Regulation Of Cyclic AMP Levels Research Articles

Enkephalin dimers: Regulation of cyclic AMP levels in NG108-15 cells

Intracellular cyclic AMP levels were determined for dimeric and monomeric enkephalins interacting with PGE 1-stimulated NG108-15 cells. The dimeric pentapeptide enkephalin (DPE 2), [D-Ala 2, Leu 5 -NH-CH 2] 2, displaying very high affinity (K = 4.2 ± 0.3 nM −1) for the δ-opiate receptor, inhibited cyclic AMP production by 70%. Its IC 50-value was between 0.1 and 0.2 nM, similar to that of the potent δ-agonist [D-Ala 2, D-Leu 5] enkephalin (DADLE) with K = 1.0 ± 0.1 nM −1. [D-Ala 2, Leu 5] enkephalin amide (DALEA), which is the monomer of DPE 2, showed an IC 50 = 4 nM. The dimeric tetrapeptide enkephalin (DTE 12), [D-Ala 2, des-Leu 5-NH-(CH 2) 6] 2 and its monomer [D-Ala 2, desLeu 5] enkephalin amide (DAPEA) showed IC 50 = 2 and 20 nM, respectively. These results indicate that the DPE 2 and DTE 12 enkephalin dimers are potent δ-agonists.

Relationships between norepinephrine and cyclic nucleotides in brain and seizure activity

To characterize further the roles of norepinephrine (NE) and cyclic nucleotides in seizure mechanisms, an examination was made of the effects of several drugs purported to depress noradrenergic influence in the CNS on pentylenetetrazol-induced seizure activity and regulation of cyclic AMP levels in the cerebral cortex and hippocampus in mice. Depletion of brain stores of NE with reserpine or treatment of neonatal mice with 6-hydroxy-dopamine decreased seizure latency and/or threshold and diminished seizure-induced accumulation of cyclic AMP in brain. Propranolol, a β-adrenergic receptor antagonist, and yohimbine, an α 2-adrenergic receptor antagonist, had effects qualitatively similar to reserpine and 6-hydroxy-dopamine, but phentolamine, a mixed α-adrenergic antagonist, increased seizure threshold and latency and did not reduce the accumulation of cyclic AMP. None of the drugs tested had any consistent effect on the regulation of cyclic GMP levels in brain during seizures. These data are consistent with the hypothesis that cyclic AMP in brain may be mediating an inhibitory influence of NE on seizure activity.

Alpha 2 adrenergic amines, adenosine and prostaglandis inhibit lipolysis and cyclic AMP accumulation in hamster adipocytes in the absence of extracellular sodium.

The accumulation of cyclic AMP due to adenosine deaminase plus theophylline and either isoproterenol or ACTH in the presence of adenosine deaminase plus theophylline, was inhibited by clonidine, N 6-(phenylisopropyl)-adenosine and prostaglandin E 2. The inhibition was nearly identical in medium containing sodium ions or in medium in which sodium and its accompanying anion were substituted by an isosmotic amount of sucrose. Consistent with this, lipolysis induced by adenosine deaminase and theophylline was significantly inhibited by clonidine, N 6-(phenylisopropyl)-adenosine and prostaglandin E 2 regardless of the presence or absence of Na + in the medium. The results do not support the suggestion that extracellular Na + is required for the regulation of cyclic AMP levels by hormones and neurotransmitters that inhibit adenylate cyclase.

Regulation of cyclic AMP levels in human lymphocytes and lymphoblasts by prostaglandins

The sensitivity of human lymphocytes to prostaglandins was compared to that of two lines of cultured lymphoblasts. It was found that 25 nM PGE 1 increased lymphocyte cyclic AMP levels twofold, but had no effect on lymphoblasts. At 2.8 μM PGE 1 the increase of cyclic AMP in lymphocytes was 14 fold versus a 2–3 fold increase in lymphoblasts. Lymphoblasts maintained a response to 6-keto-PGE 1, isoproterenol and histamine similar to that observed in lymphocytes. Thus, it appears that the rapidly proliferating lymphoblasts escape signals inhibiting proliferation by impairing the function of the PGE receptor.

