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PubMed1980

Epinephrine effects on cyclic AMP-dependent protein kinases from rat diaphragms.

Diaphragm extracts were subjected to electrophoresis on polyacrylamide gels to separate the different molecular species of th cyclic AMP-dependent protein kinase. Using cyclic [3H]AMP, three peaks of binding activity were observed. The peak closest to the origin (peak I) was associated with cyclic AMP-dependent protein kinase activity and was abolished by incubation of the extracts with cyclic AMP prior to electrophoresis. The peak farthest from the origin (peak III) was devoid of kinase activity and was increased by incubation of extracts with cyclic AMP before electrophoresis; furthermore, when extracts were incubated with cyclic [3H]AMP before electrophoresis, essentially all the radioactivity appeared in peak III. Peak II, in an intermediate position, was also abolished by preincubation of the extracts with cyclic AMP and both its binding capacity and cyclic AMP-dependent protein kinase activity were lower than in Peak I. A peak of cyclic AMP-independent protein kinase (peak 0) that migrated more slowly than peak II was also detected. From these and other data it is concluded that peaks I and II are cyclic AMP-dependent protein kinase and that peak III is the dissociated regulatory subunit, respectively. Peak 0 is cyclic AMP-independent protein kinase together with free catalytic subunits from cyclic AMP-dependent protein kinase. Incubation of rat diaphragms with epinephrine resulted in dose- and time-dependent decrease in peak I and increase in peak III. These changes correlated with the decrease of cyclic AMP-dependent protein kinase associated with peak I. No changes in Peak II were observed with epinephrine, but an increased peak 0 was noted. Changes in peak I and peak III correlated with the modification of glycogen synthase and glycogen phosphorylase activities. No regulatory subunits (peak III) were detected as phosphorylated forms in diaphragms previously equilibrated with 32P. Treatment with epinephrine produce no noticeable phosphorylation of these regulatory subunits.

Animals↗
PubMed2006

Dose-related antiallodynic effects of cyclic AMP response element-binding protein-antisense oligonucleotide in the spared nerve injury model of neuropathic pain.

A transcription factor known as cyclic AMP response element-binding protein has been shown to be involved in the central sensitization in neuropathic pain and inflammation pain. The present study examined the roles of cyclic AMP response element-binding protein and of the phosphorylated cyclic AMP response element-binding protein in the maintenance of mechanical and cold allodynia induced by a neuropathic pain model, "spared nerve injury," in rats. First, the results of immunohistochemical study showed that phosphorylated cyclic AMP response element-binding protein, but not cyclic AMP response element-binding protein, increased bilaterally in the spinal dorsal horn 14 days following spared nerve injury, indicating a possible contribution of phosphorylated cyclic AMP response element-binding protein in spared nerve injury. Second, chronic intrathecal application of cyclic AMP response element-binding protein antisense oligodeoxynucleotide with three doses (10 microg/day, 20 microg/day and 40 microg/day) for 5 days demonstrated that the higher doses (20 and 40 microg) significantly attenuated both mechanical (bilaterally) and cold (ipsilaterally) allodynia, compared with sense oligodeoxynucleotide and the lower dose (10 microg). Western blot results showed that the alleviation in intensity of behavioral performance was accompanied by a significant reduction of total cyclic AMP response element-binding protein and phosphorylated cyclic AMP response element-binding protein in the spinal dorsal horn. Moreover, there were no differences in cyclic AMP response element-binding protein and phosphorylated cyclic AMP response element-binding protein between ipsilateral and contralateral dorsal horns. Our data demonstrate a close association between the expression of behavioral hypersensitivity and cyclic AMP response element-binding protein activation in the spinal dorsal horn following spared nerve injury, supporting the notion that phosphorylated cyclic AMP response element-binding protein may play an important role in the maintenance of chronic neuropathic pain.

Animals↗
PubMed1997

Involvement of protein kinase C in the UTP-mediated potentiation of cyclic AMP accumulation in mouse J774 macrophages.

