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PubMed1986

Effects of several newer cardiotonic drugs on cardiac cyclic AMP metabolism.

The purpose of this study was to investigate the possible roles of selective inhibition of cyclic nucleotide phosphodiesterase (PDE) isozymes, adenylate cyclase activation, and tissue cyclic 3',5'-adenosine monophosphate (cyclic AMP) elevation in the positive inotropic action of five new cardiotonic drugs. Three PDE isozymes (PDE I, II and III), homogenates, and slices of guinea pig ventricles were used. The inotropics amrinone, milrinone, AR-L 115BS, MDL 17,043, and RMI 82,249 all inhibited cyclic AMP hydrolysis by PDE III in a concentration-dependent manner, as did the PDE inhibitors aminophylline and 1-methyl-3-isobutylxanthine (MIX). All drugs except for AR-L 115BS inhibited PDE III at concentrations lower than those producing a standard inotropic response. A significant correlation (r = 0.80, P less than 0.05) was observed between PDE III inhibition and inotropic activity for six of the drugs. Only aminophylline and MIX, but none of the cardiotonic drugs, inhibited cyclic AMP hydrolysis by PDE I and II and cyclic 3',5'-guanosine monophosphate (cyclic GMP) hydrolysis (amrinone not tested) by PDE I. Further, none of the cardiotonic drugs inhibited the calmodulin-stimulated cyclic AMP hydrolysis by PDE I, indicating their lack of calmodulin antagonist activity. These drugs also did not stimulate adenylate cyclase activity but all increased net cyclic AMP formation from ATP in guinea pig ventricular homogenates through inhibition of cyclic AMP breakdown. Amrinone, milrinone, MDL 17,043 and RMI 82,249, but not AR-L 115BS, raised cyclic AMP levels significantly (P less than 0.05) in guinea pig ventricular slices. Also, amrinone, MDL 17,043 and RMI 82,249, but not AR-L 115BS, potentiated forskolin-induced cyclic AMP increase. These data taken together suggest that the specific inhibition of cyclic AMP PDE III isozyme and the consequent elevation of tissue cyclic AMP levels in cardiac tissue are an important mechanism of action of amrinone, milrinone, MDL 17,043 and RMI 82,249. Because AR-L 115BS did not increase cyclic AMP levels, it is likely that another mechanism may participate in the inotropic response to AR-L 115BS.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Melatonin biosynthesis in photoreceptor-enriched chick retinal cell cultures: role of cyclic AMP in the K(+)-evoked, Ca(2+)-dependent induction of serotonin N-acetyltransferase activity.

The roles of cyclic AMP and calcium in the regulation of serotonin N-acetyltransferase (NAT) activity were studied in low density monolayer cultures of chick retinal photoreceptors and neurons. Photoreceptor-enriched retinal cell cultures were prepared from embryonic day 6 retinas and cultured for 6 days. NAT activity in these cultures could be induced by treatment with cyclic AMP protagonists, 8Br-cyclic AMP, forskolin, and 3-isobutyl-1-methylxanthine (IBMX), or by treatment with depolarizing concentrations of extracellular K+. The stimulatory effect of K+, which involves Ca2+ influx through dihydropyridine-sensitive channels, was mediated at least in part by cyclic AMP, as indicated by the following observations. Depolarizing concentrations of K+ stimulated the formation of cyclic AMP, and the stimulatory effects of K+ on both cyclic AMP formation and on NAT activity were synergistically potentiated by the cyclic nucleotide phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). MDL 12,330A, a putative adenylate cyclase inhibitor, inhibited K(+)-evoked cyclic AMP accumulation and induction of NAT activity over the identical concentration range. In contrast, MDL 12,300A failed to inhibit the induction of NAT elicited by 8Br-cyclic AMP. H-89, an inhibitor of cyclic AMP-dependent protein kinase, antagonized the induction of NAT activity by either forskolin or K+ with equal potency for both stimuli. These results suggest that cyclic AMP plays an essential role in the induction of NAT activity that occurs as a consequence of membrane depolarization. Cyclic AMP and Ca2+ may also interact at a step distal to adenylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1977

Rat pancreatic adenylate cyclase. IV. Effect of hormones and other agents on cyclic AMP level and enzyme release.

