INHIBITION OF CELL GROWTH IN THE G1 PHASE BY ADENOSINE 3'',5''-CYCLIC MONOPHOSPHATE

Incorporation of tritiated thymidine into acid-precipitable material was used to measure the rate of DNA synthesis in secondary cultures of human diploid fibroblasts. Confluent cultures of human diploid fibroblasts, which are synchronized in the G₁ phase due to contact inhibition, were released from growth inhibition either by the addition of fresh medium to the cultures or by trypsinization and replating at nonconfluent densities. Either treatment resulted in a synchronous wave of DNA synthesis beginning 10–15 h after treatment and peaking at 20–25 h. In confluent cultures stimulated by fresh medium, either the addition of 0.25 mM N⁶, O²-dibutyryl-adenosine 3',5'-cyclic monophosphate (db-cAMP) to the medium in the interval 4–8 h after stimulation or the replacement of the fresh medium in that same 4 h interval with the depleted medium present on the cells for the 2 day period before stimulation delayed the synchronous onset of DNA synthesis in the cultures by about 4 h. In nonconfluent cultures freshly seeded from trypsinized confluent cultures, this same depleted medium obtained after a 2 day incubation of fresh medium on confluent cultures is shown to support the progress of the cells into S phase; however, the addition of 0.25 mM db-cAMP to the medium 3½ h after replating still partially prevented the initiation of DNA synthesis in the cultures. The results are discussed in terms of the role of serum and cAMP in the control of cell growth in fibroblast cultures.     

THE ONTOGENY OF DOPAMINE-DEPENDENT INCREASE OF ADENOSINE 3'',5''-CYCLIC MONOPHOSPHATE IN THE CHICK RETINA

The adenosine 3′,5′-cyclic monophosphate level of chick embryonic retina changes during the course of development. In retinas from 6- to 15-day-old embryos the cAMP level is approximately 7 pmol/mg protein. A sharp 3-fold increase is observed between the 16th and 18th embronic day and remains constant thereafter. A dopamine-dependent increase in cAMP of the chick retina is already present in 7-day-old embryos, and by the 8th embryonic day maximal response is attained. Glutamate promotes a 2-fold stimulation. Carbachol, γ-aminobutyric acid and glycine do not cause any significant change in the level of cAMP of the embryonic tissue. Guanosine 3′,5′-cyclic monophosphate also accumulates during development. Its concentration is approx 0.5 pmol/mg protein from the 8th to the 14th embryonic day, then increases gradually until the 19th day of development when the level observed is approx 14 pmol/mg protein.     

EFFECT OF ADENOSINE 3''-5''-CYCLIC MONOPHOSPHATE ON CELL PROLIFERATION

Secondary cultures of human diploid fibroblasts, which demonstrate density-dependent inhibition of cell growth, were used to study the effect of adenosine 3'-5'-cyclic monophosphate (cAMP) on cell proliferation. DNA synthesis in nonconfluent cultures and in contact-inhibited cultures stimulated to grow by refeeding with fresh medium was found to be inhibited by exogenous cAMP. The properties of this inhibition of DNA synthesis, together with the alterations in cAMP metabolism observed in confluent cultures of cells stimulated with fresh medium to resume growth, strongly suggest that cAMP is involved in contact-inhibition of cell proliferation.     

A RADIOISOTOPIC METHOD FOR MEASURING THE FORMATION OF ADENOSINE 3'',5''-CYCLIC MONOPHOSPHATE IN INCUBATED SLICES OF BRAIN

Abstract— A simple and sensitive method for measuring the effect of neurohormones and other chemical agents on the formation of adenosine 3',5'-cyclic monophosphate (cyclic 3',5'-AMP) in incubated slices of brain was developed. The principle of the method depends on pulse-labelling of adenosine-5'-triphosphate in slices of brain with [8-¹⁴C]adenine, followed by incubation in a medium containing the test substance, separation by thin-layer chromatography of the cyclic nucleotide formed in the slices, and radioassay. The purity of the cyclic nucleotide was confirmed by chromatography in a variety of systems and by hydrolysis with a specific, bovine-heart phosphodiesterase. The method was used to study the effect of histamine, norepinephrine, and adenosine on the accumulation of adenosine 3',5'-cyclic monophosphate in incubated slices of brain.     