1. We have investigated the effects of nucleotide analogues on cyclic AMP formation in mouse J774 macrophages and the mechanisms involved. 2. UTP, in the concentration range 0.1-100 microM, induced concentration-dependent potentiation of prostaglandin E1 (PGE1)-induced cyclic AMP formation, but had no effect on basal cyclic AMP formation. UDP showed an equal potency, while 2-methylthio ATP, alpha, beta-methylene ATP and beta,gamma-methylene ATP gave either a slight increase or had no effect at concentrations up to 100 microM. ATP, although 100 fold less effective than UTP, also caused cyclic AMP potentiation, but had no effect on agonist-stimulated or basal cyclic AMP levels. 3. The cyclic AMP potentiation effect of UTP correlated with increased [Ca2+]i and inositol phosphate (IP) formation over the same concentration range. 4. Ionomycin, which evokes an increase in [Ca2+]i without affecting IP formation, did not cause an increase in cyclic AMP content, indicating that UTP-induced cyclic AMP regulation is not due to activation of Ca(2+)-sensitive adenylyl cyclase isoforms. 5. Although reduced, UTP potentiation was seen in cells incubated in a Ca(2+)-free and/or BAPTA-containing medium. Under these conditions, the UTP-increased IP accumulation was similarly reduced. 6. Exposure of cells to phorbol 12-myristate 13-acetate (PMA) also increased PGE1 stimulation of cyclic AMP levels, and the UTP-induced potentiation of cyclic AMP formation was inhibited by either staurosporine or Ro 31-8220. Pretreatment of cells with PMA for 4-24 h resulted in marked attenuation of UTP-stimulated cyclic AMP potentiation. 7. Pretreatment with pertussis toxin (24 h, 100 ng ml-1) did not significantly affect UTP-induced cyclic AMP potentiation and IP formation, although it increased the cyclic AMP response to PGE1. 8. Analysis of J774 cells by Western blotting with antibodies specific for different protein kinase C (PKC) isoforms shows the presence of the beta I, beta II, delta, epsilon, eta, mu, lambda and zeta isoforms. Moreover, UTP significantly increased the level of PKC beta I, beta II, delta, epsilon, mu, lambda and zeta immunoreactivity in the membrane fraction and decreased the cytosolic reactivity of PKC beta II, delta, epsilon and zeta. 9. Immunoblot studies also indicate the presence of type II adenylyl cyclase. 10. These results indicate that PKC is required for the potentiation of adenylyl cyclase activity by macrophage pyrimidinoceptors, which exhibit a higher specificity for UTP and UDP than for ATP.

Adenylate Cyclase Toxin↗
PubMed1992

Control of cyclic AMP levels in primary cultures of human tracheal smooth muscle cells.

1. [3H]-adenosine 3':5'-cyclic monophosphate ([3H]-cyclic AMP) responses were studied in primary cultures of human tracheal smooth muscle cells derived from explants of human trachealis muscle and in short term cultures of acutely dissociated trachealis cells. 2. Isoprenaline induced concentration-dependent [3H]-cyclic AMP formation with an EC50 of 0.2 microM. The response to 10 microM isoprenaline reached a maximum after 5-10 min stimulation and remained stable for periods of up to 1 h. After 10 min stimulation, 1 microM isoprenaline produced a 9.5 fold increase over basal [3H]-cyclic AMP levels. The response to isoprenaline was inhibited by ICI 118551 (10 nM), (apparent KA 1.9 x 10(9) M-1) indicating the probable involvement of a beta 2-adrenoceptor in this response in human cultured tracheal smooth muscle cells. However, with 50 nM ICI 118551 there was a reduction in the maximum response to isoprenaline. Prostaglandin E2 also produced concentration-dependent [3H]-cyclic AMP formation (EC50 0.7 microM, response to 1 microM PGE2 6.4 fold over basal). 3. Forskolin (1 nM - 100 microM) induced concentration-dependent [3H]-cyclic AMP formation in these cells. A 1.6 fold (over basal) response was also observed following stimulation with NaF (10 mM). 4. The nonselective phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (0.1 mM) and the type IV, cyclic AMP selective, phosphodiesterase inhibitor rolipram (0.1 mM) both elevated basal [3H]-cyclic AMP levels by 1.8 and 1.5 fold respectively. IBMX (1-100 microM) and low concentrations of rolipram (< 10 microM), also potentiated the response to 1 microM isoprenaline. Inhibitors of the type III phosphodiesterase isoenzyme (SK&F 94120 and SK&F 94836) were without effect upon basal or isoprenaline-stimulated cyclic AMP responses in these cells.5. Carbachol (1 nM-I 00 microM) produced concentration-dependent inhibition of the [3H]-cyclic AMP response to 1 microM isoprenaline in human cultured tracheal smooth muscle cells (IC50 0.24 JM). Carbachol(1 JM) inhibited the [3H]-cyclic AMP response to 1 JM isoprenaline by 60%. This effect of carbachol was itself inhibited by atropine (50 nM) (KA 2.3 x 109 M-') indicating the involvement of a muscarinic receptor.6. These results show that primary cultures of human tracheal smooth muscle cells demonstrate cyclic AMP responses to direct receptor stimulation, adenylyl cyclase activation and inhibition with nonselective and type IV-selective cyclic AMP phosphodiesterase isoenzyme inhibitors, and that the cyclic AMP response to isoprenaline can be inhibited by muscarinic receptor stimulation.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Effects of cyclic AMP and analogues on neurogenic transmission in the rat tail artery.

