Action Of Dibutyryl Cyclic AMP Research Articles

Actions of neuropoietic cytokines and cyclic AMP in regenerative conditioning of rat primary sensory neurons

A conditioning lesion to peripheral axons of primary sensory neurons accelerates regeneration of their central axons in vivo or neurite outgrowth if the neurons are grown in vitro. Previous evidence has implicated neuropoietic cytokines and also cyclic AMP in regenerative conditioning. In experiments reported here, delivery through a lentivirus vector of ciliary neurotrophic factor to the appropriate dorsal root ganglion in rats was sufficient to mimic the conditioning effect of peripheral nerve injury on the regeneration of dorsal spinal nerve root axons. Regeneration in this experimental preparation was also stimulated by intraganglionic injection of dibutyryl cyclic AMP but the effects of ciliary neurotrophic factor and dibutyryl cyclic AMP were not additive. Dibutyryl cyclic AMP injection into the dorsal root ganglion induced mRNAs for two other neuropoietic cytokines, interleukin-6 and leukemia inhibitory factor and increased the accumulation of phosphorylated STAT3 in neuronal nuclei. The in vitro conditioning action of dibutyryl cyclic AMP was partially blocked by a pharmacological inhibitor of Janus kinase 2, a neuropoietic cytokine signaling molecule. We suggest that the beneficial actions of increased cyclic AMP activity on axonal regeneration of primary sensory neurons are mediated, at least in part, through the induction of neuropoietic cytokine synthesis within the dorsal root ganglion.

An increase in intracellular levels of cyclic AMP produces trophic effects on striatal neurons developing in culture

Cyclic AMP-dependent kinases have been suggested to constitute signal transduction pathways involved in the regulation of neuronal development and survival. The present study examined whether elevated levels of cyclic AMP exhibit trophic activities on striatal neurons, in the rat, grown under serum-free culture conditions. Treatment with dibutyryl cyclic AMP, a permeable cyclic AMP, increased GABA uptake and immunocytochemically detectable levels of proteins such as c-Fos and Calbindin-D28k. Neuronal survival was promoted by dibutyryl cyclic AMP only in lower density cultures. Chronic exposure of neurons to dibutyryl cyclic AMP enhanced the morphological development of Calbindin-D28k-positive neurons. Furthermore, pretreatment with dibutyryl cyclic AMP afforded neuroprotection against N-methyl-d-aspartate-induced excitotoxicity. The dibutyryl cyclic AMP-induced trophic effects above were blocked by adenosine 3′,5′-cyclic monophosphothioate, a specific inhibitor of cyclic AMP-dependent kinases. We also examined whether cyclic AMP is involved in trophic effects provided by membrane depolarization induced by high K+ and growth factors such as basic fibroblast growth factor and insulin-like growth factor-1. Depolarization, but not the growth factors, increased intracellular levels of cyclic AMP. Adenosine 3′,5′-cyclic monophosphothioate diminished depolarization increases in GABA uptake, whereas it did not affect the trophic effect of the growth factors. Co-treatment with the growth factors and dibutyryl cyclic AMP produced additive effects on both increases in GABA uptake and neuroprotection against excitotoxicity.The present results indicate that cyclic AMP-dependent kinases play roles in mediating differentiation and survival of developing striatal neurons. Signalling pathways activated by either basic fibroblast growth factor or insulin-like growth factor-1 are independent of those involving cyclic AMP. In contrast, depolarization-induced trophic effects are mediated, at least in part, by cyclic AMP-dependent pathways. Protective actions of dibutyryl cyclic AMP against excitotoxic injury as well as the additive effects with the growth factors are of potential interest in the experimental therapy of acute or chronic neurodegenerative diseases.

Influence of Mn Ion the Action of Dibutyryl Cyclic AMP and Forskolin on Contraction, Membrane Response, and Cyclic AMP-Dependent Protein Kinase Activity in Rat Myometrium.

