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PubMed1986

The phorbol ester TPA inhibits cyclic AMP phosphodiesterase activity in intact hepatocytes.

Treatment of intact hepatocytes with the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) potentiated the ability of glucagon to increase intracellular cyclic AMP concentrations. This effect was dose-dependent upon TPA, exhibiting an EC50 of 0.39 ng/ml and such activation was observed at both saturating and sub-saturating concentrations of glucagon. However, this stimulatory effect of TPA was completely abolished by the presence of the cyclic AMP phosphodiesterase inhibitor 1-isobutyl-3-methylxanthine, when TPA now inhibited the glucagon-stimulated increase in intracellular cyclic AMP concentrations. It is suggested that, as well as inhibiting glucagon-stimulated adenylate cyclase activity, TPA also inhibits cyclic AMP phosphodiesterase activity in intact hepatocytes. Treatment of either hepatocyte homogenates or purified cyclic AMP phosphodiesterase with TPA failed to show any direct inhibitory effect of TPA on activity showing that TPA did not exert any direct inhibitory action on phosphodiesterase activity. However, homogenates made from hepatocytes that had been pre-treated with TPA did show a reduced cyclic AMP phosphodiesterase activity. It is suggested that TPA might inhibit cyclic AMP phosphodiesterase activity through phosphorylation by C-kinase.

1-Methyl-3-isobutylxanthine↗
PubMed1979

Cyclic 3',5'-AMP phosphodiesterase in Physarum polycephalum. I. Chemotaxis toward inhibitors and cyclic nucleotides.

The effect of several inhibitors of the enzyme cyclic 3',5'-AMP phosphodiesterase as chemoattractants in Physarum polycephalum was examined. Of the compounds tested, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Roche 20-1724/001) and 1-ethyl-4-(isopropylidinehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid ethyl ester, hydrochloride (Squibb 20009) were the most potent attractants. 3-Isobutyl-1-methyl xanthine, theophylline, and morin (a flavanoid) were moderate attractants and sometimes gave negative chemotaxis at high concentrations. Cyclic 3',5'-AMP was an effective, but not potent attractant. A repellent effect following the positive chemotactic action was sometimes observed with cyclic 3',5'-AMP at concentrations as high as 1 . 10(-2) M. Dibutyryl cyclic AMP appeared to be a somewhat more potent attractant than cyclic 3',5'-AMP. The 8-thiomethyl and 8-bromoderivatives of cyclic AMP, which are poorly hydrolyzed by the phosphodiesterase, were not attractants in Physarum. Possible participation of cyclic 3',5'-AMP in the directional movement in P. polycephalum is discussed.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1990

19F-n.m.r. studies of ligand binding to 5-fluorotryptophan- and 3-fluorotyrosine-containing cyclic AMP receptor protein from Escherichia coli.