1 The effects of two 8-substituted analogues of adenosine 3':5'-cyclic monophosphate (cyclic AMP) were compared with those of forskolin and isoprenaline on [3H]-noradrenaline release and vasoconstriction induced by electrical field stimulation (24 pulses at 0.4 Hz, 200 mA, 0.3 ms duration) in the rat tail artery, in the absence and in the presence of protein kinase inhibitors. 2 8-Bromo-adenosine 3':5'-cyclic monophosphate (8-bromo-cyclic AMP, 10-300 microM), 8-(4-chlorophenyl-thio)-adenosine 3':5' cyclic monophosphate (8-pCPT-cyclic AMP, 3-300 microM), forskolin (0.3-10 microM) and isoprenaline (1 nM-1 microM) all concentration-dependently enhanced stimulation-induced [3H]-noradrenaline release. The effect of cyclic AMP analogues was larger (2.5 fold at 300 microM) than those of cyclic AMP elevating drugs (1.6 fold at 10 microM for forskolin and 1.5 fold at 30 nM for isoprenaline). 3 At concentrations active at the prejunctional level, the four drugs had differential effects on stimulation-induced vasoconstriction, which was enhanced by the two cyclic AMP analogues, decreased by forskolin and not significantly altered by isoprenaline. 4 The [3H]-noradrenaline release-enhancing effects of 8-bromo-cyclic AMP, forskolin and isoprenaline were significantly decreased by the cyclic AMP-dependent protein kinase (PKA) inhibitor (N-[2-((3-(4-bromophenyl)-2-propenyl)-amino)-ethyl]-5- isoquinolinesulphonamide, di-hydrochloride) (H-89; 100 nM). By contrast they were unaffected by the cyclic GMP-dependent protein kinase (PKG) inhibitor, 8-bromo-guanosine 3':5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-bromo-cyclic GMPS; 10 microM). By contrast they were unaffected by the cyclic GMP-dependent protein kinase (PKG) inhibitor,8-bromo-guanosine 3':5'-cyclic monophosphorothioate, Rp-isomer (Rp-8-bromo-cyclic GMPS; 10 MicroM).At the same concentrations the PKA inhibitor attenuated only the nerve-induced vasoconstrictor responses obtained in the presence of 8-bromo-cyclic AMP, whereas the PKG inhibitor did not modify that obtained in the presence of 8-bromo-cycic AMP or forskolin.5. Exposure to the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (1 MicroM) enhanced nerve-evoked [3H]-noradrenaline release, and this effect was decreased by the PKC inhibitor, 2-[1-(3-dimethylaminopropyl)-indol-3-yl]-3-(-indol-3-yl)-maleimide (GF 109203X; 100 nM). However, the latter drug did not modify the enhancing effect of 8-bromo-cyclic AMP on [3H]-noradrenaline release.6. It is concluded that activation of cyclic AMP-dependent protein kinase is involved in the enhancing effect of cyclic AMP-elevating compounds on prejunctional release of noradrenaline. In addition the results provide no clear-cut evidence for a vasodilator role of PKA.

Animals↗
PubMed2005

Relationship between 5-aminoimidazole-4-carboxamide-ribotide and AMP-activated protein kinase activity in the perfused mouse heart.

AMP-activated protein kinase (AMPK) is a cellular energy sensor whose activity responds to AMP concentration ([AMP]). An agent that activates AMPK in cells is 5-aminoimidazole-4-carboxamide-1-riboside (AICA-riboside). Phosphorylated AICA-riboside or AICA-ribotide (ZMP) is an AMP analog. It is generally assumed that ZMP accumulation does not alter [AMP]. Additionally, the effect of AICA-riboside on AMPK activity of the heart is uncertain. Two hypotheses were tested in the isolated mouse heart: 1) sufficient ZMP concentration ([ZMP]) forms to increase AMPK activity, and 2) [ZMP] accumulation increases [AMP]. Perfusion of isolated mouse hearts with Krebs-Henseleit buffer containing 0.15-2 mM AICA-riboside concentration resulted in [ZMP] of 2-8 mM. ZMP accumulation reduced phosphocreatine concentration, which increased cytosolic [AMP]. In hearts with [ZMP] less than approximately 3 mM, in vivo AMPK allosteric activity effects of ZMP were observed; AMPK phosphorylation and [AMP] were not increased. With [ZMP] between 3 and 5 mM, in vitro AMPK activity and phosphorylation increased with unchanged [AMP]. This occurred in hearts perfused with 0.25 mM AICA-riboside for 48 min and 0.5 mM AICA-riboside for 24 min. The [ZMP] resulting in 50% AMPK activity (covalent phosphorylation of AMPK) was 4.1 +/- 0.6 mM. Hearts with [ZMP] >5 mM displayed increased [AMP] and AMPK activity that was not different from hearts with similar [AMP] with no [ZMP]; the half-maximal activity of AMP was 5.6 +/- 1.6 microM. Thus, in mouse hearts, AICA-riboside was metabolized to [ZMP] adequately to increase AMPK activity. Higher [ZMP] also increased cytosolic [AMP], which affects AMPK activity.