DIBUTYRYL CYCLIC ADENOSINE 3'' 5'' MONOPHOSPHATE, SUGAR TRANSPORT, AND REGULATORY CONTROL OF CELL DIVISION IN NORMAL AND TRANSFORMED CELLS

Swiss 3T3 cells exhibit contact-regulated cell growth and have a lower ability to transport 2-deoxyglucose than polyoma (Py)-transformed 3T3 cells. Py3T3 cells treated with dibutyryl cyclic adenosine 3'5' monophosphate (dBcAMP) and theophylline have reduced cell growth and transport 2-deoxyglucose at the same rate as normal 3T3 cells. Evidence that the cessation of cell growth and reduced transport abilities in Py3T3 cells does not represent a return to contact-regulated growth comes from the following observations. First, treating high density Py3T3 cells with dBcAMP allows more than two doublings of cell number, even though ability to transport 2-deoxyglucose is returned to levels equal to those of normal 3T3 cells. Second, dBcAMP prevents serum-stimulated increases in 2-deoxyglucose transport in Py3T3 but not in 3T3 cells.     

EFFECTS OF ATP ON THE ACTIVITY OF NUCLEOSIDE 3'',5''-CYCLIC MONOPHOSPHATE PHOSPHODIESTERASE FROM BRAIN

ATP is known to inhibit the phosphodiesterase activity in the supernatant fraction of the brain homogenate. Results showed that, when enzyme activity was assayed by determining the change in the concentration of substrate, the magnitude of the inhibition by 2 ～ 3 mm -ATP was not more than 20% and this effect of ATP can be explained mainly, if not entirely, on the basis of chelation of ATP with Mg²⁺ and Ca²⁺in vitro, both of which are necessary for enzyme activity. When brain phosphodiesterase was assayed by measuring 5′-AMP (product), the effect of ATP was erroneously exaggerated. This is due to ATP-dependent conversion of 5′-AMP to inosinic acid catalysed by adenylate deaminase in the crude preparation.     

THE INFLUENCE OF SECRETIN, GLUCAGON AND OTHER PEPTIDES, OF AMINO ACIDS, PROSTAGLANDIN ENDOPEROXIDE ANALOGUES AND DIAZEPAM ON THE LEVEL OF ADENOSINE 3'',5''-CYCLIC MONOPHOSPHATE IN NEUROBLASTOMA GLIOMA HYBRID CELLS

Abstract— Neuroblastoma glioma hybrid cells display many properties of neurons. A series of compounds, among them a number of amino acids, peptides and peptide hormones were tested for their ability to influence the level of adenosine 3',5'-cyclic monophosphate (cyclic AMP) in the hybrid cells. Two prostaglandin endoperoxide analogues exhibit a weak stimulatory action, if applied in at least micromolar concentrations. At nanomolar concentrations, only the gastrointestinal hormones secretin and glucagon stimulate the formation of cyclic AMP, as detected in the presence of a phosphodiesterase inhibitor. The effect of secretin but not that of glucagon is antagonized by secretin-(5–27), suggesting that secretin and glucagon act on the cells via different receptors. These results appear to be noteworthy since (a) an effect of secretin or glucagon on a cell with neuronal characteristics has not yet been described, and (b) many peptide hormones have been detected both in the gastrointestinal tract and the nervous system.     

维生素A酸和双丁酰基环腺苷单磷酸对小鼠胚胎干细胞的诱导分化

我们用建系的小鼠胚胎干细胞ES-5细胞为材料,研究了维生素A酸(RA)和双丁酰基环腺苷单磷酸(dBcAMP)对该胚胎干细胞的体外诱导分化。在ES-5细胞单层培养的条件下,RA单独作用或RA与dBcAMP共同作用,都产生神经元样和成纤维样两种细胞;在后一种作用情况下分化细胞可达90—95%,通过对细胞形态特征表型标志(GFAP,层粘蛋白等)的分析,初步证明绝大部分细胞为神经胶质细胞。除单层培养外,我们还研究了RA对ES-5细胞聚集体贴壁培养的诱导分化,在这种条件下,分化细胞类型较多,其是最突出的是有节律收缩的心肌样细胞。本文讨论了ES-5细胞单层培养与细胞聚集体贴壁培养两种条件下,RA诱导的差异及其可能原因。     