1. The effects of secretin and pancreozymin-C-octapeptide and phosphodiesterase inhibitors on the concentration of adenosine 3',5'-cyclic monophosphate (cyclic AMP) and on the release of enzymes from rat pancreas have been studied. 2. In determininging cyclic AMP by means of the saturation assay of Brown et al. ((1971) Biochem. J. 121, 561-563) it is found essential to purify the pancreatic tissue extract by ion-exchange chromatography prior to the assay. 3. Injection of synthetic secretin or pancreozymin-C-octapeptide in anaesthetized rats in a secretory active dose (0.1 nmol) has no effect on the pancreatic cyclic AMP level. 4. Incubation for up to 10 min of pancreatic slices in Krebs-Ringer bicarbonate glucose medium containing 10(-2) M theophylline as phosphodiesterase inhibitor does not result in an increase of the cyclic AMP level. With 10(-2) M 1-methyl-3-isobutylxanthine as phosphodiesterase inhibitor the level is more than doubled after the first min of incubation and remains constant thereafter. 5. Addition of 3-10(-7) M secretin to slices incubated in the presence of 10(-2) M theophylline causes 84% increase of the cyclic AMP level above control, whereas the addition of 3-10(-7) M pancreozymin-C-octapeptide has no significant effect. In the presence of 10(-2) M 1-methyl-3-isobutylxanthine the latter hormone causes significant increases of up to 34% above control during 10 min of incubation. Secretin in this condition augments the cyclic AMP level by up to 296% above control during a 10 min incubation period. Addition of secretin and pancreozymin-C-octapeptide together has no greater effect than of secretin alone. 6. A broken cell fraction of rat pancreas contains adenylate cyclase activity which can be stimulated to 457 and 600% above the basal activity by 3-10(-7) M pancreozymin-C-octapeptide and secretin, respectively. Incubation of pancreatic slices with either hormone has no effect on the cyclic AMP phosphodiesterase activity in the homogenate of these slices. 7. Pancreozymin-C-octapeptide, dibutyryl cyclic AMP, 1-methyl-3-isobutylxanthine and carbamylcholine cause an elevated release of chymotrypsin from pancreatic slices incubated for 2 h in Krebs-Ringer bicarbonate medium, containing 10 mM glucose, while secretin, cyclic AMP and butyric acid have no significant effect. The release of the cytoplasmic enzyme lactate dehydrogenase is also elevated by dibutyryl cyclic AMP, 1-methyl-3-isobutylxanthine and carbamylcholine, but not significantly by pancreozymin-C-octapeptide. 8. The results support the role of cyclic AMP in the action of secretin, and do not exclude a mediating function of this nucleotide in the actions of pancreozymin in rat pancreas.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

Cyclic AMP potentiates growth hormone-dependent differentiation of 3T3-F442A preadipocytes: possible involvement of the transcription factor CREB.

We have examined the effects of cyclic AMP on the differentiation of 3T3-F442A preadipocytes. High concentrations of intracellular cyclic AMP potently inhibited differentiation whereas low concentrations of intracellular cyclic AMP, induced by a number of different agents, promoted differentiation. To analyse these effects of cyclic AMP more closely, we developed a two-phase protocol for the differentiation of 3T3-F442A cells. Growth hormone (GH) was necessary to prime confluent cells during the first phase, following which, the addition of insulin and other adipogenic agents then promoted terminal differentiation. Cyclic AMP potentiated the priming action of GH but exerted an inhibitory effect on terminal differentiation when added to cells which had previously been primed with GH showing that the effects of cyclic AMP on preadipocyte differentiation are stage-dependent. We analysed the stimulatory effects of cyclic AMP during GH priming and found that cyclic AMP induced phosphorylation of the cyclic AMP response element (CRE) binding protein CREB and activated transcription of a CRE-linked reporter gene. Furthermore, GH also stimulated CREB phosphorylation and activation and this effect was potentiated by cyclic AMP. These results suggest a mechanism for the synergistic priming of preadipocytes for terminal differentiation by cyclic AMP and GH via the activation of differentiation genes containing CREs.

1-Methyl-3-isobutylxanthine↗
PubMed1998

Rat striatal adenosinergic modulation of ethanol-induced motor impairment: possible role of striatal cyclic AMP.