AMP-Activated Protein Kinases↗
PubMed1976

A possible role of cyclic AMP in mediating the effects of thyrotropin-releasing hormone on prolactin release and on prolactin and growth hormone synthesis in pituitary cells in culture.

Thyrotropin-releasing hormone (TRH) has 3 effects on clonal strains of rat pituitary cells in culture (GH-cells). Two long-term effects of TRH on GH-cells, which are measurable after 3 h or longer, have been previously reported; these are an increase in prolactin synthesis and a decrease in growth hormone production. We report here that TRH also stimulates the rapid release of stored intracellular prolactin. We have investigated the role of cyclic AMP as a possible mediator of the effects of TRH on GH-cells. Cyclic AMP concentrations are higher in cells treated with TRH compared with paired controls; a maximum difference of greater than 150% of control values is detected at 15 min if the incubation is performed in serum-free medium in the presence of 1 mM theophylline. The concentration of TRH required to give half-maximum increases in both prolactin release and cyclic AMP accumulation is 0.3 nM; half-maximal increases in prolactin synthesis occur at 3 nM TRH. Exogenous cyclic AMP (1 mM) causes only a slight increase in prolactin release; 8-bromo-cyclic AMP and 8-methylthio-cyclic AMP (1 mM) do not cause significant release. Phosphodiesterase inhibitors (0.3 mM theophylline, 0.03 mM isobutyl-methylxanthine) increase prolactin release but their effects on hormone synthesis are more complicated. Isobutylmethylxanthine, 8-bromo-cyclic AMP and 8-methylthio-cyclic AMP (0.4 MM) increase prolactin synthesis, but do not significantly affect growth hormone synthesis. Theophylline increases the synthesis of both hormones. Dibutyryl cyclic AMP (0.5 mM or more) increases prolactin release and both growth hormone and prolactin synthesis, but equivalent amounts of sodium butyrate have the same effects. We conclude that in GH-cells under carefully defined experimental conditions: 1) TRH causes an increase in intracellular cyclic AMP concentrations; 2) the increase in endogenous cyclic AMP and the effects of phosphodiesterase inhibitors are consistent with a model with cyclic AMP as a mediator of the effects of TRH on prolactin release; however, they do not prove this model, because the interpretation of these results depends on assumptions which may not all be valid; and 3) none of the analogs of cyclic AMP or the phosphodiesterase inhibitors tested mimic the decrease in growth hormone production caused by TRH.

Animals↗
PubMed1984

Control of cell mobility by cyclic AMP.

Cyclic AMP concentrations have been measured in a number of different cell types under a variety of culture conditions in an attempt to define the relationship between the endogenous concentration of cyclic AMP and cell mobility. In previous work it was shown that agents that increase the intracellular concentration of cyclic AMP can effectively suppress cell movement. In Balb/c 3T3 cells, which have a very low mobility in cellular aggregates, the intracellular concentration of cyclic AMP was elevated only transiently soon after the formation of the three-dimensional cell masses. In contrast, in the highly mobile virally transformed counterpart of Balb/c 3T3 cells, called SVT-2, the concentration of cyclic AMP was relatively low soon after the cell masses were formed, but later rose to a level that was higher than that in Balb/c 3T3 cells. Using NIL B cells, SV40-transformed NIL B cells, and several lines of tumour cells derived from NIL B cells, it was found that the average intracellular concentration of cyclic AMP did not vary significantly from one population of cells to another. Finally, the intracellular concentration of cyclic AMP was measured in chick embryo ventricle cells. The mobility of these cells had previously been found to decrease as embryonic development progressed; furthermore, it had been shown that dibutyryl cyclic AMP plus theophylline produced nearly complete inhibition of their movement in cell masses. In the series of experiments reported here we found that the endogenous concentration of cyclic AMP in aggregates and fragments of chick embryo ventricle cells decreases as development proceeds; these data are consistent with preliminary experiments reported by other investigators. In a separate set of experiments, the intracellular concentration of cyclic AMP was measured in cells that had been cultured in a medium containing 1.2 mM-dibutyryl cyclic AMP plus 1.0 mM-theophylline. This drug treatment has previously been shown to inhibit the movement of cells both in aggregates and in monolayers; it also produces striking effects on cell shape and ultrastructure. In aggregates of chick embryo ventricle cells, treatment with these drugs resulted in increases in the intracellular concentrations of cyclic AMP from approximately 10 picomol/mg protein to approximately 500 picomol/mg protein. In Balb/c 3T3 and SVT-2 cells this treatment increased cyclic AMP concentrations from 3.7 to 160 and from 6.4 to 470 picomol/mg protein, respectively.

Animals↗
PubMed1987

Differentiation of alpha 1-adrenergic receptors linked to phosphatidylinositol turnover and cyclic AMP accumulation in rat brain.