Two fluorine-containing analogues of the cyclic AMP receptor protein (CRP) from Escherichia coli were prepared by biosynthetic incorporation of 5-fluorotryptophan (5-F-Trp) and 3-fluorotyrosine (3-F-Tyr). The 19F-n.m.r. spectrum of the [5-F-Trp]CRP showed two signals corresponding to the two tryptophan residues, and that of the [3-F-Tyr]CRP showed six signals (two overlapping) corresponding to the six tyrosine residues: these results are as expected for a symmetrical dimer. A comparison of the 19F-n.m.r. spectra of the CRP analogues in the presence and in the absence of cyclic AMP reveals that the chemical shifts of both tryptophan residues and of two of the six tyrosine residues show differences. Since none of these residues is in direct contact with the bound nucleotide (although Trp-85 is fairly close), these shift changes must arise from induced conformational effects. The 19F-n.m.r. spectra of complexes with cyclic GMP showed chemical-shift perturbations different from those caused by cyclic AMP, indicating that different conformational changes are induced by the binding of cyclic GMP. The 19F-n.m.r. spectrum of the complex of [3-F-Tyr]CRP with tubercidin 3',5'-(cyclic)monophosphate (which can activate transcription) showed essentially the same chemical-shift changes as seen for the cyclic AMP complex, indicating that similar conformational changes have been induced by the nucleotide binding. [3-F-Tyr]CRP in the presence of an equimolar amount of the 20 bp self-complementary DNA oligomer 5'-AATGTGAGTTAACTCACATT-3' and excess cyclic AMP gave an 19F-n.m.r. spectrum that was almost identical with that for the [3-F-Tyr]CRP-cyclic AMP complex, indicating that the binding of DNA does not induce significant conformational changes involving the tyrosine residues. Proteolysis of [3-F-Tyr]CRP with chymotrypsin produced a 31 kDa fragment that is a dimer containing the cyclic AMP-binding domain. This fragment contains five of the six tyrosine residues, and its 19F-n.m.r. chemical shifts were essentially the same as those of the intact protein except for one missing signal (signal F): this signal could be assigned to Tyr-206 and shown to be unperturbed by the binding of cyclic nucleotide to the intact [3-F-Tyr]CRP. The similarity of the 19F-n.m.r. chemical shifts in the alpha-fragment and the intact CRP indicates that the alpha-fragment retains the same structure as found in the intact protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Cyclic AMP↗
PubMed1994

Bradykinin-dependent activation of adenylate cyclase activity and cyclic AMP accumulation in tracheal smooth muscle occurs via protein kinase C-dependent and -independent pathways.

Treatment of cultured tracheal smooth-muscle cells (TSM) with phorbol 12-myristate 13-acetate (PMA) (100 nM) or bradykinin (100 nM) elicited enhanced basal and guanosine 5'-[beta gamma-imido]-triphosphate-stimulated adenylate cyclase activities in subsequently isolated membranes. Combined stimulation of cells was non-additive, indicating that both agents activate adenylate cyclase via similar routes. Both PMA (100 nM) and bradykinin (100 nM) allowed the alpha subunit of Gs to act as a more favourable substrate for its cholera-toxin-catalysed ADP-ribosylation in vitro. PMA was without effect on intracellular cyclic AMP in control cells. However, constitutive activation of Gs by treatment in vivo with cholera toxin (0.5 ng/ml, 18 h) sensitized the cells to PMA stimulation, resulting in a concentration-dependent increase in intracellular cyclic AMP accumulation (EC50 = 7.3 +/- 2.5 nM, n = 5). Bradykinin also elicited a concentration-dependent increase in intracellular cyclic AMP (EC50 = 63.3 +/- 14.5 nM, n = 3). Constitutive activation of Gs resulted in an increased maximal response (10-fold) and potency (EC50 = 6.17 +/- 1.6 nM, n = 3) to bradykinin. This response was not affected by the B2-receptor antagonist, NPC567 [which selectively blocks bradykinin-stimulated phospholipase C (PLC), with minor activity against phospholipase D (PLD) activity]. Des-Arg9-bradykinin (a B1-receptor agonist) was without activity. These results suggest that the receptor sub-type capable of activating PLD may also be stimulatory for cyclic AMP accumulation. Furthermore, pre-treatment of the cells with butan-l-ol (0.3%, v/v), which traps phosphatidate derived from PLD reactions, blocked the bradykinin-stimulated increase in intracellular cyclic AMP. These studies suggest that there may be a causal link between PLD-derived phosphatidate and the positive modulation of adenylate cyclase activity. In support of this, the concentration-dependence for bradykinin-stimulated adenylate cyclase activity was identical with that of bradykinin-stimulated phospholipase D activity (EC50 = 5 nM). Bradykinin, but not PMA, was also capable of eliciting the inhibition of cyclic AMP phosphodiesterase activity in TSM cells (EC50 > 100 nM) via an unidentified mechanism. These studies indicate that cross-regulation between the cyclic AMP pathway and phospholipid-derived second messengers in TSM cells does not occur as a consequence of PLC-catalysed PtdIns(4,5)P2 hydrolysis, but may involve, in part, PLD-catalysed phosphatidylcholine hydrolysis.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1996

Diminished noradrenergic stimulation reduces the activity of rolipram-sensitive, high-affinity cyclic AMP phosphodiesterase in rat cerebral cortex.

