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PubMed1973

The relation of adenyl cyclase to the activity of other ATP utilizing enzymes and phosphodiesterase in preparations of rat brain; mechanism of stimulation of cyclic AMP accumulation by NaF.

1. A method for measuring the rate of production of (14)C-labelled adenine nucleotides, including cyclic adenosine 3',5'monophosphate (cyclic AMP), from [(14)C]-adenosinetriphosphate (ATP) was developed and used to study the effects of ATP, adenosine diphosphate (ADP) and adenosine 5'-monophosphate (AMP) on the rate of accumulation of cyclic AMP in cell-free preparations of adenyl cyclase from rat brain.2. The mechanism by which NaF increases cyclic AMP accumulation was studied by comparing its effect on adenine nucleotide metabolism with that of an ATP regenerating system.3. ADP and ATP are potent inhibitors of phosphodiesterase (PDE) and it is the sum of the concentrations of these two nucleotides which controlled the rate of destruction of cyclic AMP. The effect of these nucleotides was significant even in the presence of 6.7 mM theophylline; theophylline itself inhibited PDE only 50-60%.4. Fluoride ion had no direct effect on PDE but it inhibited the rate of hydrolysis of ADP and ATP and thus indirectly inhibited PDE. The effect of fluoride ion on cyclic AMP accumulation can be explained, at least in part, by this indirect inhibition of PDE.5. Studies on a more purified preparation of adenyl cyclase clearly demonstrated a direct action of NaF on adenyl cyclase.

Adenosine Diphosphate↗
PubMed1974

Dissociation of 5-hydroxytryptamine effects on myocardial contractility and cyclic AMP accumulation.

1 5-Hydroxytryptamine increased the contractility of the isolated rat atrium and produced a small rise in cyclic 3',5'-adenosine monophosphate (cyclic AMP) accumulation in rat heart slices. These effects were similar to those of tyramine.2 Incubation with phenoxybenzamine prevented the inotropic effect of 5-hydroxytryptamine but did not inhibit the rise in cyclic AMP accumulation.3 A single injection of reserpine 24 h before the experiment did not inhibit the inotropic effect of 5-hydroxytryptamine but prevented the rise in cyclic AMP accumulation.4 It is concluded that in the rat 5-hydroxytryptamine increases cardiac contractility chiefly by a direct effect which is blocked by phenoxybenzamine, whereas it increases myocardial cyclic AMP accumulation indirectly by releasing endogenous catecholamines. Cardiac contractility may thus be increased without a rise in cyclic AMP accumulation, and cyclic AMP accumulation may be increased without a rise in contractility.

Adenine↗
PubMed1986

Studies on the receptor mediating cyclic AMP-independent enhancement by adenosine of IgE-dependent mediator release from rat mast cells.

Adenosine produced a concentration-related enhancement of antigen-induced 5-hydroxytryptamine (5-HT) release from rat serosal mast cells. This potentiation was maximal following the simultaneous addition of adenosine with antigen. Enhancement of 5-HT release was accompanied by potentiation of the adenosine 3':5'-cyclic monophosphate (cyclic AMP) response to challenge. The cyclic AMP response, which was antagonized by 8-phenyltheophylline, was characterized as an A2-purinoceptor-mediated effect by the use of 5'-N-ethylcarboxamideadenosine (NECA) and L-N6-phenylisopropyladenosine (L-PIA). Enhancement of 5-HT release, conversely, was not blocked by 8-phenyltheophylline suggesting it to be mediated by a cyclic AMP-independent mechanism. The effect of adenosine on 5-HT release was not reduced by the inhibition of the facilitated uptake of adenosine with dipyridamole, hexobendine or p-nitrobenzylthioguanosine, therefore, suggesting it to be mediated by a cell surface receptor. The receptor mediating enhancement of 5-HT does not appear to belong to the P2-purinoceptor subtype as adenosine was more potent than both adenosine monophosphate (AMP) and adenosine diphosphate (ADP) and alpha, beta-methylene ATP was inactive. Furthermore, the effects of AMP were blocked by alpha, beta-methylene ADP, which inhibits the conversion of AMP to adenosine. Adenosine, NECA, L- and D-PIA were all of equal potency in enhancing 5-HT release. Inosine and 3-deazaadenosine were also active. The rank order of potency of these adenosine analogues is not consistent with an effect at A1- or A2-purinoceptors. There appear to be two adenosine receptors on rat mast cells, an A2-purinoceptor which stimulates adenylate cyclase and a separate purinoceptor, stimulation of which produces enhancement of mediator release by an unknown mechanism. The effects mediated by these receptors appear to be independent of each other.