Contractile response, membrane activity, and protein kinase A (PKA) activity were measured on the longitudinal muscle taken from the estrogen-treated rat uterus, and the influence of Mn ion on the inhibitory effects caused by db cAMP and forskolin was investigated. Phasic contractions generated in the muscle taken from the middle portion of uterus were depressed to 48 and 83% by 30 microM db cAMP and 0.1 microM forskolin in Mg-free Krebs solution, respectively; phasic contractions were more strongly depressed by the agents in the solution containing 0.2 mM Mn. Action potentials consisted of spike and plateau components, and the duration of the plateau potential was reduced by the application of the agents; membrane activity was more strongly depressed in the presence of 0.2 mM Mn. The contractile depression caused by db cAMP was reduced and by forskolin was enhanced by pretreatment of the tissue with 0.6 mM Mn for 30 min. The PKA activity was increased by 39 and 6% of the control, when 30 microM db cAMP and 0.1 microM forskolin were applied, respectively; the PKA activity in response to db cAMP and forskolin was reduced and enhanced, respectively, when the tissues were pretreated with 0.6 mM Mn. It was proposed that Mn ions permeated into cell interior when the muscle was exposed to 0.6 mM Mn, so that the effects of the agents were differently affected. It was also shown that plateau potential dominated in the muscle taken from the ovarian portion, and the contractile inhibition caused by the agents was far weaker.

Noradrenaline stimulates glucose transport in rat brown adipocytes by activating thermogenesis. Evidence that fatty acid activation of mitochondrial respiration enhances glucose transport

The mechanisms by which noradrenaline, lipolytic agents and long-chain fatty acids stimulate glucose transport were investigated in rat brown adipocytes. Glucose transport was evaluated with tracer D-[U-14C]glucose and cell respiration was measured polarographically. Noradrenaline increased basal oxygen consumption (8-10-fold) and glucose transport (4-5-fold) in a dose-dependent manner, with a maximal stimulation at 100 nM. The stimulatory effects of noradrenaline on respiration and glucose transport were selectively mimicked by dibutyryl cyclic AMP (DBcAMP), 3-isobutyl-1-methylxanthine, cholera toxin and physiological concentrations of palmitic acid. Cytochalasin B completely blocked the effects of these agents on glucose transport. The beta-adrenergic antagonist propranolol inhibited noradrenaline-induced glucose transport, but did not affect the action of DBcAMP, palmitic acid or cholera toxin on this process. The specific inhibitor of mitochondrial carnitine palmitoyltransferase, 2-tetradecylglycidic acid (McN 3802) (50 microM), inhibited the stimulatory effects of noradrenaline (100 nM) and palmitic acid (0.5 mM) on both glucose transport and mitochondrial respiration. Significantly, McN 3802 failed to affect insulin (1 nM) action under identical experimental conditions. These results demonstrate that (a) the stimulatory effects of noradrenaline on brown-adipocyte respiration and glucose transport can be dissociated from those induced by insulin, and (b) noradrenaline increases glucose transport indirectly, by activating adenylate cyclase via beta-adrenergic pathways and by stimulating mitochondrial oxidation of fatty acids.

Regulation of aromatase in estrogen-producing cells

Human adipose stromal cells in monolayer culture aromatize androstenedione to estrone. The rate of aromatization is stimulated 20- to 30-fold by glucocorticoids when fetal calf serum is present in the culture medium and by dibutyryl cyclic AMP in the absence of serum. The action of dibutyryl cyclic AMP to stimulate aromatase activity is potentiated markedly by phorbol esters and inhibited by growth factors, such as EOF. In order to investigate the mechanisms underlying this multifactorial regulation, we have prepared polyclonal and monoclonal antibodies specific for aromatase cytochrome P-450. By use of these antibodies it was demonstrated that the action of these various factors to regulate aromatase activity was caused by alterations in the rate of synthesis of aromatase cytochrome P-450, whereas the synthesis of the reductase component of the aromatase enzyme complex was relatively unaffected. The changes in the rate of synthesis of aromatase cytochrome P-450 were, in turn, reflective of changes in the levels of translatable mRNA specific for this protein. In order to analyze the levels of aromatase cytochrome P-450 mRNA directly, we have isolated a cloned cDNA insert complementary to the mRNA encoding aromatase cytochrome P-450, by screening a λgt 11 human placental cDNA library utilizing the polyclonal anti-aromatase P-450 IgG. Use of this cDNA probe in Northern analysis of RNA extracted from human adipose stromal cells revealed that the changes in translatable mRNA resulting from incubation of the cells with the various regulatory factors were due to changes in the absolute levels of mRNA encoding this protein.