Activation of alpha 1-adrenergic receptors in slices of rat brain increases inositol phosphate accumulation, increases basal cyclic AMP accumulation, and potentiates the increase in cyclic AMP caused by adenosine. We compared these three responses to determine whether they are mediated by the same receptors. The increase in inositol phosphates and the potentiation of cyclic AMP accumulation in cerebral cortex were largely blocked by chelation of extracellular calcium, whereas the increase in basal cyclic AMP was not affected. The magnitude of the increase in inositol phosphates in different brain regions correlated with the magnitude of the potentiation of cyclic AMP accumulation (r = 0.80), but neither of these correlated with the magnitude of the increase in basal cyclic AMP. Although other alkylating agents inactivated all of the alpha 1-adrenergic receptor-binding sites labeled with 125IBE 2254 in membrane preparations of cerebral cortex, chlorethylclonidine (CEC) potently and selectively inactivated only half of these sites. Pretreatment with CEC partially blocked the increase in basal cyclic AMP, but not the increase in inositol phosphates or potentiation of cyclic AMP accumulation in slices of cerebral cortex. Comparing different brain regions, there was a better correlation between the density of 125IBE 2254-binding sites not inactivated by CEC with the magnitude of the increase in inositol phosphates or potentiation of cyclic AMP accumulation than with the increase in basal cyclic AMP. Although the largest increase in inositol phosphates was observed in slices of hippocampus, there was only a small increase in basal cyclic AMP in this region, and CEC did not inactivate any 125IBE-binding sites in hippocampus. Phentolamine and WB 4101 were significantly more potent in inhibiting specific 125IBE 2254 binding in hippocampus than in cerebral cortex. After treatment of cerebral cortical membranes with CEC, however, these drugs had potencies similar to those observed in hippocampus. The results suggest that the alpha 1-adrenergic receptors mediating increases in basal cyclic AMP accumulation can be differentiated from those mediating increases in inositol phosphate accumulation and potentiating adenosine stimulated cyclic AMP accumulation by their binding properties, calcium dependency, regional distribution, and sensitivity to the alkylating agent CEC.

1-Methyl-3-isobutylxanthine↗
PubMed1996

RNA folding topology and intermolecular contacts in the AMP-RNA aptamer complex.

We report below on the NMR structural characterization of the complex between AMP and a 40-mer RNA aptamer in aqueous solution. Resonance assignments are based on multinuclear multidimensional NMR studies on complexes uniformly 13C, 15N-labeled with either AMP or the RNA aptamer. AMP binds to an internal loop (labeled G7-G8-A9-A10-G11-A12-A13-A14-C15-U16-G17) and bulge (G34 positioned opposite the internal loop) segment in the RNA aptamer, and our NMR study provides insights into features of the RNA folding topology and the molecular recognition events in the AMP binding pocket on the RNA. Specifically, the helical stems are extended by G-G mismatch formation from either direction into the internal loop/bulge segment of the RNA aptamer on complex formation. The internal loop adopts a unique fold with the purine ring of AMP intercalated between A10 and G11 in the complex. The G8-A9-A10-AMP segment adopts certain stacking features in common with a GNRA turn and is closed by the G7.G11 mismatch pair. The purine rings of A12 and G34 (syn) are stacked on each other and participate in stablizing the AMP intercalation site. A large number of intermolecular NOEs have been identified between the AMP ligand and the G8, A10, G11, G17, U18, and G34 residues on the RNA aptamer in the complex. The Watson-Crick edge of the AMP is oriented toward the exocyclic amino group of G8, suggestive of a hydrogen-bonding alignment between G8 and AMP in the complex. The AMP sugar ring is positioned in the minor groove of the rightward helical stem centered about the G17.G34 mismatch and U18.A33 Watson-Crick pairs. The AMP binds to one face of the folded internal loop/bulge segment of the RNA aptamer while the opposite face is capped by a stacked alignment of the A13-A14-C15-U16 segment located toward the 3'-end of the internal loop segment. Globally, the two helical stems of the RNA aptamer are aligned approximately orthogonal to each other with tertiary interactions centered about the internal loop/bulge segment generating the AMP binding site on the RNA.

Adenosine Monophosphate↗
PubMed1984

AMP-dependence of the cyanide-insensitive pathway in the respiratory chain of Paramecium tetraurelia.