1-Isoamyl-3-isobutylxanthine: A remarkably potent agent for the potentiation of norepinephrine, histamine, and adenosine-elicited accumulations of cyclic AMP in brain slices

1-Isoamyl-3-isobutylxanthine (EC 50 t 5 μM) potentiates by 2 to 6-fold the accumulations of cyclic AMP elicited in guinea pig cerebral cortical slices by norepinephrine, histamine, and adenosine. In addition, the xanthine derivative causes a 2 to 3-fold elevation of basal levels of cyclic AMP. 1-Isoamyl-3-isobutylxanthine has no effect on accumulations of cyclic AMP elicited by histamine or adenosine in the presence of a potent phosphodiesterase inhibitor, ZK 62771. The xanthine derivative retards the disappearance of cyclic AMP after a prior stimulation by adenosine. The results indicate that 1-isoamyl-3-isobutylxanthine is an extremely potent and effective inhibitor of phosphodiesterases involved in the regulation of cyclic AMP levels in guinea pig cerebral cortical slices. The 1-benzyl, 1-isoamyl, and 1-isobutyl derivatives of 3-isobutylxanthine potentiate the accumulation of cyclic AMP elicited by adenosine, while the 1-methyl derivative and 1-isoamyl-3-methylxanthine are inhibitory undoubtedly because of blockade of adenosine-receptors by these compounds. Xanthines with bulky 1- and 3- substituents appear to be relatively weak adenosine-antagonists and relatively specific and potent agents for inhibition of phosphodiesterases involved in cyclic AMP metabolism in brain tissue.

Regulation of cyclic amp level and synthesis of DNA, RNA and protein by quercetin in ehrlich ascites tumor cells

Quercetin (3.3, 4′, 5.7-pentahydroxy flavone) at the concentration of 10 −4 M as well as 10 −2 M theophylline and 10 −3 M dibutyryl cyclic AMP caused at least 85 per cent inhibition of [ 3H]thymidine incorporation in Ehrlich ascites tumor cells. At the same concentrations, these drugs decreased [ 3H]uridine and [ 3H]- L-leucine incorporation by 50–60 per cent and 35–45 per cent respectively. Ouabain (10 −3 M), the specific inhibitor of Na +-K + pump system, did not alter the incorporation of [ 3H]thymidine and [ 3]uridine, but decreased the incorporation of [ 3H]- L-leucine in these cells. Treatment of Ehrlich ascites tumor cells with the polyanion dextran sulfate did not change the inhibitory effect of quercetin, theophylline and dibutyrlyl cyclic AMP on [ 3H]thymidine incorporation. On the other hand, this polyanion decreased the inhibitory effect of these drugs on incorporation of [ 3H]uridine and abolished completely their effect on incorporation of [ 3H]-L-leucine.

Regulation of cyclic AMP levels in Arthrobacter crystallopoietes and a morphogenetic mutant

The extracellular levels of cyclic AMP (cAMP), cAMP phosphodiesterase activity, and adenylate cyclase activity were measured at various intervals during growth and morphogenesis of Arthrobacter crystallopoietes. There was a significant rise in the extracellular cAMP level at the onset of stationary phase, and this rise coincided with a decrease in intracellular cAMP. The phosphodiesterase activity measured in vitro increased in the early exponential phase of growth as intracellular cAMP decreased, and, conversely, prior to the onset of stationary phase the phosphodiesterase activity decreased as the intracellular cAMP levels increased. Adenylate cyclase activity was greater in cell extracts prepared from cells grown in a medium where morphogenesis was observed. Pyruvate stimulated adenylate cyclase activity in vitro. A morphogenetic mutant, able to grow only as spheres in all media tested, was shown to have altered adenylated cyclase activity, whereas no significant difference compared to the parent strain was detectable in either the phosphodiesterase activity or the levels of extracellular cAMP. The roles of the two enzymes, adenylate cyclase and phosphodiesterase, and excretion of cAMP are discussed with regard to regulation of intracellular cAMP levels and morphogenesis.