Effects of cyclic AMP and protein synthesis inhibitors on in vitro progesterone synthesis by the granulosa cells of the quail ( Coturnix coturnix japonica)

1. 1. During 3 hr of in vitro incubation, the production of progesterone by granulosa cells from the largest preovulatory follicle (F1) of the Japanese quail was stimulated by 1 mM dibutyryl cyclic AMP. 2. 2. On the other hand, the progesterone production of the granulosa cells from the second largest and the third largest follicles (F2 and F3) were autonomous, since these cells produced significant amounts of progesterone in the incubation without dibutyryl cyclic AMP, and addition of dibutyryl cyclic AMP did not further stimulate the progesterone production. 3. 3. Cycloheximide or puromycin inhibited not only the progesterone production of the granulosa cells of F1, which was due to the action of dibutyryl cyclic AMP, but also the autonomous progesterone production of the granulosa cells of F2. 4. 4. These results suggest that in the quail ovary, the labile protein may play a role for the progesterone synthesis, but is not a mediator for the action of cyclic AMP.

Differences in the mode of regulation of ornithine decarboxylase and tyrosine aminotransferase in H-35 rat hepatoma cells shown by varying the medium composition

The effects of dibutyryl cyclic AMP, dexamethasone, and asparagine as inducers of ornithine decarboxylase (EC 4.1.1.17) and tyrosine aminotransferase (EC 2.6.1.5) in H-35 rat hepatoma cells were studied; a comparison was made between cells incubated in Eagle's minimal essential medium and cells incubated in Earle's balanced salt solution (salts/glucose solution). The addition of either 1 mM dibutyryl cyclic AMP or 1 μM dexamethasone increased both ornithine decarboxylase and tyrosine aminotransferase activities of H-35 hepatoma cells maintained in Eagle's minimal essential medium, whereas 10 mM asparagine increased the activity of ornithine decarboxylase and had no effect on tyrosine aminotransferase activity. The actions of dibutyryl cyclic AMP and dexamethasone but not asparagine, as inducers of ornithine decarboxylase activity, appeared to be dependent on the incubation medium used; dibutyryl cyclic AMP and dexamethasone were ineffective in increasing ornithine decarboxylase activity of H-35 hepatoma cells maintained in the salts/glucose solution, while asparagine was fully effective. Parallel studies on tyrosine aminotransferase demonstrated that both dibutyryl cyclic AMP and dexamethasone elicited maximal increases of tyrosine aminotransferase activity of H-35 hepatoma cells maintained in the salts/glucose solution. Experiments on the activation of cyclic AMP-dependent protein kinase and the uptake/binding of [ 3H]dexamethasone to whole cells revealed no significant difference between H-35 hepatoma cells maintained in Eagle's minimal essential medium and cells maintained in the salts/glucose solution. The half-lives of ornithine decarboxylase of cells maintained in minimal essential medium and salts/glucose solution were 45 and 38 min, respectively; the corresponding values for tyrosine aminotransferase were 2.5 and 1.5 h, respectively. It is proposed that the actions of dibutyryl cyclic AMP and dexamethasone as inducers of ornithine decarboxylase may involve the participation of component(s), possibly amino acids, present in the Eagle's minimal essential medium but not in the salts/glucose solution. These components may either mediate or modulate the actions of dibutyryl cyclic AMP and dexamethasone in the induction of ornithine decarboxylase activity.

Opposing actions of dibutyryl cyclic AMP and GMP on temperature in conscious guinea-pigs

Intracerebroventricular administration of dibutyryl cyclic AMP (Db-cAMP), induced hyperthermia in guinea-pigs which was not mediated through prostaglandins (PG) or norepinephrine since a prostaglandin synthesis inhibitor, indomethacin, and an α-adrenergic receptor blocking agent, phenoxybenzamine did not antagonize the hyperthermia. In contrast, the hyperthermic response to dibutyryl cyclic AMP was attenuated by central administration of a β-adrenergic receptor antagonist, sotalol, indicating that cyclic AMP may be involved, through β-adrenergic receptors, in the central regulation of heat production/conservation. Central administration of dibutyryl cyclic GMP (Db-cGMP) produced hypothermia which was not mediated via histamine H 1- or H 2-receptors and serotonin since the H 1-receptor antagonist, mepyramine, the H 2-receptor antagonist, cimetidine, and the serotonin antagonist, methysergide, had no antagonistic effects. The antagonism of hypothermia induced by dibutyryl cyclic GMP and acetylcholine + physostigmine, by central administration of a cholinergic muscarinic receptor antagonist, atropine, and not a cholinergic nicotinic receptor antagonist, d-tubocurarine, suggests that cholinoceptive neurons and endogenous cyclic GMP may regulate heat loss through cholinergic muscarinic receptors. These results support a regulatory role in thermoregulation provided by a balance between opposing actions of cyclic AMP and cyclic GMP in guinea-pigs.