The AMP-dependent stimulation of the cyanide-insensitive respiration of Paramecium mitochondria was investigated. The nucleotides exhibiting a stimulatory effect on the cyanide-insensitive oxidation of pyruvate (+ malate) in a medium supplemented with EDTA or carboxyatractyloside were, in decreasing order of efficiency, AMP, GMP, IMP, UMP and TMP. On the other hand, ADP, ATP and cyclic AMP were ineffective. In the presence of carboxyatractyloside, addition of AMP to Paramecium mitochondria incubated with pyruvate (+malate) led to an increase in membrane potential. In the absence of light, the photoactivable derivative of AMP, 3'-[4-[N-(4-azido-2-nitrophenyl)amino]butyryl]-AMP (NAP4-AMP) added to Paramecium mitochondria opposed the stimulatory effect of AMP on the cyanide-insensitive respiration; the Ki for NAP4-AMP was much lower than the Km for AMP, 0.2 microM compared with 120 microM. The ADP-stimulated respiration was not affected. Photoirradiation of Paramecium mitochondria in the presence of NAP4-AMP resulted in irreversible inhibition of the AMP-stimulated cyanide-insensitive respiration. No effect on the ADP-stimulated respiration was observed. A heatlabile cyanide-insensitive ubiquinol oxidase was extracted from Paramecium mitochondria with the detergent NN-dimethyl-N-(3-laurylamidopropyl)amine oxide. The quinol oxidase activity was slightly stimulated by AMP.

Adenosine Diphosphate↗
PubMed1992

The effect of AMP on the NAD-dependent glutamate dehydrogenase during activation and morphogenesis in the cellular slime moulds.

In extracts from vegetative Dictyostelium discoideum V12 the basal NAD-dependent glutamate dehydrogenase (NAD-GDH) activity was low, but it increased on standing at 4 degrees C. When 0.1 mM-AMP was included in the assay mix, enzyme activity was stimulated nearly 30-fold. As the extract was allowed to age, the enzyme rapidly lost its ability to be stimulated by AMP. The response of NAD-GDH to AMP was also dependent on the stage of morphogenesis. The ratios of NAD-GDH activity assayed with and without AMP (+AMP/-AMP ratios) in freshly prepared extracts from cells at 0, 4, 8 and 12 h of development were similar, but declined later in morphogenesis. The +AMP/-AMP ratio decreased sharply during activation at 4 degrees C in extracts from cells at 0, 4, 16 and 20 h of development. By contrast, extracts from cells starved for 8 and 12 h remained more responsive to AMP throughout activation. Analysis of Western blots showed that vegetative NAD-GDH did not undergo any detectable proteolytic cleavage during 96 h of activation at 4 degrees C. Also, no change in molecular mass appeared to take place within the cells until culmination (20-24 h), when some breakdown products appeared. Activation of NAD-GDH also occurred in D. discoideum strains NC4 and AX3, and in D. mucoroides. In addition, the enzyme from these four strains was stimulated by AMP and the +AMP/-AMP ratio declined with similar kinetics during activation. The enzyme from Polysphondylium violaceum was not activated on standing, but it was stimulated by AMP. The effect of activation of NAD-GDH is discussed in relation to a postulated catabolic role for this enzyme.

Adenosine Monophosphate↗
PubMed2004

Depression of cell metabolism and proliferation by membrane-permeable and -impermeable modulators: role for AMP-to-ATP ratio.

The metabolic and developmental depression commonly observed during natural states of dormancy, such as diapause and quiescence, is typically accompanied by an increase in the intracellular ratio of AMP to ATP. We investigated the impact of artificially increasing the AMP-to-ATP ratio in mouse macrophages. Evidence is presented here that the P2X7 receptor channel can be used as an effective means to load cells with membrane-impermeable compounds. Intracellular loading of adenosine-5'-O-thiomonophosphate (AMPS), a nonhydrolyzable analog of 5'-AMP and potent activator of AMP-activated protein kinase, significantly depresses metabolism and proliferation of macrophages. The intracellular effective AMP-to-ATP ratio obtained (the sum of AMPS plus endogenous 5'-AMP) was 0.073, well above that reported to activate AMP-activated protein kinase in vitro. Optimizing both the conditions under which the P2X7 receptor channel is opened and the duration of opening facilitates high analog uptake and approximately 98% survivorship. An advantage to AMPS is its minimal impact on other components of the nucleotide pool, most notably the unchanged concentration of ADP. An alternative way to shift the effective AMP-to-ATP ratio is by incubation with the membrane-permeable compound 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), which is phosphorylated intracellularly to form the 5'-AMP analog ZMP. Despite a rapid intracellular accumulation of AICAR, conversion to ZMP was slow and inefficient. Furthermore, AICAR incubation increased cellular ADP, and, although cell proliferation was depressed, the overall cellular energy flow was unchanged. The rapid action of AMPS avoids upregulation of compensatory metabolic pathways and may provide a viable approach for promoting cell stasis.

Adenosine Monophosphate↗
PubMed1980

Dibutyryl cyclic AMP decreases glutamine synthetase in cultured 3T3-L1 adipocytes.