The present study examined the in vivo regulation of rolipram-sensitive, high-affinity cyclic AMP phosphodiesterase (PDE4) in rat cerebral cortex. The hydrolysis of cyclic AMP, formed by stimulation of beta-adrenergic receptors, was measured in cerebral cortical slices. Hydrolysis of cyclic AMP formed under these conditions was inhibited by the PDE4-selective inhibitor rolipram but not by selective inhibitors of other PDE families. Intraventricular infusion of 6-hydroxydopamine (6-OHDA; 200 micrograms) decreased the rate constant of cyclic AMP hydrolysis and increased the cyclic AMP half-life 17 days, but not 1 or 7 days, following the treatment. A reduction in norepinephrine (NE) content occurred first; the NE level was reduced to 42, 24, and 6% of control at 1, 7, and 17 days after 6-OHDA infusion, respectively. This was followed by the development of supersensitivity of beta-adrenergic receptor-linked adenylyl cyclase, which occurred 7 days after the infusion. The reduction in PDE4 activity occurred last. When a higher dose of 6-OHDA (300 micrograms) was used, the reduction in the rate constant of cyclic AMP hydrolysis occurred by 7 days; at this time NE content was depleted to 6% of control. Similar to 6-OHDA treatment, continuous blockade of beta-adrenergic receptors, produced by chronic propranolol infusion, decreased the rate constant of cyclic AMP hydrolysis. Therefore, the current results indicate that diminished stimulation of beta-adrenergic receptors, either by loss of noradrenergic innervation or by receptor blockade, reduces the activity of PDE4. This suggests that PDE4 regulation may contribute in the homeostasis of the noradrenergic receptor-effector system in the brain.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1976

Extracellular cyclic AMP-phosphodiesterase accelerates differentiation in Dictyostelium discoideum.

Extracellular cyclic AMP-phosphodiesterase accelerates the development of aggregation competence in Dictyostelium discoideum when present during the preaggregation stage. The effect on development appears to depend only on hydrolysis of extracellular cyclic AMP and not on other properties of the phosphodiesterase molecule. Extracellular cyclic AMP-phosphodiesterase, as a promoter of differentiation, acts mainly throughout the first half of interphase. Our evidence supports the proposal that cyclic AMP oscillations control the rate and possibly the initiation of development. Since extracellular cyclic AMP-phosphodiesterase acts from the beginning of interphase cyclic AMP oscillations may also occur from early interphase, at least in the presence of this enzyme. This would imply that the cyclic AMP oscillator is a determinant, but not a product, of the developmental programme.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

Altered cyclic-AMP receptor activity and morphogenesis in a chemosensory mutant of Dictyostelium discoideum.

The functional properties of the cell-surface cyclic-AMP receptor that controls chemotaxis were found to be altered in an aggregation mutant of Dictyostelium discoideum. The mutant aggregated without stream formation and had a tenfold increased cell-density requirement for the initiation of aggregation. After aggregation, mounds formed multiple tips and subsequently subdivided to give multiple fruits that were small and abnormally proportioned. Cyclic-AMP-induced light-scattering changes in cell suspensions indicated that the mutant had a diminished response to external cyclic-AMP signals. Associated with these altered functional responses was a physical change in the cyclic-AMP sensory system. Cyclic-AMP-binding studies showed that the parent had two classes of cyclic-AMP binding sites, i.e., Kd = 32 and 110 nM. In contrast, the mutant had two- to threefold or more high-affinity sites (Kd = 25 nM) and altered low-affinity sites (Kd less than 3 microM). These results indicate that both affinity classes of binding site are independently mutable. This observation suggests that the two affinity classes can be interconverted by mutation, or the mutation alters a single molecular species and its equilibrium between binding sites with different affinities for cyclic AMP, as postulated in receptor cycling models.

