Neurotrophic control of cyclic nucleotide levels during muscle differentiation in cell culture

The effects of chick brain–spinal cord extract on morphological development and cyclic nucleotide levels of cultured chick embryo skeletal muscle cells were determined. It had previously been shown that the extract stimulated morphological differentation, protein synthesis, and cholinesterase activity of muscle cells. Myoblasts fused earlier and an increase in number as well as diameter of myotubes were seen in the extract treated cultures. Cyclic nucleotides levels were higher (almost twice the controls for both adenosine 3′, 5′ -cyclic monophosphate and guanosine 3′, 5′ -cyclic monophosphate) and preceded their occurence in the control cultures. It was suggested that factor(s) in the extract interact with membrane receptor(s) to alter nucleotide levels which, in turn, allow the effects to be expressed.     

Cyclic nucleotide content of tobacco BY-2 cells

The cyclic nucleotide content of cultured tobacco bright yellow-2 (BY-2) cells was determined, after freeze-killing, perchlorate extraction and sequential chromatography, by radioimmunoassay. The identities of the putative cyclic nucleotides, adenosine 3',5'-cyclic monophosphate (cyclic AMP), guanosine 3',5'-cyclic monophosphate (cyclic GMP) and cytidine 3',5'-cyclic monophosphate (cyclic CMP) were unambiguously confirmed by tandem mass spectrometry. The potential of BY-2 cell cultures as a model system for future investigations of cyclic nucleotide function in higher plants is discussed.     

Identification of Adenosine-3′,5′-Monophosphate as the Bacterial Attractant for Myxamoebae of Dictyostelium discoideum

Adenosine-3',5'-cyclic monophosphate was shown to be the compound found in Escherichia coli responsible for the attraction of the amoebae of the cellular slime mold Dictyostelium discoideum. A number of other nucleotides were tested and the following were active: tubercidin-3',5'-cyclic monophosphate, N(6)-2'-O-dibutyryl-adenosine-3',5'-cyclic monophosphate, 5'-methylene adenosine-3',5'-cyclic monophosphonate, guanosine-3',5'-cyclic monophosphate, uridine-3',5'-cyclic monophosphate, cytidine-3',5'-cyclic monophosphate, inosine-3',5'-cyclic monophosphate, and thymidine-3',5'-cyclic monophosphate. They were less active than adenosine-3',5'-cyclic monophosphate. It is suggested that cyclic adenosine monophosphate secreted by the bacteria is used by the amoebae as a means of sensing and orienting towards food.     

Effects of substance P on the long-term regulation of tyrosine hydroxylase activity and catecholamine levels in cultured adrenal chromaffin cells

Acetylcholine, released from splanchnic nerve terminals innervating adrenal chromaffin cells, is known to increase synthesis of adrenal tyrosine hydroxylase, the rate-limiting enzyme in catecholamine synthesis. The neuropeptide substance P is also present in the splanchnic nerve innervating the adrenal medulla, and this study examined whether substance P has any long-term effects on tyrosine hydroxylase activity and catecholamine levels in cultures of adult bovine adrenal chromaffin cells. When cultures were incubated for 3 days with substance P and carbachol, a cholinergic agonist, substance P (10(-6) M, and greater) completely inhibited the increase in tyrosine hydroxylase activity normally induced by carbachol. Long-term stimulation with carbachol also depleted endogenous catecholamines from the cells and substance P prevented this carbachol-induced depletion of catecholamine content. Substance P by itself, in the absence of carbachol, had only a slight effect on tyrosine hydroxylase activity. 8-Bromoadenosine 3':5'-cyclic monophosphate, an analogue of adenosine 3':5'-cyclic monophosphate, also increases tyrosine hydroxylase activity in chromaffin cells; however, substance P had no effect on the increase in tyrosine hydroxylase activity induced by this analogue. These results indicate that substance P's effects are relatively specific for the carbachol-induced increased in tyrosine hydroxylase activity and that the primary site of action of substance P is not a site common to the mechanism of tyrosine hydroxylase induction by carbachol and 8-bromoadenosine 3':5'-cyclic monophosphate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of cyclic AMP in chondrocyte cultures: effects on sulfated-proteoglycan synthesis