Glutamine synthetase specific activity increases greater than 100-fold during the insulin-mediated differentiation of confluent 3T3-L1 cells into adipocytes. Incubation of the adipocytes for 22 h with 0.5 mM dibutyryl cyclic AMP plus 0.5 mM theophylline, 0.2 mM 8-bromo-cyclic AMP, 10 micro M epinephrine, or 1 microgram of alpha 1-24 adrenocorticotropic hormone/ml decreased glutamine synthetase by greater than 60%. During the same incubation period, there was no effect of these compounds on protein or on the specific activities of glucose-6-P dehydrogenase or hexokinase. In the presence of 0.5 mM theophylline, the dibutyryl cyclic AMP-mediated decrease in glutamine synthetase activity was half-maximal at 50 micro M dibutyryl cyclic AMP. Furthermore, between 10 micro M and 5 mM dibutyryl cyclic AMP, the dibutyryl cyclic AMP-mediated decrease in glutamine synthetase was similar in the absence or presence of 1 microgram of insulin/ml. Immunotitration of glutamine synthetase activity from 3T3 adipocytes indicates that the dibutyryl cyclic AMP-mediated decrease in the activity is due to a decrease in the cellular content of glutamine synthetase molecules. We studied the effects of dibutyryl cyclic AMP on the synthesis and degradation of glutamine synthetase. Synthesis rate was estimated from the incorporation of L-[35S]methionine into glutamine synthetase during a 60-min incubation period. Degradation rate was estimated from the first order disappearance of radioactivity from glutamine synthetase in 3T3 adipocytes previously incubated with L-[35S]methionine. Glutamine synthetase was isolated by immunoprecipitation followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Incubation of 3T3 adipocytes with dibutyrl cyclic AMP resulted in a rapid decline in the apparent synthesis rate of glutamine synthetase. In addition, dibutyryl cyclic AMP treatment increased the initial rate of glutamine synthetase degradation. The half-life of glutamine synthetase was 24.5 h in control cultures and 16 h in dibutyryl cyclic AMP-treated cultures. In contrast, dibutyryl cyclic AMP had little effect on the synthesis or degradation of soluble protein. Our data indicate that the dibutyryl cyclic AMP-mediated decrease in 3T3 adipocyte glutamine synthetase activity results from a decrease in the synthesis rate and an increase in the initial degradation rate of the enzyme.

Adipose Tissue↗
PubMed1980

Adenylate degradation in Escherichia coli. The role of AMP nucleosidase and properties of the purified enzyme.

Extracts of Escherichia coli K12 degrade AMP to hypoxanthine, adenine, adenosine, and inosine. Degradation experiments with mutants which lack purine nucleoside phosphorylase or both purine nucleoside phosphorylase and adenosine deaminase demonstrate that hypoxanthine formation is dependent on purine nucleoside phosphorylase. These findings are consistent with an absence of adenine deaminase activity in E. coli. Adenine is formed from AMP in extracts of the E. coli mutants as well as the wild type cells. This activity is due to AMP nucleosidase. Purified, homogeneous AMP nucleosidase gives a subunit Mr = 52,000 on denaturing gel electrophoresis and an oligomer molecular weight of approximately 280,000 by comparative gel filtration. Kinetic studies with this enzyme give cooperative initial rate curves with AMP as substrate, with MgATP2- as an activator, and with Pi as an inhibitor. Phosphate inhibition is competitive with McATP2- (Ki = 0.2 mM) and reverses the activation by MgATP2-. In the absence of MgATP2-, the apparent S0.5 for AMP is 15 mM and decreases to 90 microM at saturating MgATP2-. The maximum rate of AMP hydrolysis is not affected by MgATP2-. Kinetics of MgATP2- activation give a constant for half-maximum activation varying from 120 microM in the presence of low AMP to approximately 2 microM when AMP is present at near saturation. Formycin 5'-PO4 is a powerful competitive inhibitor with respect to AMP, giving a Kis of 72 nM and a Km/Kis ratio of 1,200. Adenylate degradation experiments indicate that AMP nucleosidase is the major enzyme of AMP catabolism in E. coli. The kinetic properties of the purified enzyme indicate that regulation occurs by the intracellular MgATP2- /Pi ratio and the concentration of AMP.

Adenine↗
PubMed1994

The allosteric site of human liver fructose-1,6-bisphosphatase. Analysis of six AMP site mutants based on the crystal structure.

