome components and restored growth on glucose to the normal rate observed with the parental strain. Cytochrome synthesis in the parental strain was also stimulated by exogenous cyclic AMP. These studies have provided evidence that cyclic AMP participates in regulating cytochrome synthesis in E. coli, and, coupled with other observations, have suggested a role for this cyclic nucleotide in controlling the construction and operation of the organism's membrane system.

Effects of hormone treatment and age on the stimulation of rat anterior pituitary adenylate cyclase and cyclic adenosine 3′,5′ monophosphate by hypothalamic extract

Crude ovine hypothalamic extract in vitro significantly stimulated adenylate cyclase activity and LH release by pituitary gland obtained from newborn rats (0.25–0.5 days old) and increased cyclic adenosine 3′,5′-monophosphate (cyclic AMP) in 2-day-old rats. In adult rats, the stimulation of adenylate cyclase activity and cyclic AMP formation, but not LH release, induced by hypothalamic extract was inhibited by adrenalectomy. In vivo administration of pharmacologic doses of 1 mg dexamethasone asd 100 μg thyroxine for 1 day markedly reduced the effect of 0.2 mg/ml but not 1 mg/ml hypothalamic extract on cyclic AMP concentrations. The action of the latter dose of hypothalamic extract on cyclic AMP was inhibited by injection of dexamethasone and thyroxine for 3 days, while the stimulation of LH release was preserved. Such dissociation between activation of the adenylate cyclase-cyclic AMP system and LH release suggests that cyclic AMP formation may not be essential for increased release of LH. Adrenalectomy did not inhibit stimulation of adenylate cyclase activity or cyclic AMP formation caused by prostaglandin E 1 (PGE 1). Hypothalamic extract or PGE 1 added to intact pituitary in vitro increased protein kinase activity and decreased the protein binding of exogenous cyclic AMP, reflecting the increased generation of endogenous cyclic AMP.