Cyclic 3′:5′-nucleotide phosphodiesterase of rabbit sinoatrial node

The sinoatrial node contains pacemaker cells which control heart rate. Changes of heart rate correlate closely to cyclic adenosine 3′:5′-monophosphate (cyclic AMP) levels in sinoatrial node. In order to elucidate the mechanism of regulation of cyclic AMP level in sinoatrial node, we investigated cyclic 3′:5′-nucleotide phosphodiesterase (3′:5′-cyclic-nucleotide 5′-nucleotidohydrolase, EC 3.1.4.17) of isolated rabbit sinoatrial node. Sinoatrial node of rabbit heart contains at least three kinetically distinct forms of cyclic nucleotide phosphodiesterase (F I, F II and F III) which were clearly separated by DEAE-cellulose column chromatography. Although phosphodiesterase F I and F II hydrolyzed cyclic GMP faster than cyclic AMP at low substrate concentration, phosphodiesterase F III hydrolyzed equally cyclic GMP and cyclic AMP at a low substrate concentration. Phosphodiesterase F I displayed normal kinetics both for cyclic AMP hydrolysis, with a K m of 8.3 μM, and for cyclic GMP hydrolysis, with a K m of 2.0 μM. Phosphodiesterase F II and F III displayed abnormal kinetics both for cyclic AMP, with a K m for 1.2 and 0.8 μM, respectively, and for cyclic GMP, with a K m of 2.0 and 0.1 μM, respectively. The effects of the activator, Ca 2+, cyclic AMP and cyclic GMP on these forms were also studied. The cyclic GMP and cyclic AMP hydrolytic activities in phosphodiesterase F I and F II were stimulated by micromolar concentration of Ca 2+ in the presence of the activator. Phosphodiesterase F III was not affected by addition of Ca 2+ in the presence of the activator. Cyclic AMP hydrolysis by phosphodiesterase F I and F II was activated by micromolar amounts of cyclic GMP, whereas cyclic GMP hydrolysis by these fractions was inhibited by micromolar amounts of cyclic AMP. Cyclic AMP hydrolytic activity in phosphodiesterase F III was inhibited by micromolar amounts of cyclic GMP, and cyclic GMP hydrolysis by phosphodiesterase F III was also inhibited by cyclic AMP.

Regulation of cyclic AMP levels in canine thyroid slices by α-adrenergic action

In an attempt to clarify the role of adrenergic receptors in metabolic responses, interaction of norepinephrine with TSH was studied in canine thyroid slices with regard to cyclic AMP levels. Norepinephrine caused a very rapid (within 1 min), but quite transient increase in cyclic AMP levels. The elevation of cyclic AMP levels induced by TSH was markedly inhibited by norepinephrine. Phentolamine, an α-adrenergic blocker, not only prevented the decline of cyclic AMP levels that followed the rise by norepinephrine, but also abolished the norepinephrine effect on the TSH-induced elevation of cyclic AMP levels. Propranolol, a β-adrenergic blocker, exhibited no such effects. These results indicate that the α-adrenergic receptors control cyclic AMP levels in the thyroid gland.