The effects of N6,O2'-dibutyryladenosine 3':5'-cyclic monophosphate (DBcAMP), 8-bromoadenosine 3':5'-cyclic monophosphate (8Br-cAMP), 3':5'-cyclic monophosphate (cAMP), L-isoproterenol and L-epinephrine on sulfated-proteoglycan synthesis by rabbit articular chondrocytes were compared. DBcAMP and 8Br-cAMP in the presence or absence of 3-isobutyl-1-methylxanthine (IBMX) stimulated sulfated-proteoglycan biosynthesis after 20 h of incubation. cAMP had no significant effect. Both DBcAMP and 8Br-cAMP increased the hydrodynamic size of the newly synthesized proteoglycan monomer (A1D1) relative to control cultures. By contrast, although isoproterenol and epinephrine stimulated total cAMP synthesis, neither stimulated sulfated-proteoglycan synthesis. Whereas intracellular cAMP accumulated after incubation with DBcAMP and 8Br-cAMP, this was not the case with isoproterenol whether IBMX was present or not. Thus, stimulation of sulfated-proteoglycan synthesis by cAMP analogues in chondrocyte cultures appears to be dependent on increased intracellular cAMP accumulation rather than total cAMP biosynthesis.     

Intracellular Levels of Cyclic Nucleotides during Cell Enlargement and Cell Division in Excised Tobacco Pith Tissues

Two lines of evidence, one of which is based on the radioimmunoassay and the other on adenosine 3', 5'-cyclic monophosphate (cyclic AMP)-dependent histone phosphorylation, are presented to demonstrate the presence of cyclic AMP in excised tissues of higher plant species. Intracellular levels of this cyclic nucleotide appear to be determined by auxin and a positive correlation has been found to exist between cell enlargement and chromosomal DNA replication, both auxin-dependent processes, and the level of cyclic AMP in tobacco pith cells. Intracellular guanosine 3', 5'-cyclic monophosphate (cyclic GMP) levels, while measurable, did not appear to be affected by either auxin or kinetin, or both, during the cell enlargement or cell division phases of the cell cycle in the tobacco pith system.     

Effects of oxytocin and methacholine on cyclic nucleotide levels of rabbit myometrium

The effects of oxytocin and methacholine on cyclic nucleotide levels in estrogen-primed rabbit myometrium were studied in the presence and absence of 1-methyl-3-isobutyl xanthine (MIX), a phosphodiesterase inhibitor. In the absence of MIX, methacholine increased guanosine 3',5'-cyclic monophosphate (cGMP) levels at a time when contraction was decreasing, but had no influence on adenosine 3',5'-cyclic monophosphate (cAMP) levels. In contrast, oxytocin did not elevate cGMP, but rapidly decreased cAMP levels. MIX (1 mM) increased both cAMP and cGMP levels. Oxytocin or methacholine further increased cGMP, indicating activation of guanylate cyclase. Oxytocin- but not methacholine-induced stimulation of guanylate cyclase was abolished in Ca2+-free solution. Oxytocin increased cAMP over the levels produced by MIX alone, whereas methacholine decreased cAMP below the MIX control values; these effects were insensitive to indomethacin. Tissue levels of cGMP and cAMP did not directly correlate with isometric tension. The results also indicate that both oxytocin and methacholine stimulate guanylate cyclase but have opposing effects on adenylate cyclase of rabbit myometrium.     

Biogenesis of liver delta-aminolevulinate synthase. The role of cAMP in the induction

In primary cultures of chick embryo hepatocytes pulse labeled with [35S]methionine, immunochemical analyses indicated that adenosine 3':5'-cyclic monophosphate (cAMP) did not affect either the rate of production or the maturation of delta-aminolevulinate synthase (ALA synthase). In addition, allylisopropylacetamide caused a slight drop in intracellular cAMP while testosterone caused the levels of cAMP to rise to 260% of the basal levels measured in hepatocytes in culture. Thus the results of this study did not indicate a direct short-term role for cAMP in the regulation of production of ALA synthase.     

cAMP suppresses CA2+-dependent electrical activity of airway smooth muscle induced by TEA