1-Methyl-3-isobutylxanthine↗
PubMed1988

Long-term regulation of synaptic acetylcholine release and nicotinic transmission: the role of cyclic AMP.

1. Using the rat superior cervical ganglion in vitro, the relative efficacy of nicotinic synaptic transmission was estimated by recording the postganglionic compound action potential and the amount of endogenous acetylcholine (ACh) released. These two parameters were correlated in individual ganglia by sampling the bathing medium for the assay of ACh while simultaneously recording the postganglionic response. 2. The beta-adrenoceptor agonist isoprenaline potentiated both the evoked release of ACh and the postganglionic response by about 20% during preganglionic stimulation at 0.2 Hz. 3. The adenosine receptor agonist 2-chloroadenosine inhibited ACh release and the postganglionic response by about 35%. 4. Tetanic preganglionic stimulation for a few seconds induced a long-term potentiation of nicotinic responses and of ACh release. Both of these potentiations were dependent upon extracellular Ca2+ during the tetani. 5. Forskolin and analogues of cyclic AMP also caused a long-lasting potentiation of both the evoked release of ACh and the postganglionic response, indicating that cyclic AMP may regulate transmission by a presynaptic mechanism. The specificity of the cyclic AMP analogues was tested using various butyryl- and bromo-purine nucleotides. 6. The effects of forskolin and 8-bromo-cyclic AMP did not appear to be dependent upon extracellular Ca2+. 7. The potentiation caused by forskolin was consistently augmented by three phosphodiesterase inhibitors--AH 21-132, papaverine and SQ 20-006. However, the effect of forskolin was not consistently enhanced by theophylline, nor was it reduced by the adenylate cyclase inhibitor SQ 22-536. 8. The neurogenic long-term potentiation was augmented by two of the phosphodiesterase inhibitors that also augmented the forskolin-induced potentiation--papaverine and SQ 20-006. 9. It was concluded that cyclic AMP can enhance nicotinic transmission, and can do so by increasing the evoked release of ACh. However, it was not possible to prove that cyclic AMP mediates the long-term potentiation induced by tetanic preganglionic stimulation.

Acetylcholine↗
PubMed1989

Differences in the adenosine receptors modulating inositol phosphates and cyclic AMP accumulation in mammalian cerebral cortex.

1. 2-Chloroadenosine stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation and potentiated (guinea-pig) or inhibited (mouse) the histamine H1-receptor-stimulated hydrolysis of inositol phospholipids in slices of guinea-pig and mouse cerebral cortex. 2. Two xanthine-based adenosine receptor antagonists were identified which were one order of magnitude more potent at the adenosine receptor mediating augmentation of the histamine-stimulated inositol phospholipid hydrolysis than at the receptor linked to cyclic AMP formation in guinea-pig cerebral cortical slices. 3. These compounds, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and 7-benzyl-3-(2-methylpropyl)xanthine (BMPX) retained their selectivity under near-identical incubation conditions in both assays. 4. These compounds also showed similar affinities and selectivity for the adenosine receptor mediating inhibition of histamine-stimulated inositol phospholipid hydrolysis in mouse cerebral cortical slices. 5. Inclusion of agents which give rise to, or mimic, high levels of cyclic AMP (forskolin (1 microM), 8-bromo-cyclic AMP (1 mM) and vasoactive intestinal polypeptide (1 microM] in the incubation medium failed to mimic the action of 2-chloroadenosine on inositol phospholipid turnover in cerebral cortical slices from either species. 6. These data suggest that the adenosine receptor modulating the hydrolysis of inositol phospholipid in mouse and guinea-pig cerebral cortical slices is different from the adenosine receptor linked to cyclic AMP formation, and, furthermore, that the modulation of inositol phospholipid metabolism in either species is not mediated via alterations in cyclic AMP levels.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Cyclic AMP counteracts mitogen-induced inositol phosphate generation and increases in intracellular Ca2+ concentrations in human lymphocytes.