Striatal Dopamine Release In Vitro: A β‐Adrenoceptor‐Regulated Response Not Mediated Through Cyclic AMP

The effects of (-)isoproterenol (10(-6) M), dibutyryl cyclic AMP (10(-3) M), and the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX) (10(-4) M) on in vitro [3H]dopamine ([3H]DA) efflux and synthesis were studied in rat striatal slices continuously superfused with [3H]tyrosine. The beta-adrenoceptor agonist (-)isoproterenol induced an immediate and significant facilitation of [3H]DA efflux but did not alter [3H]DA synthesis as measured by [3H]H2O formation. In contrast, both dibutyryl cyclic AMP and IBMX enhanced [3H]DA synthesis as well as efflux. The presence of IBMX in the superfusing medium did not potentiate the augmentation of [3H]DA efflux caused by (-)isoproterenol. Additionally, the blockade of [3H]DA synthesis by alpha-methyl-p-tyrosine (10(-4) M) completely prevented the action of dibutyryl cyclic AMP on [3H]DA efflux. However, under similar conditions, (-)isoproterenol was still able to increase [3H]DA efflux. The results suggest that (-)isoproterenol can modify striatal DA release through a mechanism not involving cyclic AMP.

Mechanisms of action of various hormones and vasoactive substances on glomerular ultrafiltration in the rat

Angiotensin II (AII) and arginine vasopressin are capable of triggering glomerular mesangial cell contraction in vitro. A similar mechanism acting in vivo to reduce glomerular capillary surface area could account for the decline in the ultrafiltration coefficient (Kf)( that occurs in single glomeruli in response to infusion of these substances. Less clear is the mechanism whereby similar declines in Kf are induced with infusions of dibutyryl cyclic AMP (DBcAMP), parathyroid hormone (PTH), and prostaglandins, because PTH and PGE2, at least, are incapable of eliciting mesangial cell contraction in vitro. To further explore the factors that regulate Kf in vivo, we performed micropuncture experiments in 47 euvolemic Munich-Wistar rats. Infusions of DBcAMP, PTH, prostaglandins I2 and E2 led to lower mean values for plasma flow rate (QA) and Kf in superficial glomeruli than were found in animals given vehicle alone (control group), whereas average values for glomerular transcapillary hydraulic pressure difference (delta P) and total renal arteriolar resistance (RTA) tended to be higher. These increases in delta P and RTA, and decreases in QA and Kf, with DBcAMP, PTH, PGI2, and PGE2 are typical of changes induced by AII. Indeed, when saralasin, a competitive AII antagonist, was infused together with these various vasoactive substances, the effects on delta P, QA, RTA, and Kf were largely abolished. Therefore, the actions of DBcAMP, PTH, PGI2, and PGE2 on the glomerular microcirculation appear to depend on an intermediate action of AII. By contrast, although pitressin (ADH) infusion also led to a significant decline in Kf, saralasin administration did not reverse this change, suggesting that the action of ADH on the glomerular microcirculation is independent of a pathway involving AII. Based on these studies, it seems reasonable to propose that AII and ADH are both potentially important regulators of mesangial cell contraction, and thereby, glomerular capillary filtering surface area and Kf.

Effects of noradrenaline and N 6,O 2 -dibutyryl 3′,5′-cyclic AMP on adipocyte glycerolipid-synthesizing enzymes

1. 1. Adipocytes from rat epididymal fat pads were incubated with glucose and albumin. 2. 2. Under these conditions adrenaline decreased diacylglycerol acyltransferase activity without affecting CDP choline phosphotransferase activity. 3. 3. Adrenaline and insulin had no effect upon phosphatidate cytidylyl transferase activity. 4. 4. 1 and 2mM dibutyryl cyclic AMP decreased Mg 2+ -dependent phosphatidate phosphohydrolase activity. 5. 5. Insulin was unable to oppose the action of dibutyryl cyclic AMP on phosphatidate phosphohydrolase activity.