Using intracellular microelectrodes, we investigated whether exogenous dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) or forskolin influenced the electrical effects of tetraethylammonium (TEA) on canine tracheal smooth muscle. We found that 20 mM TEA depolarized airway smooth muscle cells from a resting membrane potential (Em) of -59 +/- 4 mV (mean +/- SD) to -45 +/- 2 mV and caused spontaneous action potentials (AP's) to develop, which were 33 +/- 2 mV in amplitude. These were totally abolished in 0 Ca2+ solution. DBcAMP (1 mM) suppressed the development of this TEA-induced electrical activity and the phasic contractions electrically coupled to it. DBcAMP had no significant effect on Em in the absence of TEA however. Forskolin (1 microM) produced similar effects. Our findings suggest that Ca2+ is the principal ion responsible for the inward current associated with the TEA-induced AP's in airway smooth muscle, and that adenosine 3',5'-cyclic monophosphate may suppress the electrogenesis of this current.     

Effect of cold exposure on liver and muscle cAMP content and cAMP phosphodiesterase activity

Adenosine 3',5'-cyclic monophosphate (cAMP) concentration and 3',5'-cyclic-nucleotide phosphodiesterase (PDE) activity were measured in skeletal muscle, heart, and liver of rats exposed to 1, 3, 5, and 7 days of cold. Cyclic nucleotide concentration increased in fast-twitch red muscle at the same time that PDE activity was decreasing. Nucleotide concentration and enzyme activity of slow-twitch red muscle were not altered by the cold exposure. The PDE activity of fast-twitch white muscle was elevated approximately 50% above control after 1 and 3 days of cold exposure. By the 5th day in the cold, white muscle PDE activity had returned to control levels and remained there through the 7th day of experimentation. cAMP concentration in hearts of cold-exposed rats was significantly (P less than 0.01) elevated above control at all time points measured. Myocardial PDE activity was elevated above control (P less than 0.05) at 1 and 3 days of cold exposure but returned to control levels by the 5th day in the cold. Hepatic cAMP and PDE activity were elevated above control at all time points analyzed. These data suggest that changes in cyclic nucleotide metabolism play a role in attaining homeostasis during acute cold exposure.     

Independent regulation of pyruvate kinase expression by cyclic AMP and prostaglandin F2 alpha in mouse mastocytoma cells

P-815 mouse mastocytoma cells express the K isozyme of pyruvate kinase and the specific activity of this enzyme is increased in response to N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate, 8-bromoadenosine 3':5'-cyclic monophosphate, cholera toxin, and epinephrine, all of which also elevate the intracellular concentration of adenosine 3':5'-cyclic monophosphate. Prostaglandin F2 alpha also increases the cellular activity of this enzyme, but does not increase the adenosine 3':5'-cyclic monophosphate levels. Under all these conditions, the increase in enzymatic activity is accompanied by an equivalent increase in the pyruvate kinase protein level. However, neither the rate of enzyme synthesis nor the level of pyruvate kinase mRNA is elevated by N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate. On the other hand, it does increase the enzyme's half-life. In contrast, prostaglandin F2 alpha increases the rate of synthesis and the level of pyruvate kinase K mRNA, but has no influence on the rate of degradation. Therefore, these cells have two mechanisms which increase pyruvate kinase K levels. One operates via an increase in cAMP level and results in a decrease in the rate of degradation, whereas the other minimizes an upsurge in cAMP levels but still increases pyruvate kinase K activity by increasing its rate of synthesis.     

Cyclic AMP and serum arrest of the mitotic activity of human diploid fibroblasts.

Mitotic activity in confluent cultures of human diploid fibroblasts was arrested by the reduction of the serum concentration of the incubation medium to 0.5% or by the addition of 0.5 mM 6-N, 2'-O-dibutyryl-adenosine 3':5'-cyclic monophosphate (db cAMP). Under either of these conditions, cultures maintained a constant cell number for 14 days; cultures continuously exposed to medium containing 10% serum doubled their cell number during this 14-day period. The protein cotent per cell decreased by 20% when cells were maintained with 0.5% serum whereas that of cells exposed to db cAMP remained constant. Ultrastructural studies revealed that cells exposed to db cAMP exhibited a morphology typical of cells cultures with 10% serum alone, whereas cells incubated with 0.5% serum showed the ultrastructural changes in mitochondria, endoplasmic reticulum and Golgi complex previously identified with low-serum arrest. Cellular adenosine 3':5'-cyclic monophosphate (cAMP) levels remained constant during the 7-day growth period in which confluency was attained, as well as during the 14-day arrested period with 0.5% serum. These results indicated that the mitotic inhibition induced by reducing the serum concentration of the incubation medium was not mediated by increased intracellular levels of cAMP and differed from that induced by the addition of exogenous db cAMP.     