1. The effects of increases in intracellular adenosine 3':5'-cyclic monophosphate (cyclic AMP) on mitogen-induced generation of inositol phosphates and increases in intracellular Ca2+ concentration were investigated in human peripheral blood mononuclear leukocytes (MNL). 2. The mitogens concanavalin A (Con A), pokeweed mitogen (PWM) and phytohaemagglutinin (PHA) concentration-dependently stimulated generation of inositol phosphates. Catecholamines inhibited this process with an order of potency: isoprenaline greater than adrenaline greater than noradrenaline indicating involvement of beta 2-adrenoceptors. This order of potency was also consistent with the catecholamine potencies for stimulating the generation of cyclic AMP. 3. In addition to catecholamines, the cyclic AMP formation-stimulating agents prostaglandin E1 (PGE1) and forskolin concentration-dependently inhibited mitogen-induced inositol phosphate generation, too. Moreover, the inhibitory effect of isoprenaline was potentiated by co-incubation with the phosphodiesterase inhibitor isobutylmethylxanthine demonstrating that these inhibitory effects were mediated by cyclic AMP. 4. Con A and PHA concentration-dependently increased the intracellular Ca2+ concentration in human MNL (assessed by the fluorescent indicator dye Fura-2). This increase was almost completely blocked by chelation of extracellular Ca2+, demonstrating influx rather than mobilization from intracellular stores. 5. The elevation of intracellular Ca2+ was not blocked by pretreatment with pertussis toxin, 100 ng ml-1, for 16 h. 6. Isoprenaline, PGE1, and forskolin, however, inhibited the mitogen-stimulated elevation of intracellular Ca2+. This inhibition was enhanced by the phosphodiesterase inhibitors isobutylmethylxanthine and Ro 20-1724, demonstrating mediation by cyclic AMP. 7. We conclude that catecholamines and other cyclic AMP increasing agents can inhibit mitogen-stimulated generation of inositol phosphates and elevation of intracellular Ca2+ in resting human MNL.

Adult↗
PubMed1992

Cholinoceptor regulation of cyclic AMP levels in bovine adrenal medullary cells.

1. The regulation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels by cholinoceptors has been studied in cultured bovine adrenal medullary cells. 2. Acetylcholine (100 microM), nicotine (10 microM) and dimethylphenylpiperazinium (20 microM) each increased cellular cyclic AMP levels 2 to 4 fold over 5 min in the absence of phosphodiesterase inhibitors. The muscarinic agonist acetyl-beta-methylcholine (100 microM) had no effect either on its own or on the response to nicotine. The responses to acetylcholine and nicotine were unaffected by atropine (1 microM) but were abolished by mecamylamine (5 microM). 3. Cellular cyclic AMP increased transiently during continuous exposure to nicotine (1-20 microM), with the largest response seen after 5 min, a smaller response after 20 min, and no change in cyclic AMP levels seen after 90 or 180 min. The maximal response after 5 min stimulation was seen with 5-10 microM nicotine and the EC50 was about 2 microM. In contrast, extracellular cyclic AMP levels did not change after 5 or 20 min stimulation with nicotine, but increased slightly after 90 min and further after 180 min. 4. The cellular cyclic AMP response to nicotine (10 microM) was unchanged or weakly enhanced in the presence of the unselective phosphodiesterase inhibitor, isobutylmethylxanthine, and was unchanged in the presence of rolipram. Nicotine did not interact synergistically with low concentrations of forskolin. The response was however completely abolished in the absence of extracellular Ca2+.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Correlation of cyclic AMP accumulation and relaxant actions of salmeterol and salbutamol in bovine tracheal smooth muscle.