INCREASE IN THE CONTENT OF ADENOSINE 3''5''- MONOPHOSPHATE IN MOUSE FOREBRAIN DURING SEIZURES AND PREVENTION OF THE INCREASE BY METHYLXANTHINES

Abstract— Seizures produced significant elevations of the content of adenosine 3',5'-monosphosphate (cyclic AMP) in mouse forebrain in vivo. The content of cyclic AMP doubled at 5 s and had increased four- to five-fold at 90 s after the onset of tonus. Smaller increases were observed when mice were placed in an 0₂-enriched atmosphere. The increase in the content in forebrain of cyclic AMP during seizures was significantly reduced in mice that had previously been injected with heophylline or caffeine. The methylxanthines did not alter the initial (pre-ictal) level of cycIicXMP in mouse forebrain. On the basis of these and previous data, the following hypothesis is proposed to account for the present observations : When seizures produce asphyxia in the forebrain, the consequent dephosphorylation of adenine nucleotides liberates small amounts of free adenosine which may then diffuse out of cells and activate an adenyl cyclase-linked receptor that is located on plasma membranes and is accessible from the extracellular space.     

INHIBITION OF THYROXINE-INDUCED RESORPTION OF TADPOLE TAIL BY ADENOSINE 3', 5'-CYCLIC MONOPHOSPHATE

Small segments of tail of Bufo bufo japonicus tadpoles were cultured in medium containing thyroxine (T₄) and dibutyryl cyclic AMP (dbcAMP). Like prolactin, the cyclic nucleotide blocked T₄-induced shrinkage or tail pieces. Histological study of the segments after 4-days culture revealed that dbcAMP suppressed degenerative changes induced by T₄. The inhibitory effect of prolactin on T₄-induced tail regression was promoted by caffeine, an inhibitor of adenosine 3', 5'-cyclic monophosphate (cyclic AMP)-phosphodiesterase.
The effect of prolactin on the level of cyclic AMP in the tail was also studied in vivo . Sixty min after prolactin injection, the cyclic AMP level was 2–3 times the control value. Possible involvement of cyclic AMP in the action of prolactin, which blocks tail resorption induced by T₄, was discussed.     

2'',3''-CYCLIC NADP AS A SUBSTRATE FOR 2'',3''-CYCLIC NUCLEOTIDE 3''-PHOSPHOHYDROLASE

Abstract– 2',3'-Cyclic NADP has been prepared by cyclization of NADP at pH 6 in the presence of l-ethyl-(3-dimethylaminopropyl)-carbodiimide. The NADP derivative is readily hydrolyzed to NADP by the enzyme in brain and nerve that hydrolyzes 2',3'-cyclic nucleotides to 2'-phospho esters. The K _m for this substrate is the same as that for 2',3'-cyclic AMP (0.22 m m ) at pH 6 and 25°C. The two substrates are hydrolyzed by the phosphohydrolase at similar maximum velocities. The nicotinamide moiety in cyclic NADP thus has little effect on the enzyme-substrate interaction. This synthetic substrate can be used in a rapid (2 min) and sensitive (10 ng of 31-fold purified enzyme) spectrophotometric coupled enzyme assay for 2',3'-cyclic nucleotide 3'-phosphohydrolase; in this assay the hydrolysis proceeds in the presence of glucose-6-phosphate dehydrogenase and its substrate and the NADPH formed is measured by the increase in absorbance at 340 nm. The assay is applicable to tissue extracts as well as to purified preparations of the enzyme. There is no interference from nucleases of the pancreatic RNase A type.     

CYCLIC ADENOSINE 3'', 5''-MONOPHOSPHATE IN GUINEA PIG CEREBRAL CORTICAL SLICES: POSSIBLE REGULATION OF PHOSPHODIESTERASE ACTIVITY BY CYCLIC ADENOSINE 3'', 5''-MONOPHOSPHATE AND CALCIUM IONS