Cyclic nucleotide function in trachealis muscle of dogs with and without airway hyperresponsiveness

We examined basal adenosine 3',5'-cyclic monophosphate (cAMP) levels, isoproterenol (ISO)-stimulated cAMP responses, basal cAMP, and guanosine 3',5'-cyclic monophosphate (cGMP) phosphodiesterase (PDE) activities and protein-kinase (PK) activities in trachealis muscle from five Basenji-greyhound (BG) and four greyhound dogs to determine whether the inverse relationship between in vivo and in vitro airway responsiveness could be due to altered cyclic nucleotide metabolism. Basal cAMP levels were not significantly different (PNS) in muscle from BG (11.6 +/- 0.53 pmol/mg protein) and greyhound dogs (10.30 +/- 1.60 pmol/mg protein). The cAMP responses to stimulation with ISO were enhanced in BG compared with greyhound dogs. The low Michaelis constant (1) for Km-cAMP PDE activity (Km = 0.63 microM) was significantly less (P less than 0.005) in BG dogs (1.54 +/- 0.28 pmol.min-1.mg protein-1) than greyhounds (11.76 +/- 2.48). Endogenously active PK activity was significantly greater (P less than 0.005) in BG (54.74 +/- 5.39 pmol.min-1.mg protein-1) than in greyhound dogs (15.50 +/0 2.20). Increases in PK activity with 5 microM cAMP added were not significantly different between BG (14.79 +/- 6.00) and greyhound dogs (7.04 +/- 2.14). Approximately 90% of both endogenous PK activity and cAMP-activated PK activity in BG and greyhound dogs was inhibited by a cAMP-dependent PK inhibitor (PKI'). These data suggest that decreased cyclic nucleotide degradation due to decreased cyclic nucleotide PDE activity with increased PK could account for the in vitro hyporesponsiveness of airway smooth muscle in BG dogs as a protective adaptive mechanism.     

Effects of intracellular cyclic AMP and cyclic GMP levels on DNA synthesis of young-adult rat hepatocytes in primary culture.

Possible roles of dibutyryladenosine 3',5'-cyclic monophosphate (cAMP) and dibutyryl-guanosine 3',5'-cyclic monophosphate (cGMP) in regulation of hepatocyte DNA synthesis were examined using primary cultures of young-adult rat hepatocytes maintained in arginine-free medium. Throughout the experimental period, nonparenchymal cells were hardly observed in the selective medium. When epidermal growth factor (EGF) was added to the cultures, a transient increase in the intracellular cAMP level preceded the elevation of hepatocyte DNA synthesis. EGF-stimulated hepatocyte DNA synthesis was remarkably enhanced by the elevation of the intracellular cAMP level induced by treatment with cAMP alone or a combination of cAMP and theophylline, an inhibitor of cyclic nucleotide phosphodiesterase. Furthermore, the early elevation of intracellular cAMP alone, which was induced by treatment with the combination of cAMP and theophylline, caused a remarkable increase in hepatocyte DNA synthesis. On the other hand, addition of EGF to the cultures caused a rapid decrease in the intracellular cGMP level followed by an increase in hepatocyte DNA synthesis. EGF-stimulated hepatocyte DNA synthesis was severely suppressed or completely inhibited by the elevation of the intracellular cGMP level induced by treatment with cGMP alone or a combination of cGMP and dipyridamole, a specific inhibitor of cGMP phosphodiesterase. These findings indicate that cAMP and cGMP act oppositely on the regulation of DNA synthesis of young-adult rat hepatocytes in primary culture: cAMP plays a positive role, whereas cGMP plays a negative role. Also it is strongly suggested that an early elevation of the intracellular cAMP level is essential for the onset of DNA synthesis in hepatocyte primary cultures.     