Correlation of cyclic AMP and cyclic GMP levels with changes in contractile force of dog ventricular myocardium during cholinergic antagonism of positive inotropic actions of histamine, glucagon, theophylline and papaverine

On the dog isolated right ventricular muscle, experiments were carried out in order to elucidate the characteristics of the cholinergic antagonism against the positive inotropic action (PIA) induced via different subcellular mechanisms. The relationship between cyclic AMP and cyclic GMP levels, and contractile force during cholinergic antagonism was assessed. Carbachol (10 μM) by itself decreased only slightly the tension developed, but inhibited prominently the PIAs of isoprenaline, histamine, glucagon, theophylline and papaverine. The action of dibutyryl cyclic AMP was inhibited less than PIAs of other agents mentioned above. In contrast, carbachol did not affect the PIAs of calcium and g-strophanthin. The antagonism by carbachol of PIAs of isoprenaline, histamine, glucagon, theophylline and papaverine was accompanied by a reduction of the intracellular cyclic AMP level elevated previously by these agents, and by an elevation of the intracellular cyclic GMP level. A good correlation was found between changes in the tension developed, and cyclic AMP and cyclic GMP levels during the cholinergic antagonism of PIAs induced by these agents in the dog ventricular myocardium.

Correlation of cyclic AMP and cyclic GMP levels with changes in contractile force of dog ventricular myocardium during cholinergic antagonism of positive inotropic actions of histamine, glucagon, theophylline and papaverine.

On the dog isolated right ventricular muscle, experiments were carried out in order to elucidate the characteristics of the cholinergic antagonism against the positive inotropic action (PIA) induced via different subcellular mechanisms. The relationship between cyclic AMP and cyclic GMP levels, and contractile force during cholinergic antagonism was assessed. Carbachol (10 microM) by itself decreased only slightly the tension developed, but inhibited prominently the PIAs of isoprenaline, histamine, glucagon, theophylline and papaverine. The action of dibutyryl cyclic AMP was inhibited less than PIAs of other agents mentioned above. In contrast, carbachol did not affect the PIAs of calcium and g-strophanthin. The antagonism by carbachol of PIAs of isoprenaline, histamine, glucagon, theophylline and papaverine was accompanied by a reduction of the intracellular cyclic AMP level elevated previously by these agents, and by an elevation of the intracellular cyclic GMP level. A good correlation was found between changes in the tension developed, and cyclic AMP and cyclic GMP levels during the cholinergic antagonism of PIAs induced by these agents in the dog ventricular myocardium.

Vinblastine potentiates the secretagogue action of dibutyryl cyclic AMP on the exocrine pancreas

The effects of VB 4 4 VB = Vinblastine sulphate. It was a generous gift from Eli Lilly (St-Cloud, France). on both structure and function of the pancreatic acinar cell have been examined in vitro. VB potentiates the secretagogue effect of DbcAMP 5 5 DbcAMP = N 6-2′-0-Dibutyryl-adenosine 3′,5′-monophosphate, cyclic. but fails to affect the spontaneous release of enzymes. This potentiation is parallel to the disappearance of cellular microtubules and depends on the dose and the time of exposure to VB. The potentiation is energy dependent; it is more obvious in calcium containing medium than in absence of calcium. A damaging effect of VB an acinar cells is ruled out. These findings suggest the participation of microtubules in the secretory cycle of the pancreatic acinar cell.

Plasma membrane phosphorylation by endogenous phosphate donors in human blood platelets Selectivity of the action of dibutyryl cyclic AMP