Cyclic AMP↗
PubMed1981

Inhibitory effect of prostaglandins on the stimulation by glucagon and adrenaline of formation of cyclic AMP in rat hepatocytes.

Several prostaglandins were found to inhibit hormone-induced cyclic AMP accumulation in suspensions of intact rat hepatocytes. Prostaglandin E1 in concentrations of 0.05--25 micrometers inhibited the cyclic AMP response to glucagon. Maximal inhibition was about 50%. The effect was rapid, being evident within 30 s. Prostaglandins E2, F1 alpha, F2 alpha, A1 and A2 also inhibited the glucagon effect on cyclic AMP in hepatocytes. In cells made highly responsive to adrenaline, by pretreatment of the animals with the carcinogen 2-acetylaminofluorene, and inhibitory effect of prostaglandin E1 was seen also on adrenaline-induced cyclic AMP accumulation. The mechanism of the inhibitory effect of prostaglandins on hormone-stimulated cyclic AMP accumulation was not clarified. Prostaglandin E1 did not inhibit glucagon binding to intact hepatocytes, and so far we have not been able to demonstrate any effect of the prostaglandins on the adenylate cyclase or phosphodiesterase in broken cell preparations. It is concluded that while several previous studies have shown that stimulatory effects of prostaglandins on cyclic AMP are only marginal or lacking in parenchymal liver cells the present data indicate that several prostaglandins exert strong inhibitory interference with hormone-induced cyclic AMP accumulation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

Purification of the AMP-activated protein kinase on ATP-gamma-sepharose and analysis of its subunit structure.

The AMP-activated protein kinase has been purified by affinity chromatography on ATP-gamma-Sepharose. A proportion of the activity can be eluted using AMP, while the remainder is eluted using ATP. The AMP eluate contains three polypeptides of 63, 38 and 35 kDa (p63, p38 and p35) in a molar ratio (by Coomassie blue binding) close to 1:1:1. p63 was previously identified as the AMP-binding catalytic subunit [Carling, D., Clarke, P. R., Zammit, V. A. & Hardie, D. G. (1989) Eur. J. Biochem. 186, 129-136]. All three polypeptides exactly comigrate both on native gel electrophoresis and on gel filtration, suggesting that p38 and p35 are additional subunits. Estimation of Stokes radius (5.4-5.8 nm) by gel filtration, and sedimentation coefficient (7.9-8.4 S) by glycerol gradient centrifugation, suggest that the kinase has an asymmetric structure with a native molecular mass for the complex of 190 +/- 10 kDa. Thus the native enzyme appears to be a heterotrimer with a p63/p38/p35 (1:1:1) structure. Despite the fact that the ATP eluate has a higher specific activity than the AMP eluate (3.5 +/- 0.2 vs 2.3 +/- 0.2 mumol.min-1.mg-1), it appears to be less pure, containing p63, p38 and p35 plus other polypeptides. Experiments examining the effects of protein phosphatase-2A and kinase kinase, and analysis by Western blotting with anti-p63 antibody, suggests that the AMP eluate is entirely in the low-activity dephosphorylated form, while the ATP eluate is a mixture of that form and the high-activity phosphorylated form. As well as establishing the subunit structure of the AMP-activated protein kinase, these results suggest that the kinase can bind to ATP-gamma-Sepharose through either the allosteric (AMP/ATP) site or the catalytic (ATP) site, and that phosphorylation by the kinase kinase increases the affinity for the latter site.

AMP-Activated Protein Kinase Kinases↗
PubMed1989

Effect of isozyme-selective inhibitors of phosphodiesterase on histamine-stimulated cyclic AMP accumulation in guinea-pig hippocampus.