Cyclic adenosine 3′, 5′-monophosphate (cyclic AMP) accumulates in guinea pig cerebral cortical slices during incubation with histamine, histamine + noradrenaline and adenosine. Noradrenaline does not enhance cyclic AMP formation. In the absence of Ca²⁺ ions and presence of 1 mM-EGTA in the Krebs-Ringer bicarbonate medium the effects of histamine, histamine + noradrenaline and adenosine are significantly enhanced and noradrenaline elicits an increase in cyclic AMP over control levels. When histamine is used as stimulant, cyclic AMP levels start to decline after only 5 min. However, in the absence of calcium and in the presence of EGTA in the medium this decline is not observed and cyclic AMP levels continue to rise for a considerable period of time. In normal medium, responses to restimulation by histamine or histamine + noradrenaline are greatly reduced in magnitude after a prior stimulation by these putative neurotransmitters. In contrast, when calcium is omitted from the incubation medium and 1 mM-EGTA is included, cyclic AMP levels increase to normal values at a second stimulation with histamine or histamine + noradrenaline. When slices are preincubated for various periods of time with histamine before addition of noradrenaline, the accumulation of cyclic AMP is significantly reduced as compared to levels obtained when histamine + noradrenaline were added simultanously. This decline in the overall response to histamine + noradrenaline is not observed when preincubation with histamine and subsequent incubations with histamine + noradrenaline are performed in Ca²⁺-free, 1 mM-EGTA containing buffer. Also preincubation with noradrenaline in normal, calcium-containing medium does not affect the total amount of cyclic AMP accumulating in the brain slices. The results are discussed in terms of an activation of phosphodiesterase within the cerebral cortical slices by increased levels of intracellular, freely available calcium which is mediated by the elevation of cyclic AMP concentration following hormonal stimulation.     

IN VIVO CHANGES OF CEREBRAL CYCLIC ADENOSINE 3'',5''-MONOPHOSPHATE INDUCED BY BIOGENIC AMINES: ASSOCIATION WITH PHOSPHORYLASE ACTIVATION

—The intravenous injection of adrenaline, isoprenaline and histamine to 4-6-day-old chicks resulted in a rapid increase in the cyclic AMP content of cerebral hemispheres that had been removed and frozen within 0·5 s using a freeze-blowing technique. Noradrenaline, dopamine, adenosine, 5-HT and acetylcholine did not significantly alter the nucleotide concentration in vivo. Addition of adrenaline, isoprenaline and histamine to incubated chick cerebral cortex slices also increased the cyclic AMP content of the tissue. Noradrenaline was considerably less potent than these amines and adenosine was ineffective. Low phosphorylase a levels (16 per cent of total activity) were observed in instantaneously frozen cerebral hemispheres of untreated chicks. The injection of adrenaline, isoprenaline and histamine resulted in a rapid conversion of phosphorylase b to a and a significant fall in tissue glycogen. Administration of noradrenaline was without effect on the relative forms of phosphorylase and also failed to influence cerebral glycogen. Phosphorylase activation was not observed in chick cerebral slices under conditions producing large increases in cyclic AMP. It is suggested that in vivo phosphorylase activation and subsequent glycogenolysis may occur, at least in part, in glia and that these cells may be damaged during preparation of cerebral slices. The results provide evidence of a metabolic role for cyclic AMP in cerebral tissue.     

CATECHOLAMINE INDUCED INCREASE OF CYCLIC ADENOSINE 3'',5''-MONOPHOSPHATE IN RAT BRAIN IN VIVO

Abstract— Four catecholamines injected into the cerebral ventricles increased the content of cyclic adenosine 3',5'-monophosphate (cAMP) in vivo in the whole brain of rats. The highest rise (2.6-fold) was measured 2 min after an injection of 100 μg epinephrine. Isoproterenol and norepinephrine were less active and dopamine hardly increased the cAMP level. These results are compatible with the view that physiological actions of catecholamines in the nervous system may be mediated by an increase of CAMP.     

CYCLIC NUCLEOTIDES IN MURINE BRAIN: EFFECT OF HYPOTHERMIA ON ADENOSINE 3'',5'' MONOPHOSPHATE, GLYCOGEN PHOSPHORYLASE, GLYCOGEN SYNTHASE AND METABOLITES FOLLOWING MAXIMAL ELECTROSHOCK OR DECAPITATION

Abstract —The accumulation of adenosine-3',5'-cyclic monophosphate (cyclic AMP) has been investigated in murine brain following electroconvulsive shock and decapitation. Animals were made hypothermic (20°C) to minimize the freezing time of the brain and to delay metabolic events. Cyclic AMP concentrations were decreased in the cerebral cortex of hypothermic rats and mice. Furthermore, the changes in cyclic AMP elicited by electroconvulsive shock and decapitation were delayed. In hypothermic animals, the metabolic rate as determined by high energy phosphate use was decreased to 65% of control values. The interconversions of the active and inactive forms of glycogen phosphorylase and glycogen synthase were sufficiently retarded in hypothermic animals to correlate with changes in cyclic AMP concentrations. The conversion of phosphorylase b to a and synthase a to b occurred when cyclic AMP concentrations had increased from 2 to 5 μmol/kg, following either electroconvulsive shock or decapitation. The results indicate that cyclic AMP plays a role in regulation of glycogen metabolism in cerebral cortex.     