We have previously reported the involvement of the striatum in acute ethanol-induced motor incoordination and the striatal adenosinergic modulation of ethanol-induced motor incoordination through A1 receptor-mediated mechanism(s). The present study, a continuation of our previous work, was carried out to investigate the possible functional correlation between striatal cyclic AMP and ethanol-induced motor incoordination, and its modulation by striatal adenosine in Sprague-Dawley rats. Forskolin (0.1, 0.5 and 1.0 pmol), a known activator of adenylate cyclase, significantly attenuated ethanol-induced motor incoordination in a dose-dependent manner following its direct intrastriatal microinfusion. Forskolin also antagonized the accentuating effect of intrastriatal N6-cyclohexyladenosine on ethanol-induced motor incoordination. These results suggested that ethanol-induced motor incoordination might be functionally correlated to a decrease in the striatal cyclic AMP levels and that the striatal adenosine A1 receptors might modulate ethanol-induced motor incoordination through cyclic AMP signaling mechanism(s). Further support to this hypothesis was obtained by the actual measurement of the striatal cyclic AMP levels in the same experimental conditions as in motor coordination studies using high-performance liquid chromatography with fluoroscence detection. Regardless of the method (focused microwave irradiation, cervical dislocation or decapitation into a dry ice-ethanol mixture) used to kill the animals, a significant decrease in the striatal cyclic AMP levels was observed due to ethanol. Intrastriatal adenosine A1-selective agonist, N6-cyclohexyladenosine (24 ng), caused a further significant decrease in the striatal cyclic AMP levels in the ethanol- but not in the vehicle-treated animals. The further enhancement in the ethanol-induced decrease in the striatal cyclic AMP levels by intrastriatal N6-cyclohexyladenosine, therefore, functionally correlated with the observed potentiating effect of intrastriatal N6-cyclohexyladenosine on ethanol-induced motor incoordination. The effects of intrastriatal N6-cyclohexyladenosine+ethanol and of ethanol alone on the striatal cyclic AMP levels were blocked by intrastriatal pertussis toxin (500 ng) pretreatment, indicating the involvement of pertussis toxin-sensitive G-proteins (Gi, Go) and possibly of the adenosine A1 receptor coupled to the G-proteins in the striatum. Furthermore, ethanol alone significantly decreased the basal as well as the cyclic AMP-stimulated catalytic activities of the striatal cyclic AMP protein kinase, which were further reduced by intrastriatal N6-cyclohexyladenosine. The results of the present study therefore support an involvement of a cyclic AMP signaling pathway in the striatal adenosinergic modulation of ethanol-induced motor incoordination at the post-adenosine A1 receptor level.

Adenosine↗
PubMed1984

The effects of alkylated xanthines on cyclic AMP accumulation in dog thyroid slices exposed to carbamylcholine.

In dog thyroid slices stimulated by thyrotropin (TSH), activation of muscarinic cholinergic receptors leads to a decrease in cyclic AMP levels. Previous studies have established that carbamylcholine enhances cyclic GMP levels and inhibits cyclic AMP accumulation. Several experimental data have suggested that these effects are mediated by an increase in intracellular Ca2+ levels. The inhibition of cyclic AMP accumulation results in accelerated catabolism. Dog thyroid phosphodiesterase activity is due to a mixture of three enzyme forms: a calmodulin-sensitive form, a cyclic GMP-stimulated form and a cyclic AMP-specific form. This report is concerned with the comparison of the effects of several phosphodiesterase inhibitors on cell-free phosphodiestease activity and on cyclic nucleotide accumulation in intact cells. Alkylated xanthines, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724), and 2-O-propoxyphenyl-8-azapurin-6-one (M & B 22948) were studied as inhibitors of partially purified dog thyroid phosphodieterases and for their ability to alter cyclic AMP and cyclic GMP accumulation in dog thyroid slices that had been stimulated with TSH and/or carbamylcholine. 1-Methyl-3-isobutylxanthine (MIX) and 7-benzyl MIX were the most potent inhibitors of phosphodiesterase activities in the crude soluble and particulate fractions but exhibited no selectivity for inhibiting cyclic AMP or cyclic GMP hydrolysis. In dog thyroid slices stimulated by TSH and in the absence of carbamylcholine, Ro 20-1724 and 1-isoamyl-3-isobutylxanthine (IIX) were the most effective compounds to potentiate the accumulation of cyclic AMP. The rank order of abilities to potentiate cyclic AMP accumulation in dog thyroid slices stimulated by TSH paralleled the rank order of potencies to inhibit the cyclic AMP-specific phosphodiesterase. In the presence of carbamylcholine, the observed decrease in cyclic AMP levels was attenuated by MIX, 8-methoxymethyl MIX (8-MeOMe MIX), 7-benzyl MIX, and M & B 22948, the most potent inhibitors of the calmodulin-sensitive phosphodiesterase. MIX, 8-MeOMe MIX, and 7-benzyl MIX inhibited the cyclic GMP-stimulated phosphodiesterase in the same rank order of potencies as the calmodulin-sensitive enzyme, but M & B 22948 did not significantly inhibit the cyclic GMP-stimulated enzyme activity. IIX and Ro 20-1724 did not alter the carbamylcholine-induced inhibition of cyclic AMP accumulation.(ABSTRACT TRUNCATED AT 400 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1979