Regulation of cyclic AMP level and lactic acid production in Ehrlich ascites tumor cells

1. 1.Quercetin (3,3′,4′,7-pentahydroxy flavone) at the concentration of 10 −4M, as well as 2·10 −2 M theophylline and 1.5·10 −4 M prostaglandin E 2 caused maximal rise of cyclic AMP in Ehrlich ascites tumor cells. 2. 2. No additional increase of cyclic AMP level in these cells was found when both quercetin (10 −4 M) and theophylline (2·10 −2 M) were present in the incubation medium. while combination of quercetin (10 −4 M) and prostaglandin E 2 (1.5·10 −4 M) has a synergistic effect on the level of cyclic AMP. 3. 3. Degradation of cyclic AMP by homogenate pf Ehrlich ascites tumor cells was inhibited by both quercetin and theophylline. 4. 4. Quercetin, and to a smaller but significant extent theophylline, inhibited the lactic acid production in Ehrlich ascites tumor cells while prostaglandin E 2 did not change the glycolytic rate in these cells. No synergistic inhibitory effect on lactic acid production was found when combinations of quercetin and prostaglandin E 2, quercetin and theophylline or prostaglandin E 2 and theophylline were tested. 5. 5. Treatment of Ehrlich ascites tumor cells with dextran sulfate abolished the inhibitory effect of quercetin on lactic acid production, while the effect of the bioflavonoid on cyclic AMP levels was not altered.

On the regulation of cyclic AMP level in bacteria II. In vitro regulation of adenylate cyclase activity. Solubilization and reconstitution of a functional membrane-bound adenylate cyclase system responsive to regulation by glucose

1. 1. The in vitro regulation of the membrane bound adenylate cyclase of Escherichia coli B/r by a variety of carbohydrates and one mammalian hormone was examined. 2. 2. The membrane bound adenylate cyclase was found responsive to regulation by the various growth substrates and to glucagon. 3. 3. Solubilization of the bacterial membrane preparation by a procedure specific for the solubilization of the phosphotransferase enzyme E 1 1 to its E 1 1 A and E 1 1 B subunits was found to be accompanied by the loss of the adenylate cyclase regulation by glucose. 4. 4. Reconstitution of the membrane was found to result in recovery of the regulative response of the adenylate cyclase to glucose. 5. 5. A model for the intermediate steps in the interaction between glucose, the phosphotransferase E 1 1 and the adenylate cyclase is discussed.

Cyclic AMP-mediated induction of the cyclic AMP phosphodiesterase of C-6 glioma cells.

Long-term regulation of the cyclic nucleotide phosphodiesterase of the C-6 rat glioma cell line has been studied. Both the low K(m) and high K(m) activities can be induced by elevation of intracellular cyclic AMP levels following either dibutyryl cyclic AMP or norepinephrine treatment of the cells. The enzymes are maximally induced by 3-4 hr. The presence of either cycloheximide or actinomycin D prevents induction by either dibutyryl cyclic AMP or norepinephrine. Evidence is presented that the norepinephrine effect is mediated by the beta-catecholamine receptor. The increased phosphodiesterase activity causes a partial refractoriness to a second challenge with norepinephrine, which can be overcome by blockade of the induction with cycloheximide. The results suggest that just as short-term regulation of cyclic AMP levels occurs via changes in the rates of synthesis or degradation, long-term alterations of the system may also involve either the adenylate cyclase or the phosphodiesterase.

Inhibition of 3′,5′-nucleotide phosphodiesterase in guinea pig cerebral cortical slices

Several compounds have been tested for their activity as inhibitors of 3′,5′-nucleotide phosphodiesterase in brain cortical slices from guinea pig. SQ 20,009 (1-ethyl-4-isopropylidenehydrazino)-1H-pyrazolo (3,4-b)pyridine-5-carboxylate, ethylester, hydrochloride), a very potent inhibitor of 3′,5′-nucleotide phosphodiesterase from rat and rabbit brain shows only moderate activity as 3′,5′-nucleotide phosphodiesterase inhibitor when tested in brain slices. It enhances cyclic AMP accumulation only when slices are stimulated by histamine. It does not affect cyclic AMP levels when histamine/norepinephrine are used as stimuli of cyclic AMP formation and decreases the activity of adenosine as stimulant slightly. Ro 20–1724 (4-(3-butoxy-4-methoxy)-2-imidazolidinone) a potent inhibitor of canine cerebral cortex PDE activity effectively augments the increase in cyclic AMP under all stimulating conditions mentioned, as does to a somewhat smaller extent the more water soluble Ro 20–2926 (4-(3-ethoxy-ethoxy-4-methoxy)-2-imidazolidinone). Dose-response curves for Ro 20–1724 under three stimulating conditions of increased cyclic AMP formation (0.1 m m histamine, 0.1 m m histamine/0.1 m m norepinephrine, 0.1 m m adenosine) yield an ED 50 of about 20 μ m in all instances. A significant increase over respective controls is seen even at 1 μ m Ro 20–1724 (histamine/norepinephrine). The drugs may be useful as tools for studying the regulation of cyclic AMP levels in the central nervous system.