REGULATION OF GUANOSINE 3'',5'' CYCLIC MONOPHOSPHATE IN THE RAT PINEAL AND POSTERIOR PITUITARY GLANDS

Potassium and norepinephrine stimulate the accumulation of cyclic AMP and cyclic GMP in rat pineal glands and their efflux into the medium. The efflux of both cyclic nucleotides was blocked by probenecid. The accumulation and efflux of cyclic GMP, but not of cyclic AMP, depends upon the presence of intact nerve endings and extracellular calcium. The calcium-dependent release of norepinephrine caused by veratridine was accompanied by the efflux of both cyclic AMP and cyclic GMP. In contrast, the calcium-independent release of norepinephrine caused by tyramine was accompanied by the efflux of cyclic AMP but not cyclic GMP. Changes in cyclic GMP therefore, may be related to exocytosis from the sympathetic nerve endings in the gland. High concentrations of potassium also increased tissue levels of cyclic GMP in the posterior pituitary gland. Veratridine and potassium, but not norepinephrine, stimulated the efflux of cyclic GMP from this neurosecretory gland. Thus, the relationship between cyclic GMP and exocytosis may extend beyond sympathetic nerve endings. The enhanced accumulation of cyclic GMP in the pineal gland after potassium does not appear to be mediated by extracellular (released) norepinephrine. Desmethylimipramine blocked the norepinephrine-stimulated changes in cyclic GMP, but not those caused by potassium. Investigation of the possible relationship between cyclic GMP and release of neurotransmitters is complicated by the apparent seasonal variation in the response of pineal cyclic GMP to potassium or norepinephrine.     

PURIFICATION AND COMPARISON OF 2'',3''-CYCLIC NUCLEOTIDE 3''-PHOSPHOHYDROLASES FROM BOVINE BRAIN AND SPINAL CORD

The membrane-bound enzyme 2′,3′-cyclic nucleotide 3′-phosphohydrolase has been purified from acetone powders of bovine white matter and spinal cord. Affinity chromatography on AMP-Sepharose has been used as the final step in the chromatographic purifications. The yield was about 3 mg of purified enzyme per 100 g of tissue in each instance. The enzymes from the two sources were indistinguishable by chromatography, gel electrophoresis, and amino acid analysis; the enzyme from spinal cord, however, has shown a specific activity of 225 units/mg compared to 342 units/mg for the enzyme from white matter. Both proteins had a molecular weight of 100,000 by gel filtration and 50,000 by sodium dodecyl sulfate-gel electrophoresis under reducing conditions. The properties of the enzyme, including amino acid composition determined on the purified soluble protein and on the protein purified by sodium dodecyl sulfate-gel electrophoresis, were those of a basic hydrophobic protein.     

ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE IN CHLAMYDOMONAS REINHARDTII : Influence on Flagellar Function and Regeneration

Adenosine 3',5'-cyclic monophosphate (cAMP) influences both flagellar function and flagellar regeneration in Chlamydomonas reinhardtii. The methylxanthine, aminophylline, which can cause a tenfold increase in cAMP level in C. reinhardtii, inhibits flagellar movement and flagellar regeneration by wild-type cells, without inhibiting cell multiplication. Caffeine, a closely related inhibitor, also inhibits flagellar movement and regeneration, but it inhibits cell multiplication too. Regeneration by a mutant lacking the central pair of flagellar microtubules was found to be more sensitive than wild type to inhibition by caffeine and to be subject to synergistic inhibition by aminophylline plus dibutyryl cAMP. Regeneration by three out of seven mutants with different flagellar abnormalities was more sensitive than wild type to these inhibitors. We interpret these results to mean that cAMP affects a component of the flagellum directly or indirectly, and that the responsiveness of that component to cAMP is enhanced by mutations which affect the integrity of the flagellum. The component in question could be microtubule protein.