Studies on the source of cyclic AMP in canine gastric juice.

The possibility that cyclic AMP in gastric juice is derived from plasma by simple clearance was evaluated. The effect of exogenous cyclic AMP administration (1 mg/min) on radioimmunoassayable cyclic AMP in plasma and gastric juice was studied in dog stomach during histamine (8--16 micrograms/kg/hr) infusion. Experiments were performed in vagally denervated fundic (Heidenhain) and in innervated gastric pouches. During infusion of cyclic AMP with histamine, plasma cyclic AMP concentration rapidly increased 500-fold. Simultaneously, gastric juice cyclic AMP levels decreased almost 50%. In contrast, dibutyryl cyclic AMP infusion increased gastric juice cAMP concentration 5-fold, suggesting that the gastric mucosa is more permeable to dibutyryl cyclic AMP. In Heidenhain pouch experiments the clearance of dibutyryl cyclic AMP into gastric juice was only 1.5% of the clearance of aminopyrine. It appears that plasma clearance of cyclic AMP fails to account for most of the cyclic AMP present in gastric juice.

Aminopyrine↗
PubMed1988

Cyclic AMP is an inhibitor of stalk cell differentiation in Dictyostelium discoideum.

Cyclic AMP and DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone) together induce stalk cell differentiation in vitro in Dictyostelium discoideum strain V12M2. The induction can proceed in two stages: in the first, cyclic AMP brings cells to a DIF-responsive state; in the second, DIF-1 alone can induce stalk cell formation. We report here that during the DIF-1-dependent stage, cyclic AMP is a potent inhibitor of stalk cell differentiation. Addition of cyclic AMP at this stage to V12M2 cells appreciably delays, but does not prevent, stalk cell formation. In contrast, stalk cell differentiation in the more common strain NC4 is completely suppressed by the continued presence of cyclic AMP. This fact explains earlier failures to induce stalk cells in vitro in NC4. We now consistently obtain efficient stalk cell induction in NC4 by removing cyclic AMP in the DIF-1-dependent stage. Cyclic AMP also inhibits the production of a stalk-specific protein (ST310) in both NC4 and a V12M2 derivative. Adenosine, a known antagonist of cyclic AMP action, does not relieve this inhibition by cyclic AMP and does not itself promote stalk cell formation. Finally, stalk cell differentiation of NC4 cells at low density appears to require factors in addition to cyclic AMP and DIF-1, but their nature is not yet known. The inhibition of stalk cell differentiation by cyclic AMP may be important in establishing the prestalk/prespore pattern during normal development, and in preventing the maturation of prestalk into stalk cells until culmination.

Cell Differentiation↗
PubMed1992

Epidermal growth factor potentiates the transmitter-induced stimulation of C-AMP and inositol phosphates in human pigment epithelial cells in culture.

Salbutamol, isoproterenol and dopamine stimulate C-AMP production in human retinal pigment epithelium (RPE) cells by activation of beta 2-type receptors. Epidermal growth factor (EGF) in contrast does not alter basal levels of C-AMP but elevates in an apparently dose-dependent manner the isoproterenol-induced stimulation of C-AMP. EGF also potentiates the forskolin-induced stimulation of C-AMP but has no effect on the elevation of C-AMP caused by NECA (5'-[N-ethyl]-carboxamido adenosine), an adenosine A2-receptor agonist. EGF, isoproterenol and NECA have no effect on basal levels of inositol phosphates (InsPs) in human RPE cells, but EGF specifically elevates the carbachol-induced stimulation of InsPs. The carbachol effect on InsPs is attenuated by the phorbol ester PMA (4 beta-phorbol 12 myrisate 13-acetate). PMA did not, however, affect the stimulation of C-AMP caused by isoproterenol. The interaction of EGF and C-AMP is further demonstrated in experiments where the incorporation of [3H]thymidine into RPE cells was studied, as an index for proliferation. EGF stimulates RPE cell proliferation while isoproterenol and dibutyryl C-AMP nullify the EGF effect. Dibutyryl C-AMP has a negative effect on RPE cell proliferation while isoproterenol is ineffective. The data presented here suggest that after stimulation of EGF receptors, tyrosine-kinase-activated products can influence secondary messenger products produced from activation of beta 2-type (linked with C-AMP formation) and muscarinic (linked with InsPs production) receptors in RPE cells. We could find no evidence of an interaction between receptors associated with C-AMP and InsPs/diacylglycerol production.