Incubation of platelet-rich plasma with 32P i leads to cellular uptake of the isotope and covalent incorporation into several cell constituents. Plasma membranes isolated from intact labelled platelets, delipidated and solubilized in sodium dodecyl sulfate, show, upon gel electrophoretic analysis, three main peaks of radioactivity: two in the molecular weight range 100 000–30 000 and an additional very slow migrating component strongly positive by the periodic acid-Schiff reaction. Treatment of the cells with dibutyryl cyclic AMP under conditions just sufficient to completely inhibit platelet aggregation leads to an increased isotope incorporation. Electrophoretic analysis of membranes isolated from dibutyryl cyclic AMP-treated cell reveals: (a) no change in the general pattern of distribution of the isotope, (b) no difference in the isotope incorporation to the two components of lower mol. wt. and (c) a marked increase(> 100%) in isotope incorporation in the slow migrating material as compared to membranes isolated from control cells. This material can be extracted from platelet plasma membranes after treatment of the membranes for 5 h with Triton X-100, at a detergent-to-protein ratio of 7.5. When the membrane material extracted with Triton X-100 is subjected to gel chromatography in Agarose (Biogel A-15m), the phosphorylated material that corresponds to the slow migrating band in polyacrylanide gel electrophoresis is eluted with or very close to the void volume of the column. Isoelectric focussing of this fraction, shows a single radioactive peak corresponding to an isoelectric point of 3.78. The isolated component is pronase-sensitive, contains 52% of carbohydrate and 15% sialic acid. Analysis of the stability of the bound phosphate suggests that about 43% of it is bound as acylphosphate. The results reported, obtained through an approach that closely resembles physiological conditions are compatible with the participation of this membrane phosphoglycoprotein in the phenomena of platelet aggregation.

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

The stimulatory effects of N 6, 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.

INFLUENCES OF AMINOPHYLLINE AND REDUCTION IN EXTERNAL Na ON THE ANTISPASMODIC ACTION OF ISOPROTERENOL IN THE ISOLATED RAT RECTUM

Effects of aminophylline and the reduction in external Na on the antispasmodic action of isoproterenol were investigated in relation to the mobilization of Ca in the isolated rat rectum. The antispasmodic action of isoproterenol on the phasic contractions by acetylcholine and K both in Ca-free and in Ca-free and Na-poor media was potentiated by treatment with aminophylline, however the antispasmodic action was attenuated by reducing Na in the Ca-free medium. Dibutyryl cyclic AMP inhibited acetylcholine- and K-induced phasic contractions in Ca-free and in Ca-free and Na-poor media and the inhibitory action was also potentiated by treatment with aminophylline, while the inhibitory action of dibutyryl cyclic AMP was attenuated by reducing Na in the Ca-free medium. From these findings, it appears that isoproterenol inhibits the release of Ca from storage sites induced by acetylcholine and K via the increase of intracellular cyclic AMP content and that the external Na may play an important role in the Ca release-inhibiting effect of cyclic AMP.

Comparisons between the antianesthetic action of dibutyryl cyclic AMP and analeptic drugs on amobarbital-induced narcosis in the rat.

The dose-related antianesthetic and antidotal property of dibutyryl cyclic AMP, devoid of toxic effects, imparts uniqueness to the nucleotide as an arousal agent. Of the analeptic drugs studied (d-amphetamine, picrotoxin, pentylenetetrazol, caffeine, theophylline, strychnine, ethamivan and doxapram), only picrotoxin demonstrated antianesthetic properties. However, picrotoxin was associated with severe toxicity at all dose levels tested. No analeptic drug is effective in reversing the central nervous system depression produced by sedative, hypnotic or tranquilizer drug overdosage.

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.

Elevation of hepatic phenylalanine: Pyruvate aminotransferase by dibutyryl cyclic AMP in rats

The ip injection of dibutyryl cyclic AMP (50 mg/kg) into rats caused a four- to five-fold elevation of hepatic l-phenylalanine: pyruvate aminotransferase activity 48 hr later, and the enzyme level was still elevated at twice the control values at 8 days. Theophylline (50 mg/kg) did not affect enzyme activity itself, but enhanced the action of dibutyryl cyclic AMP so that doses as low as 1 mg/kg were effective in inducing the enzyme. Actinomycin D injected at the same time as the dibutyryl cyclic AMP antagonized the increase in enzyme activity. Phenylalanine aminotransferase levels were not elevated by dibutyryl cyclic AMP in extrahepatic tissues (brain, heart, kidney, and skeletal muscle). A second, third, and fourth injection of dibutyryl cyclic AMP caused further rises in hepatic enzyme levels. Cyclic AMP had less effect on the enzyme than did its dibutyryl derivative, and cyclic GMP and cyclic IMP caused no significant change in the enzyme activity. The properties of dibutyryl cyclic AMP as an inducer of this enzyme are generally similar to those of glucagon and glucagon derivatives that we have reported earlier.