Demonstration of 2',3'-Cyclic Nucleotide 3'-Phosphohydrolase in Cultured Human Schwann Cells

Abstract: Schwann cell cultures were established from adult human sural nerve biopsies. 2'3'-Cyclic nucleotide 3'-phosphohydrolase (CNPase) activity was estimated in the homogenates of those cells by a sensitive isotope assay using [³H]2',3'-cyclic AMP as substrate. A high level of CNPase activity was observed in cultured Schwann cells, whereas cultured human muscle and skin fibroblasts contained negligible levels of CNPase activity. CNPase of human Schwann cells followed typical enzyme-substrate kinetics, with an apparent K _m of 1.6 m M for 2',3'-cyclic AMP, and the enzyme was stimulated by detergents such as Triton X-100 and deoxycholate. It was inhibited by p -chloromercuricbenzoate and 2'-AMP. These properties are typical of CNPase isolated from adult brain and spinal cord. CNPase can serve as a new biochemical marker of normal cultured human Schwann cells and can be useful in analyzing the properties of cultured Schwann cells from patients with dysschwannian neuropathies.     

Atriopeptin II relaxes and elevates cGMP in bovine pulmonary artery but not vein

Atriopeptin II, a 23-amino acid synthetic peptide fragment of atrial natriuretic factor, caused an endothelium-independent relaxation of isolated precontracted rings of bovine intrapulmonary artery that was accompanied by the concomitant accumulation of guanosine 3',5'-cyclic monophosphate (cGMP) but not adenosine 3',5'-cyclic monophosphate. In contrast, rings of intrapulmonary vein were unaffected by atriopeptin II whether or not endothelium was present. Whereas methylene blue, an inhibitor of soluble guanylate cyclase, abolishes endothelium-dependent and independent arterial relaxation and cGMP accumulation in response to acetylcholine and glyceryl trinitrate, respectively, methylene blue failed to alter these responses to atriopeptin II. Similarly, the effects of atriopeptin II were unaltered by propranolol, indomethacin, or atropine. These results indicate that relaxation elicited by atriopeptin II may be selective for arterial smooth muscle receptors, does not require endothelial cells, and does not involve the soluble form of guanylate cyclase, although cGMP accumulation is stimulated.     

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.     

A further study on the regulation of cyclic nucleotide phosphodiesterase activity in neuroblastoma cells: effect of growth.

Adenosine 3',5'-cyclic monophosphate (cyclic AMP) phsophodiesterase activity in mouse neuroblastoma cells in culture markedly increased during exponential growth and reached a maximal level at confluency; whereas guanosine 3'5'-cyclic monophosphate (cyclic GMP) phosphodiesterase activity only slightly but significantly increased under a similar experimental condition. The increase in cyclic AMP phosphodiesterase activity was blocked by both cycloheximide and dactinomycin, whereas the increase in cyclic GMP phosphodiesterase was blocked by only cycloheximide. When the confluent cells were replated at low density, the cyclic nucleotide phosphodiesterase activity decreased; however, when they were plated at high cell density which equaled confluency, the enzyme activity did not decrease. Unlike cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity did not change significantly in prostaglandin E1-treated cells, but decreased in cells treated with the inhibitor of phosphodiesterase. Like cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity also did not change in cells treated with serum-free medium, X-irradiation, sodium butyrate and 6-thioguanine.     

Mutants Defective in Binding Activity for Cyclic Adenosine 3′,5′-Monophosphate in Vibrio parahaemolyticus

Mutants of Vibrio parahaemolyticus defective in binding activity for adenosine 3',5'-cyclic monophosphate are described. They were selected with medium containing the nucleotide together with starch.     

The identification of cyclic nucleotides from living systems using collision-induced dissociation of ions generated by fast atom bombardment mass spectrometry

Extracts derived from rat liver and Phaseolus leaves are shown, by collision-induced dissociation of [MH]+ ions generated by fast atom bombardment mass spectrometry, to contain cytidine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate respectively, and not the 2',3'-cyclic isomers. Interference peaks, expected to be common to all mass-analysed ion kinetic energy spectra of ions generated by the fast atom bombardment process from glycerol-based matrices are identified. It is shown that unequivocal identification of cytidine 3',5'-cyclic monophosphate can be made at the microgram level. Attempts to derive a quantitative procedure based on using different cyclic nucleotides as internal standards were unsuccessful due to the poor solubility of these compounds in the matrix system.