Adolescent↗
PubMed1976

Effects of prostaglandins and other drugs on the cyclic AMP content of cultured bone cells.

Prostaglandins of the E-series (PGE1 and PGE2) may be involved in disease-related, localized loss of bone. E-prostaglandins increase the cyclic AMP content of many cells; and, to determine if their effects on bone are mediated by cyclic AMP, we examined the effects of E-prostaglandins and of other agents on the cyclic AMP content of cultured bone cells. PGE2 produced a rapid, marked and dose-related increase in the cyclic AMP content of confluent monolayers of bone cells isolated from newborn rat calvaria. At 2.8 X 10(-6) M, PGE1 and PGE2 had approximately the same effect, while the effect of PGF2alpha was much less pronounced. In the presence of theophylline, PGE2 had a more marked effect than parathyroid hormone (PTH) and the combination of PGE2 and PTH had a synergistic effect. The divalent, cationic, ionophore, A23187, produced an increase in cellular cyclic AMP and had an additive effect in combination with PGE2. Synthetic salmon calcitonin (CT), which inhibits the bone resorptive effect of PGE2, increased cellular cyclic AMP and had an additive effect in combination with PGE2. A prostaglandin antagonist, SC-19220, partially inhibited the resorptive effect of PGE2 and reduced its effect on cellular cyclic AMP. The calcium antagonist, D600, inhibited the bone resorptive effects of PGE2 but had no effect on increased cellular cyclic AMP produced by PGE2. The marked effect of PGE2 on bone cell cyclic AMP suggests that this action is involved in the mechanism of PGE2-related bone loss. The fact that agents with different effects on PGE2-induced increases in cellular cyclic AMP can inhibit its resorptive actions, suggests that PGE2-induced changes in cyclic AMP may be related less to its resorptive actions than to its inhibitory effect on bone formation.

Animals↗
PubMed1988

Regulation of growth hormone secretion and cyclic AMP metabolism in ovine pituitary cells: interactions involved in activation induced by growth hormone-releasing hormone and phorbol esters.

Growth hormone-releasing hormone (GHRH) and the phorbol ester tetradecanoylphorbol acetate (TPA) each stimulated a rapid and extensive (up to 15-fold) increase in the secretion of growth hormone from cultured ovine anterior pituitary cells. Effects of the releasing hormone on growth hormone secretion were associated with a concurrent, large increase in cellular cyclic AMP accumulation. TPA induced a much smaller (26-78%), though still significant, increase in cellular cyclic AMP levels. Forskolin and isobutylmethylxanthine (IBMX) also stimulated growth hormone secretion and cyclic AMP accumulation. When combined with a maximally effective concentration of GHRH these compounds did not further elevate growth hormone secretion even though they induced further increases in cyclic AMP concentration; this is consistent with activation occurring via a common cyclic AMP-dependent pathway. In contrast TPA when combined with maximally effective concentrations of either GHRH, forskolin or IBMX caused additional release of growth hormone, suggesting that the TPA-induced secretion involved a cyclic AMP-independent process. However, TPA also markedly potentiated the cellular cyclic AMP accumulation due to each of these agents. That TPA induced stimulation of basal and GHRH-stimulated cyclic AMP levels measured in the presence of IBMX suggests an action affecting cyclic AMP synthesis. Carbachol had no effect on basal or GHRH-stimulated growth hormone secretion or cyclic AMP levels. The two actions of TPA, one on secretion and one on cyclic AMP metabolism, may result from activation of some common event possibly involving protein kinase C. Our results suggest that GHRH and TPA activate independent pathways regulating growth hormone secretion.

1-Methyl-3-isobutylxanthine↗
PubMed1996

GABAB receptors negatively regulate transcription in cerebellar granular neurons through cyclic AMP responsive element binding protein-dependent mechanisms.