1. The ability of salmeterol to stimulate cyclic AMP accumulation and relaxation has been compared with that of salbutamol in bovine tracheal smooth muscle. In addition, the anti-spasmogenic effects of these agents and their abilities to modulate histamine-stimulated [3H]-inositol phosphate accumulation have also been investigated. 2. In tissue strips, a close temporal correlation was found to exist between salmeterol (0.1 microM)-induced relaxation of methacholine (500 nM)-induced tone and cyclic AMP accumulation, both maximal reversal of induced tone (26.2 +/- 6.0%) and maximal levels of cyclic AMP accumulation being achieved after 30-40 min. In contrast to salmeterol, salbutamol exerted greater and more rapid effects on both parameters. Maximal reversal of methacholine-induced tone (79.3 +/- 14.0%) and maximal levels of cyclic AMP accumulation were produced within 5 min. 3. Salmeterol-induced cyclic AMP accumulation (EC50 = 5.3 [1.8 - 15.2] nM) and inhibition of histamine (0.1 mM)-stimulated [3H]-inositol phosphate accumulation (IC50 = 1.4 [0.3-6.3] nM) were both more potent than those induced by salbutamol (EC50 = 169 [99 - 290] nM; IC50 = 13.8 [7.0 - 27.4] nM). However, maximal effects exerted by each of these agents were similar in magnitude. 4. Anti-spasmogenic effects were examined by beta-adrenoceptor agonist application to tissue strips prior to construction of spasmogen concentration-effect curves. Both salmeterol and salbutamol exerted more marked inhibition of the contractile response induced by histamine than that induced by methacholine, salmeterol being the more potent agent, while salbutamol produced a greater maximal inhibitory effect. 5. The results demonstrate that salmeterol is a more potent agent than salbutamol and have highlighted a close temporal correlation between promotion of cyclic AMP accumulation and tissue relaxation stimulated by each agent when both parameters are measured under identical conditions.

Adrenergic beta-2 Receptor Agonists↗
PubMed1996

Coupling of metabotropic glutamate receptors to phosphoinositide mobilisation and inhibition of cyclic AMP generation in the guinea-pig cerebellum.

1. The effects of metabotropic glutamate receptor (mGluR) agonists on cyclic nucleotide and phosphoinositide turnover were investigated in adult guinea-pig cerebellar slices by use of radioactive precursors. 2.L-Glutamate, 1-aminocyclopentane-1S,3R-dicarboxylate (1S,3R-ACPD) and RS-3,5-dihydroxyphenylglycine (DHPG) evoked concentration-dependent increases in the accumulation of [3H]-inositol phosphates with pEC50 values of 2.98 +/- 0.02, 4.45 +/- 0.06 and 4.47 +/- 0.07, respectively. Maximal responses to these agents were 43 +/- 8, 52 +/- 12 and 84 +/- 11% of the response to 1 mM histamine, respectively. 3. The phosphoinositide response to 1S,3R-ACPD was antagonized in the presence of (+)-alpha-methyl-4-carboxyphenylglycine, with a calculated pKi value of 3.55 +/- 0.03. 4. Forskolin-stimulated accumulation of [3H]-cyclic AMP was not significantly altered in the presence of 10 microM DCG-IV or 300 microM 1S,3R-ACPD. Similarly, 300 microM 1S,3R-ACPD failed to alter isoprenaline-(1 microM) or 2-chloroadenosine (2-CA, 30 microM)-stimulated accumulation of [3H]-cyclic AMP. 5. Forskolin-stimulated accumulation of [3H]-cyclic AMP was concentration-dependently inhibited in the presence of L-glutamate and L-serine-O-phosphate (L-SOP) with pIC50 values of 2.91 +/- 0.17 and 2.86 +/- 0.04 with maximal inhibitions of 47 +/- 2 and 92 +/- 3%, respectively. L-2-Amino-4-phosphonobuty-rate (L-AP4) inhibited the forskolin response without saturating, evoking an inhibition of 71 +/- 7% at 3 mM. 6. 2-CA-evoked accumulation of [3H]-cyclic AMP was also inhibited by L-glutamate and L-SOP with pIC50 values of 2.71 +/- 0.03 and 2.72 +/- 0.08 and maximal inhibitions of 51 +/- 5 and 99 +/- 0%, respectively. L-AP4 inhibited the 2-CA response without saturating, evoking an inhibition of 68 +/- 1% at 3 mM. 7. Isoprenaline-evoked accumulation of [3H]-cyclic AMP was inhibited by L-glutamate and L-SOP with pIC50 values of 3.21 +/- 0.01 and 2.96 +/- 0.08 and maximal inhibitions of 88 +/- 2 and 93 +/- 3%, respectively. 8. These results suggest that the guinea-pig cerebellum expresses Group I and Group III mGluRs coupled to phosphoinositide turnover and inhibition of cyclic AMP generation, respectively.

2-Chloroadenosine↗
PubMed1996

VIP-induced relaxation of guinea-pig intestinal smooth muscle cells: sequential involvement of cyclic AMP and nitric oxide.