Regulation of cyclic AMP levels in brain

Publisher Summary This chapter describes regulation of cyclic amp levels in brain. The rate of formation and degradation of cyclic AMP in the central nervous system depends on a variety of factors: (1) the basal levels of adenylate cyclases; (2) the degree and specificity of activation of this enzyme by biogenic amines such as norepinephrine, dopamine, serotonin and histamine; (3) the levels and availability of ATP as a substrate for adenylate cyclases; (4) the degree of stimulation of the system by adenosine, and the extent of synergistic interaction of adenosine with stimulatory biogenic amines; (5) the levels and properties of cyclic nucleotide phosphodiesterases; and (6) the levels of cyclic AMP dependent protein kinases with binding sites that can sequester cyclic AMP. The chapter also highlights that the stimulation of cyclic AMP formation by biogenic amines differs among species and brain regions both with respect to magnitude of stimulation and the properties of the amine regulatory unit of adenyl cyclase, that is, the receptor.

The involvement of cyclic AMP in the hormonal regulation of protein synthesis in rat adipocytes.

The involvement of cyclic 3′,5′ AMP has been examined in the regulation of protein synthesis by ACTH, epinephrine and insulin in fat cells isolated from rat epididymal adipose tissue. Cyclic AMP was measured in fat cell suspensions by radioimmunoassay and found to be 7.9 ± 0.63 pmoles/mg protein. The cyclic 3′,5′ GMP level was an order of magnitude lower at 0.46 ± 0.07 pmoles/mg protein and was unaffected by ACTH, epinephrine or insulin. Increasing doses of theophylline, ACTH and epinephrine produced a progressive increase in cyclic AMP levels and glycerol release coupled with a reciprocal inhibition of protein synthesis. For the same elevation of cyclic AMP levels, ACTH inhibited protein synthesis to a greater degree than epinephrine while theophylline produced an inhibition much greater than either of the lipolytic hormones. Dibutyryl cyclic AMP, 0.05–5.0 mM, produced an increasing rate of lipolysis with a concomitant decrease in the rate of protein synthesis. Time course studies revealed that cyclic AMP levels were elevated by 1 min, peaked between 5–10 min and slightly declined from the peak by 30 min in the presence of theophylline, ACTH and epinephrine. The stimulation of lipolysis and inhibition of protein synthesis by epinephrine, ACTH, theophylline and dibutyryl cyclic AMP was at constant rates, once established. Although a causal relationship may exist between cyclic AMP elevations and inhibition of protein synthesis, the data suggests that certainly theophylline and dibutyryl cyclic AMP have other actions on adipocytes that caused the greater degree of inhibition of protein synthesis. Insulin at 100, 500, or 1000 μU/ml caused no change in cyclic AMP levels of fat cells throughout a 30-min incubation period while the same doses of insulin stimulated protein synthesis reaching 150% of the control. Insulin did not alter the elevations of cyclic AMP caused by ACTH or epinephrine but completely overcame the inhibition of protein synthesis caused by these hormones. In contrast, insulin had no effect on either the elevation of cyclic AMP or inhibition of protein synthesis caused by theophylline. Insulin appeared to stimulate protein synthesis by a mechanism other than regulation of cyclic AMP levels. (Endocrinology90: 1277, 1972)