GABAB receptors affect short-term signalling in various cell types. However, nothing is known about possible long-term effects on transcription. To analyse such effects in the CNS, we studied GABAB receptor-mediated gene regulation in primary cultures of cerebellar granule neurons. Transcription was followed using a chloramphenicol acetyl transferase reporter gene driven by the minimal cyclic AMP-responsive element (TGACGTCA). Transcription was stimulated by activation of both the cyclic AMP (forskolin: 5 x 10(-6) M) and the Ca2+ dependent (KCl: 30 mM) pathways (-)-Baclofen (10(-6) M to 10(-4) M), a specific GABAB receptor agonist, reduced by 50-70% the transcriptional stimulation evoked by both forskolin and KCl, whereas isoguvacine, a GABAA receptor agonist, was without effect. Moreover, the GABAB antagonist CGP 35348 abrogated the inhibitory effects of both GABA and baclofen, indicating that GABAB receptors were specifically implicated in this response. Measurements of cyclic AMP levels suggested that (-) baclofen inhibits forskolin-initiated transcription by reducing cyclic AMP production. Direct transcriptional activation, via the cyclic AMP pathway, by overexpression of the catalytic subunit of the cyclic AMP-dependent protein kinase, was not significantly altered by (-) baclofen. This indicates again that (-) baclofen-dependent inhibitory mechanisms operate upstream of cyclic AMP-dependent protein kinase at the level of second messenger formation. Further, we used a yeast transcriptional activator GAL4-cyclic AMP-responsive element binding protein to analyse whether GABAB receptor-mediated inhibition of cyclic AMP-responsive element transcription implicated the transacting factor cyclic AMP-responsive element binding protein. We show that the negative effects of (-) baclofen implicate this transcription factor and this holds good for both the forskolin and KCl-stimulated pathways. The results indicate that GABAB receptors negatively regulate cyclic AMP-responsive element binding protein-mediated transcription in the CNS.

Animals↗
PubMed1998

The inhibitory effect of ursodeoxycholic acid and pentoxifylline on platelet derived growth factor-stimulated proliferation is distinct from an effect by cyclic AMP.

This study assessed the ability of ursodeoxycholic acid (UDCA) and one of its metabolites, tauroursodeoxycholic acid (TUDCA), to inhibit platelet derived growth factor (PDGF) stimulated fibroproliferation and compared these results to the effect of pentoxifylline and its metabolite-1 [1-(5-hydroxyhexyl)-3,7-dimethylxanthine] and assessed the potential role of cyclic AMP in this process. Fibroproliferative activity was measured by the tritiated thymidine uptake assay in human fibroblast cultures. All four compounds: pentoxifylline, metabolite-1, UDCA and TUDCA inhibited the fibroproliferative activity stimulated by PDGF (8 ng/ml). Incubation of fibroblasts with dibutyryl cyclic AMP reduced proliferation stimulated by PDGF suggesting that the PDGF stimulated proliferation was sensitive to inhibition by a membrane permeable analogue of cyclic AMP. Incubation of myofibroblasts with dibutyryl cyclic AMP significantly inhibited PDGF stimulated proliferation suggesting that cyclic AMP can regulate PDGF stimulated proliferation in the myofibroblast. To determine if the effect of pentoxifylline on fibroproliferation was mediated by cyclic AMP, we used dideoxyadenosine, a potent inhibitor of adenylyl cyclase. The effect of pentoxifylline on fibroproliferation was not prevented by dideoxyadenosine, which inhibits formation of cyclic AMP, thus suggesting that the inhibitory effect of pentoxifylline on PDGF-stimulated proliferation of fibroblasts was not mediated by cyclic AMP, arguing against a role for cyclic AMP in this process. Combinations of UDCA (250 microM) plus pentoxifylline (120 microM) or UDCA (250 microM) plus TUDCA (250 microM) inhibited fibroproliferative activity stimulated by PDGF to a greater extent than either drug alone. As UDCA has been reported to decrease cyclic AMP these results argue against a role for cyclic AMP in this process. Finally the results suggest that UDCA may inhibit PDGF-stimulated proliferation via an inhibition of C-kinase.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1993

Isolation and characterization of AMP deaminase from mammalian (rabbit) myocardium.

AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) is a ubiquitous enzyme in eukaryotes, which may play a role in ATP catabolism during myocardial ischaemia. We report isolation of AMP deaminase from rabbit myocardium with a 19% recovery and a 650-fold enrichment, using a newly devised protocol involving sequential cation-exchange, gel-permeation and affinity chromatographies. The cardiac AMP deaminase preparation described was electrophoretically and chromatographically homogeneous and contained one unique N-terminal residue (leucine). The isolated enzyme was sensitive to various cations (K+, Mg2+, Ca2+). The pH optimum of purified cardiac AMP deaminase was 6.8, its pI was 6.5, and it displayed substrate-specificity toward 5'-AMP. The subunit molecular mass of rabbit heart AMP deaminase on SDS/PAGE (81 kDa) and the holoenzyme molecular mass as estimated by non-denaturing size-exclusion h.p.l.c. (330 kDa) indicated that the native enzyme was a tetramer. Cardiac AMP deaminase displayed a sigmoidal substrate-saturation curve in the presence of 100 mM KCl. Apparent Michaelis constants were a Km of 5.8 mM AMP and a Vmax. of 11.1 mumol/min per mg of protein. ATP and ADP were positive allosteric effectors of cardiac AMP deaminase: the apparent Km was decreased to 1.7 mM by 1.0 mM ATP. The enzyme was inhibited by GTP, coformycin, coformycin 5'-phosphate, palmitoyl-CoA, inorganic phosphate compounds, and the metal chelator o-phenanthroline. No inhibition either by product nucleotide (IMP) or by nicotinamide nucleotides was detected when these agents were examined at concentrations up to 2.5 mM. We conclude that this enzyme preparation offers a means by which the kinetic mechanism and regulation of mammalian cardiac AMP deaminase may be directly investigated.

AMP Deaminase↗
PubMed1993

Modulation of mammalian cardiac AMP deaminase by protein kinase C-mediated phosphorylation.

Using AMP deaminase (AMP aminohydrolase; EC 3.5.4.6) purified from rabbit left-ventricular heart tissue, we report direct investigation of the potential for cardiac AMP deaminase activity to be regulated by kinase-mediated phosphorylation. Rabbit heart AMP deaminase served as a substrate for Ca2+/phospholipid-dependent protein kinase (protein kinase C; PKC) exclusively; no other mammalian protein kinase phosphorylated the enzyme. PKC-dependent AMP deaminase phosphorylation was rapid, linear with respect to time and the concentrations of PKC and AMP deaminase in the reaction, and inhibitable by staurosporine. Upon phosphorylation, the apparent Km of cardiac AMP deaminase decreased from 5.6 mM to 1.2 mM, without effect on the Vmax. Whether phosphorylated or not, rabbit heart AMP deaminase was inhibited by 1.0 mM GTP, which decreased the Vmax. by approximately 50% in each case. PKC-dependent phosphorylation of cardiac AMP deaminase did not alter the enzyme's allosterism toward millimolar ATP or ADP: both nucleotides at 1.0 mM concentration decreased the apparent Km to approximately 0.5 mM. Treatment of cardiac phospho-AMP deaminase with either the protein phosphatase calcineurin or alkaline phosphatase generated a dephosphorylated form which displayed molecular and kinetic properties identical with those of the originally isolated enzyme. These data raise the possibility that a phosphorylation-dephosphorylation mechanism may regulate flux through AMP deaminase in the heart under pathological conditions, such as myocardial ischaemia, characterized by PKC activation and adenylate depletion.

AMP Deaminase↗
PubMed1994

The effect of extracellular cyclic AMP on differentiation inducing factor (DIF)-dependent prestalk cell gene expression in monolayers of Dictyostelium is complex.

An earlier finding that the ecmA and ecmB prestalk cell specific genes exhibited very different responses to cyclic AMP prompted the suggestion that cyclic AMP might act as the major spatial regulator of the prestalk cell developmental pattern in Dictyostelium. A more detailed kinetic analysis in monolayers of Dictyostelium has revealed that cyclic AMP inhibits the rate of expression of all three differentiation inducing factor (DIF) inducible genes, ecmA, ecmB and pDd26. After prolonged incubation, however, cyclic AMP enhances the levels of both ecmA and ecmB mRNAs, and nuclear run-on experiments suggest that cyclic AMP inhibits the degradation of both mRNA species. This complex response to cyclic AMP can explain the differential effects reported previously. Thus depending upon the experimental conditions, cyclic AMP can either enhance or reduce the apparent steady state level of a specific mRNA species. These results are not compatible with the earlier proposal that cyclic AMP is a spatial regulator of the prestalk developmental pattern. Although ecmA and ecmB accumulate rapidly in response to DIF, there is a lag in the accumulation of pD26 mRNA and the induction requires protein synthesis. These results suggest that pDd26 transcription requires the accumulation of an additional factor(s). Inhibition of pDd26 mRNA accumulation by cyclic AMP also occurs during the lag period, suggesting the possibility that cyclic AMP inhibits the accumulation of the, as yet, unknown factor. The inhibitory effect of cyclic AMP on pDd26 gene expression is unaffected by caffeine, suggesting that inhibition does not involve adenylate cyclase activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1997