1. A possible interaction between cyclic AMP and nitric oxide (NO) in mediating the relaxant effect of vasoactive intestinal polypeptide (VIP) on intestinal smooth muscle cells has been investigated. The effects of the inhibitor of NO synthesis, NG-nitro-L-arginine methyl ester (L-NAME), have been studied on VIP-, forskolin-, and 8 bromo-cyclic AMP- induced relaxation of cells, dispersed by enzymatic digestion of muscle strips from the circular layer of guinea-pig ileum. 2. VIP alone did not modify the length of isolated muscle cells. By contrast, when the cells were contracted by cholecystokinin octapeptide, CCK8 (10 nM), VIP inhibited this contraction, inducing a concentration-dependent relaxation of the cells. Maximal relaxation was induced by 1 microM VIP (EC50 = 408.2 +/- 16.7 pM). 3. N-ethylmaleimide, inhibitors of adenylate cyclase or somatostatin, abolished the relaxing effect of VIP. (R)-p-cAMPs, an antagonist of cyclic AMP on protein kinase A also inhibited the VIP-induced relaxation by 92.1 +/- 6.3%. Inhibitors of nitric oxide synthase (NOS), L-NAME and L-NMMA, partially inhibited VIP-induced relaxation. The effect of L-NAME was reversed by L-arginine but not by D-arginine. 4. (R)-p-cAMPS and L-NAME also inhibited the cell relaxation induced either by forskolin which directly stimulates adenylate cyclase activity or 8-bromo-cyclic AMP, an analogue of cyclic AMP. 5. When cells were incubated for 30 min with dexamethasone 10 microM, a glucocorticoid known to decrease the synthesis of iNOS, the relaxing effect of a maximal concentration of VIP was decreased by 52 +/- 4% and L-NMMA had no further effect on this residual VIP-induced relaxation. Milrinone, a phosphodiesterase type III inhibitor, potentiated the relaxant effect of VIP. 6. These data demonstrate that the intracellular pathway mediating the relaxant effect of VIP in intestinal smooth muscle cells includes the sequential activation of adenylate cyclase, protein kinase A, activation of NOS and finally production of NO and cyclic GMP. NO could in turn regulate the cyclic AMP-dependent pathway of cell relaxation.

Animals↗
PubMed1996

Evidence that cyclic AMP phosphodiesterase inhibitors suppress TNF alpha generation from human monocytes by interacting with a 'low-affinity' phosphodiesterase 4 conformer.

1. We have investigated the inhibitory effects of RP 73401 (piclamilast) and rolipram against human monocyte cyclic AMP-specific phosphodiesterase (PDE4) in relation to their effects on prostaglandin (PG)E2-induced cyclic AMP accumulation and lipopolysaccharide (LPS)-induced TNF alpha production and TNF alpha mRNA expression. 2. PDE4 was found to be the predominant PDE isoenzyme in the cytosolic fraction of human monocytes. Cyclic GMP-inhibited PDE (PDE3) was also detected in the cytosolic and particulate fractions. Reverse transcription polymerase chain reaction (RT-PCR) of human monocyte poly (A+) mRNA revealed amplified products corresponding to PDE4 subtypes A and B of which the former was most highly expressed. A faint band corresponding in size to PDE4D was also observed. 3. RP 73401 was a potent inhibitor of cytosolic PDE4 (IC50: 1.5 +/- 0.6 nM, n = 3). (+/-)-Rolipram (IC50: 313 +/- 6.7 nM, n = 3) was at least 200 fold less potent than RP 73401. R-(-)-rolipram was approximately 3 fold more potent than S-(+)-rolipram against cytosolic PDE4. 4. RP 73401 (IC50: 9.2 +/- 2.1 nM, n = 6) was over 50 fold more potent than (+/-)-rolipram (IC50: 503 +/- 134 nM, n = 6) ) in potentiating PGE2-induced cyclic AMP accumulation. R-(-)-rolipram (IC50: 289 +/- 121 nM, n = 5) was 4.7 fold more potent than its S-(+)-enantiomer (IC50: 1356 +/- 314 nM, n = 5). A strong and highly-significant, linear correlation (r = 0.95, P < 0.01, n = 13) was observed between the inhibitory potencies of a range of structurally distinct PDE4 inhibitors against monocyte PDE4 and their ED50 values in enhancing monocyte cyclic AMP accumulation. A poorer, though still significant, linear correlation (r = 0.67, P < 0.01, n = 13) was observed between the potencies of the same compounds in potentiating PGE2-induced monocyte cyclic AMP accumulation and their abilities to displace [3H]-rolipram binding to brain membranes. 5. RP 73401 (IC50: 6.9 +/- 3.3 nM, n = 5) was 71 fold more potent than (+/-)-rolipram (IC50: 490 +/- 260 nM, n = 4) in inhibiting LPS-induced TNF alpha release from monocytes. R-(-)-rolipram (IC50: 397 +/- 178 nM, n = 3) was 5.2-fold more potent than its S-(+)- enantiomer (IC50: 2067 +/- 659 nM, n = 3). As with cyclic AMP, accumulation a closer, linear correlation existed between the potency of structurally distinct compounds in suppressing TNF alpha with PDE4 inhibition (r = 0.93, P < 0.01, n = 13) than with displacement of [3H]-rolipram binding (r = 0.65, P < 0.01, n = 13). 6. RP 73401 (IC50: 2 nM) was 180 fold more potent than rolipram (IC50: 360 nM) in suppressing LPS (10 ng ml-1)-induced TNF alpha mRNA. 7. The results demonstrate that RP 73401 is a very potent inhibitor of TNF alpha release from human monocytes suggesting that it may have therapeutic potential in the many pathological conditions associated with over-production of this pro-inflammatory cytokine. Furthermore, PDE inhibitor actions on functional responses are better correlated with inhibition of PDE4 catalytic activity than displacement of [3H]-rolipram from its high-affinity binding site, suggesting that the native PDE4 in human monocytes exists predominantly in a 'low-affinity' state.