The molecular structure of human liver fructose-1,6-bisphosphatase complexed with AMP was determined by x-ray diffraction using molecular replacement, starting from the pig kidney enzyme AMP complex. Of the 34 amino acid residues which differ between these two sequences, only one interacts with AMP; Met30 in pig kidney is Leu30 in human liver. From this analysis, six sites in which side chains of amino acid residues are in contact with AMP, Ala24, Leu30, Thr31, Tyr113, Arg140, and Met177, were mutated by polymerase chain reaction. The wild-type and mutant forms were expressed in Escherichia coli, purified, and their kinetic properties determined. Circular dichroism spectra of the mutants were indistinguishable from that of the wild-type enzyme. Kinetic analyses revealed that all forms had similar turnover numbers, Km values for fructose 2,6-bisphosphate, and inhibition constants for fructose 2,6-bisphosphate. Apparent Ki values for AMP inhibition of the Leu30 --> Phe and Met177 --> Ala mutants were similar to those of the wild-type enzyme, but the apparent Ki values for the Arg140 --> Ala and Ala24 --> Phe mutants were 7-to 20-fold higher, respectively. The Thr31 --> Ser mutant exhibited a 5-fold increase in apparent Ki for AMP, while mutation of Thr31 to Ala increased the apparent Ki 120-fold. AMP inhibition of the Tyr113 --> Phe mutant was undetectable even at millimolar AMP concentrations. Fructose 2,6-bisphosphate potentiated AMP inhibition of the mutants to the same extent as for the wild-type enzyme, except in the case of the Thr31 --> Ala and Tyr113 --> Phe mutants. Thus, the Met177 --> Ala mutant suggests that the side chain beyond C alpha is not needed for AMP binding, and that the Leu30 --> Phe mutant preserves the AMP contacts with these side chains. Thr31, Tyr113, and Arg140 form key hydrogen bonds to AMP consistent with strong side chain interactions in the wild-type enzyme. Finally, the absence of any effect of fructose 2,6-bisphosphate on AMP inhibition observed in the Thr31 --> Ala mutant may be an important clue relating to the mechanism of synergism of these two inhibitors.

Adenosine Monophosphate↗
PubMed1976

Cyclic AMP binding to intracellular receptor proteins in rat myometrium. Effect of epinephrine and prostaglandin E1.

In estrogen-pretreated rat myometrium, the relaxing effect exerted by theophylline or epinephrine has been correlated with their ability to raise cyclic AMP levels (Vesin and Harbon, 1974). The present study demonstrates that such a correlation can be quantitatively extended to the degree of saturation of intracellular cyclic AMP receptors. The rise in cyclic AMP induced by theophylline and/or epinephrine in intact myometrial strips was accompanied by a decrease in the ability of the corresponding extracts to bind exogenous 3H-labeled cyclic AMP. Total intracellular cyclic AMP binding sites were not modified and averaged a value of 0.22 muM. Accurate estimation of intracellular receptor-cyclic AMP complex has been correlated with the corresponding level of cyclic AMP in the tissue, the apparent intracellular Kd for cyclic AMP has been evaluated at 450 nm. Stimulation of myometrial strips with prostaglandin E1 (PGE1) which has been shown previously to induce contractions, although elevating cyclic AMP levels, was accompanied by a parallel increase in the saturation of the endogenous receptor, in an identical manner to that found with epinephrine or theophylline. The postulated hypothesis for a compartmentalization of cyclic AMP, or an interference of PGE1 with the intracellular cyclic AMP binding equilibrium has not been verified. The cyclic AMP system cannot be considered as the exclusive mechanism regulating uterine relaxation.

Animals↗
PubMed2003

Negative functional effects of cyclic GMP are altered by cyclic AMP phosphodiesterases in rabbit cardiac myocytes.

In this study, we tested the hypothesis that the negative functional effects of cyclic GMP on cardiac myocytes would be affected by the actions of cyclic GMP on cyclic AMP phosphodiesterases. Ventricular myocytes from eight rabbits were used to determine the functional and cyclic AMP changes caused by 10(-7), 10(-6), 10(-5) M 8-Bromo-cGMP alone and after the administration of 10(-6) M milrinone (cyclic GMP-inhibited cyclic AMP phosphodiesterase inhibitor) or 10(-6) M erythro-9-(2-Hydroxy-3-3-nonyl)adenine (EHNA, cyclic GMP-stimulated cyclic AMP phosphodiesterase inhibitor). 8-Br-cGMP dose-dependently reduced %shortening by 35+/-4% of baseline at 10(-5) M. This effect was significantly blunted by EHNA at all doses. The maximum rate of shortening was reduced by 31+/-3% by 10(-5) M 8-Br-cGMP. This effect of 8-Br-cGMP was significantly enhanced (42+/-4%) in the milrinone group. A similar pattern was observed in the maximum rate of relaxation data. Cyclic AMP levels were significantly increased from a baseline level of 4.0+/-0.8 pmol/10(5) myocytes by milrinone (+60%), EHNA (+61%) and 8-Br-cGMP (+47%). The combination of EHNA plus 8-Br-cGMP increased cyclic AMP levels significantly more that the combination of milrinone plus 8-Br-cGMP. Exogenous cyclic GMP reduces myocyte function, while raising cyclic AMP possibly through cyclic GMP-inhibited cyclic AMP phosphodiesterase effects. Blocking cyclic GMP-inhibited cyclic AMP phosphodiesterase enhances the functional effects cyclic GMP, while blocking cyclic GMP-stimulated cyclic AMP phosphodiesterase reduced these effects. The study demonstrated a functional interaction between cyclic GMP and cyclic AMP related to the cyclic GMP affected cyclic AMP phosphodiesterases.

3',5'-Cyclic-AMP Phosphodiesterases↗