Cyclic AMP-mediated enhancement of high-affinity choline transport and acetylcholine synthesis in brain.

Intracerebroventricular administration of N6, 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (db-cyclic AMP) to mice increased high-affinity choline transport (HAChT) into synaptosomal preparations from the hippocampus, striatum, and frontal cortex in a time- dose-, and brain region-dependent manner. Similar observations were made when the cyclic AMP analogue 8-bromo-cyclic AMP, the adenylyl cyclase activator forskolin, and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine were administered. Inhibition of phosphatase 1 and 2A, with okadaic acid, increased basal choline transport and enhanced the response to db-cyclic AMP. The early increase of HAChT activity induced by db-cyclic AMP was blocked by H-7 and H-89, protein kinase A inhibitors, but not by cycloheximide, a protein synthesis inhibitor. Kinetic analysis of the early changes of HAChT revealed an increase in the apparent Vmax without a change of the Km for choline. Hemicholinium-3 (HC-3) binding was not altered when studied 1 h after db-cyclic AMP administration. In contrast, HC-3 binding and HAChT activity were both elevated when estimated 3 h after the treatment, and pretreatment with cycloheximide partially prevented the db-cyclic AMP-induced HAChT rise. As evidence that enhanced HAChT is associated with a direct action of cyclic AMP-dependent pathways on the cholinergic nerve terminals, addition of 8-bromocyclic AMP to isolated hippocampal synaptosomes induced an increase of HAChT that was prevented by H-89. Choline acetyltransferase activity was not affected at any time during the studies. The synthesis of acetylcholine, however, was enhanced 1 h after db-cyclic AMP addition. Our studies show that cyclic AMP-mimetic compounds appear to modulate the choline carrier by a dual mode: an early increase of the maximal velocity without a change of the number of HC-3 binding sites and a late rise of transport that is accompanied by an increase of HC-3 binding. We postulate that HAChT and consequently acetylcholine synthesis in vivo is modulated, in part, by protein kinase A.

Acetylcholine↗
PubMed1999

Effects of cyclic AMP on components of the cell cycle machinery regulating DNA synthesis in cultured astrocytes.

Cyclic AMP is a second messenger for various hormones that inhibits cell multiplication and DNA synthesis in cultured astrocytes. We examined the effects of increasing intracellular cyclic AMP on the catalytic (cdks) and regulatory (cyclins and ckis) components of cyclin-dependent protein kinases, which regulate progression of the cell cycle before completion of DNA synthesis, in primary cultured astrocytes and in an astrocytic cell line C.LT.T.1.1. The amount of cdk4 changed little during the cell cycle and was not affected by cyclic AMP. There was little cdk1 and cdk2 in quiescent cells, and their expression increased during the G1-S phases. Cyclic AMP strongly inhibited cdk1 and cdk2 expression. Transforming growth factor beta also inhibited cdk1 expression in primary astrocytes. Cyclic AMP did not affect the two ckis p27KIP1 and p21CIP1. There was little cyclin D1 in quiescent cells, but it increased during the G1 phase and was reduced by cyclic AMP. Cyclin E increased during the G1-S phases and was not affected by cyclic AMP in primary astrocytes. The amount of cyclin A was low in quiescent cells and increased during the G1-S phases. Expression of its mRNA and protein was inhibited by cyclic AMP. The protein kinase activities associated with complexes of cyclins and cdks were increased by growth factors and prevented by cyclic AMP. We conclude that cyclic AMP inhibits progression of the cell cycle in astrocytes at least by preventing the expression of the regulatory subunits, cyclins D1 and A, and catalytic subunits, cdk1 and cdk2, of cyclin-regulated protein kinases. Key Words: Cyclin-dependent protein kinases-Glial cells-Cyclic AMP.

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