Benzamides↗
PubMed1996

Cyclic AMP-dependent regulation of K+ transport in the rat distal colon.

1. The effect of agonists of the cyclic AMP pathway and of 293B, a chromanole-derived K+ channel blocker, on K+ transport in the rat distal colon was studied by measuring unidirectional fluxes, uptake, and efflux of Rb+ in mucosa-submucosa preparations and by patch-clamp of crypt epithelia from isolated crypts. 2. 293B concentration-dependently inhibited basal and forskolin-stimulated short-circuit current. In isolated crypts 293B blocked a basal K+ conductance but had no effect on cyclic AMP-evoked depolarization induced by the opening of apical Cl- channels. When the effect of cyclic AMP on Cl- conductance was prevented by substituting Cl- with gluconate, an inhibition of total cellular K+ conductance by forskolin and a membrane-permeable cyclic AMP analogue was unmasked. 3. Unidirectional ion flux measurements revealed that 293B suppressed the increase in JRbsm induced by forskolin. This, together with the inhibition of cyclic AMP-induced anion secretion indicates that the drug blocks K+ channels, presumably both in the apical and the basolateral membrane. Forskolin caused not only inhibition of K+ absorption, but also stimulation of K+ secretion. The inhibition was diminished, but not blocked, in the presence of inhibitors of the apical H(+)-K(+)-ATPase, vanadate and ouabain. Forskolin stimulated serosal, bumetanide-sensitive Rb+ uptake, whereas mucosal, ouabain/vanadate-sensitive uptake remained unaffected. 4. Efflux experiments revealed that forskolin caused a redistribution of cellular K+ efflux reducing the ratio of basolateral versus apical Rb+ efflux. 5. These results suggest that intracellular cyclic AMP exerts its effects on K+ transport by several mechanisms: an increase in the driving force for K+ efflux due to the depolarization induced by opening of Cl- channels, a stimulation of the basolateral uptake of K+ via the Na(+)-K(+)-Cl(-)-cotransporter, and a decrease of the ratio of basolateral versus apical K+ conductance leading to an enhanced efflux of K+ into the lumen and a reduced K+ efflux to the serosal compartment.

Animals↗
PubMed1996

Identification of cyclic AMP phosphodiesterases 3, 4 and 7 in human CD4+ and CD8+ T-lymphocytes: role in regulating proliferation and the biosynthesis of interleukin-2.

1. The cyclic AMP phosphodiesterases (PDE) expressed by CD4+ and CD8+ T-lymphocytes purified from the peripheral blood of normal adult subjects were identified and characterized, and their role in modulating proliferation and the biosynthesis of interleukin (IL)-2 and interferon (IFN)-gamma evaluated. 2. In lysates prepared from both subsets, SK&F 95654 (PDE3 inhibitor) and rolipram (PDE4 inhibitor) suppressed cyclic AMP hydrolysis indicating the presence of PDE3 and PDE4 isoenzymes in these cells. Differential centrifugation and subsequent inhibitor and kinetic studies revealed that the particulate fraction contained, predominantly, a PDE3 isoenzyme. In contrast, the soluble fraction contained a PDE4 (approximately 65% of total activity) and, in addition, a novel enzyme that had the kinetic characteristics of the recently identified PDE7. 3. Reverse transcription-polymerase chain reaction (RT-PCR) studies with primer pairs designed to recognise unique sequences in the human PDE4 and PDE7 genes amplified cDNA fragments that corresponded to the predicted sizes of HSPDE4A, HSPDE4B, HSPDE54D and HSPDE7. No message was detected for HSPDE4C after 35 cycles of amplification. 4. Functionally, rolipram inhibited phytohaemagglutinin- (PHA) and anti-CD3-induced proliferation of CD4+ and CD8+ T-lymphocytes, and the elaboration of IL-2, which was associated with a three to four fold increase in cyclic AMP mass. In all experiments, however, rolipram was approximately 60 fold more potent at suppressing IL-2 synthesis than at inhibiting mitogenesis. In contrast, SK&F 95654 failed to suppress proliferation and cytokine generation, and did not elevate the cyclic AMP content in T-cells. Although inactive alone, SK&F 95654 potentiated the ability of rolipram to suppress PHA- and anti-CD3-induced T-cell proliferation, and PHA-induced IL-2 release. 5. When a combination of phorbol myristate acetate (PMA) and ionomycin were used as a co-mitogen, rolipram did not affect proliferation but, paradoxically, suppressed IL-2 release indicating that cyclic AMP can inhibit mitogenesis by acting at, or proximal to, the level of inositol phospholipid hydrolysis. 6. Collectively, these data suggest that PDE3 and PDE4 isoenzymes regulate the cyclic AMP content, IL-2 biosynthesis and proliferation in human CD4+ and CD8+ T-lymphocytes. However, the ability of rolipram to suppress markedly mitogen-induced IL-2 generation without affecting T-cell proliferation suggests that growth and division of T-lymphocytes may be governed by mediators in addition to IL-2. Finally, T-cells have the potential to express PDE7, although elucidating the functional role of this enzyme must await the development of selective inhibitors.

Cell Division↗
PubMed1980

Effect of chlorpromazine on ion transport induced by cholera toxin, cyclic AMP and cyclic GMP in isolated mucosa from hen intestine.

The isolated short circuit mucosa of chicken colon was established as an in vitro model for studies of the pathophysiology of diarrhoea and the mechanism of action of antidiarrhoeic drugs. Cholera toxin, 10(-7) M, added to the mucosal aide of the preparation, caused in a delayed reaction a pronounced increase of short circuit current (Isc). Cyclic AMP, which mediates the effect of cholera toxin (when added serosal) induced an immediate rise of Isc. Half maximal reaction was achieved at 3 mM cyclic AMP and maximal at 7 mM. The increase of Isc corresponded to the increase in the flux of chloride from serosa to mucosa. Unlike cyclic AMP, cyclic GMP almost equally stimulated sodium and chloride transport from serosa to mucosa. Unlike cyclic AMP, cyclic GMP almost equally stimulated sodium and chloride transport from serosa to mucosa, while the effect on Isc of the two nucleotides was additive. Chlorpromazine, which effectively reverses diarrhoea in cholera patients, totally normalized Isc after treatment with either cholera toxin, cyclic AMP or cyclic GMP. This reduction was achieved by a specific stimulation of transport of chloride from mucosa to serosa. The effect occurred also without previous treatment of the tissue with secretagogues (cholera toxin, cyclic nucleotides). No change in mucosal resistance was induced by chlorpromazine or cyclic GMP while it was reduced by cyclic AMP.

Animals↗
PubMed1983

Cyclic AMP in urine, kidney and liver following long-term administration of fluoride to rats.

Rats were exposed to fluoride (F) for up to 8 weeks through the drinking water containing 100 p.p.m. F. Urine samples were regularly collected, together with tissue samples of liver and kidney at termination. All samples were assayed for cyclic AMP. No F-effect was found on liver or kidney levels of cyclic AMP. In the urine from the F-exposed rats there was an overall increase in the cyclic AMP concentration, but a decreased or unaffected 24 hrs urinary excretion of cyclic AMP. However, the F-exposed rats exhibited a considerably lower diuresis than the control animals. Because a significant negative correlation was found between diuresis and urinary concentration of cyclic AMP, the material was corrected to similarity in the mean diuresis between the two groups. Then no effect of F-exposure could be detected either in the urinary concentration or the daily excretion of cyclic AMP.

Animals↗
PubMed1995

Role of cyclic AMP in stimulation-evoked release of 3H-noradrenaline from rabbit isolated ear artery.

The possible involvement of cyclic AMP in the stimulation-evoked 3H-overflow from rabbit isolated ear artery preloaded with 3H-noradrenaline was studied. Cyclic AMP (10(-5)-3 x 10(-4)M), 8-bromo-cyclic AMP (3 x 10(-4)M) and adenosine (10(-5)-3 x 10(-4)M) enhanced stimulation-evoked 3H-overflow. Dibutyryl-cyclic AMP (10(-5)-3 x 10(-4)M) had no effect. Theophylline (3 x 10(-5)M) and the phosphodiesterase inhibitor AH 21-132 (3 x 10(-5)M) did not alter the enhancement of 3H-overflow caused by either cyclic AMP or adenosine. Forskolin (3 x 10(-6)M) and the phosphodiesterase inhibitors ICI 63 197 (10(-4)M) and AH 21-132 (3 x 10(-6)-3 x 10(-5)M) increased stimulation-evoked 3H-overflow. Forskolin (10(-6)M) enhanced the effect of ICI 63 197 (3 x 10(-5)M) but it did not alter the effect of AH 21-132. It is concluded that cyclic AMP is involved in the stimulation-evoked release of noradrenaline from postganglionic sympathetic nerves in the rabbit ear artery.

Adenosine↗
PubMed1977

Effect of human leucocyte migration inhibitory factor (LIF) on 3', 5'-cyclic AMP levels of peripheral blood leucocytes.

The possible involvement of 3', 5'-cyclic AMP (cyclic AMP) in the mechanism of action of leucocyte migration inhbitory factory (LIF) was tested. LIF-treated human peripheral blood leucocytes were incubated at 37 degrees C for various times between 0 and 22 h. The concentrations of cyclic AMP in these cultures did not differ from those in controls. Furthermore, LIF did not affect the cellular release of cyclic AMP. Neither when testing purified neutrophils, which are the prime targets of LIF action, an effect of LIF was found. Cyclic AMP levels decreased with time of incubation, whether testing mononuclear cells (92 per cent lymphocytes), purified neutrophils (98 per cent), or buffy coat cells (72 per cent neutrophils; 22 per cent lymphocytes). However, a transient and as yet unexplained increase in the cyclic AMP levels of the latter mixed population of cells was manifested within 10 to 30 min of incubation.

Cells, Cultured↗
PubMed1975

Characterization of the metabolism of exogenous cyclic AMP by perfused rat heart and incubated prepubertal rat ovary.

In order to study the metabolism of extracellular 3',5'-adenosine monophosphate (cAMP), rat hearts were perfused and prepubertal rat ovaries incubated with 3H- and 32P-labelled cAMP (0.025-1 muM). The rate of disappearance of cAMP from the medium was determined by "Ba-Zn-precipitation" and degradation products of 3H- and 32P-CAMP by paper chromatography. Both tissues degraded cAMP to 5'-adenosine monophosphate (AMP), but the enzyme kinetic for this phosphodiesterase activity was different (apparent Km value for the heart 3.95 muM and for the ovary 0.2 muM). AMP was further degraded, since also other labelled substances were found in the medium. An uptake of both 3H- and 32P-labelled substance(s) into the heart and the ovary was noticed. Tissue extracts contained several labelled purines, but the amounts of labelled cAMP did not exceed expected amounts in the extracellular space. In the ovary the uptake of cAMP and AMP seemed to be low, since the uptake of labelled substances was inhibited by high concentrations of unlabelled AMP or adenosine. The degradation of 32P-cAMP was unchanged when AMP was present, strongly suggesting that the phosphodiesterase enzyme was acting extracellularly. In the heart added AMP was very rapidly degraded making it impossible to elucidate whether cAMP was degraded extracellularly or not. It is concluded that elimination of extracellular cAMP under physiological conditions can be due to degradation of cAMP by various tissues. At least for the ovary this phosphodiesterase enzyme is extracellularly active.

Adenine↗