Addition of histamine (0.1 mM) to guinea-pig hippocampal slices causes a 20- to 30-fold increase in the accumulation of cyclic AMP compared with basal levels. This accumulation represents a balance between cyclic AMP production by adenylate cyclase and cyclic AMP breakdown mediated by phosphodiesterase (PDE). However, brain tissues are known to contain several different PDE isozymes. To determine which are involved in this response to histamine, the effect of isozyme-specific PDE inhibitors on cyclic AMP accumulation was examined in the hippocampus. MB 22948 (0.1 mM), an inhibitor of PDEs I and II, had no significant effect on the response to either 1 microM or 0.1 mM histamine. SKF 94120 (0.1 mM), a PDE III inhibitor, was also without effect in the presence of 1 microM histamine, although with 0.1 mM histamine, it caused a weak (1.25-fold compared with control), but statistically significant, enhancement of cyclic AMP accumulation. However, both rolipram (0.1 mM), a PDE IV inhibitor, and 3-isobutyl-1-methylxanthine (0.1 or 1 mM), an inhibitor of all forms of PDE, significantly increased cyclic AMP accumulation (2.8- to 6.5-fold compared with controls), and the relative size of this effect decreased with increasing histamine concentration. It is concluded that PDE IV is the main PDE isozyme involved in cyclic AMP turnover in guinea-pig hippocampal slices responding to histamine.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Hypoxia increases the cyclic AMP content of the cat carotid body in vitro.

The cyclic AMP content of cat carotid bodies in vitro measured with a radioimmunoassay under control conditions (PO2: 230 torr) was 0.79 +/- 0.10 pmol/carotid body (n = 10). Lowering medium PO2 to 20 torr for 2 min significantly increased cyclic AMP content to 1.13 +/- 0.14 pmol/carotid body (n = 10). This increase was inhibited neither by propranolol (34 microM) nor by propranolol plus haloperidol (27 microM). Inhibition of the cyclic nucleotide phosphodiesterase with 1-methyl-3-isobutylxanthine (0.8 mM) provoked a fast and large increase in cyclic AMP during both control and hypoxic conditions. The cyclic AMP increase induced by hypoxia was still observed when extracellular Ca2+ was absent. Inhibition of the adenylate cyclase by N-(cis-2-phenylcyclopentyl)azacyclotridecan-2-imine hydrochloride (MDL 12330A; 20-1,000 microM) under zero-Ca2+ conditions irreversibly inhibited the cyclic AMP increase produced by hypoxia. Similarly, inhibition of the Ca2(+)-calmodulin complex by trifluoperazine (0.2 mM) or calmidazolium (R 24571; 50-200 microM) prevented the cyclic AMP response. These results suggest that cyclic AMP may be involved in the PO2-sensing mechanism of the carotid body. Hypoxia appears to activate adenylate cyclase directly and independent of any hormone-receptor interactions.

1-Methyl-3-isobutylxanthine↗
PubMed1985

Contraction and cyclic AMP-related relaxation of the intimal and medial smooth muscle layers of pig thoracic aorta.

The contractile responses of an alpha-adrenoceptor agonist, phenylephrine, and of histamine were compared in the intimal and medial smooth muscle layers of the pig aortic arch. Further, the relaxant effects evoked by some compounds influencing the cyclic AMP system were compared in the two muscle layers, as well as their effects on the cyclic AMP content and phosphodiesterase activity. Phenylephrine and histamine induced contraction of the smooth muscle layers. The increase in tension was faster in the intimal than in the medial layer. The alpha-adrenoceptor agonist phenylephrine was a more potent contractile agent in the intimal than in the medial smooth muscle. With histamine, no significant difference in the dose-response curves between the two muscle layers was found. Histamine-contracted muscle preparations were relaxed in a dose-dependent manner by the phosphodiesterase-inhibiting compound 3-isobutyl-1-methylaxanthine (MIX) and by 8-bromo-cyclic AMP. The two substances were more potent relaxants in the medial than in the intimal smooth muscle layer. The content of cyclic AMP in the intimal and the medial smooth muscle was increased by MIX. Isoprenaline had no relaxing effect on the muscle preparations and did not change the content of cyclic AMP. There were no differences in the basal levels of cyclic AMP in the intima and media. Vmax of phosphodiesterase activities differed, however, between the two preparations. This study demonstrates that the intimal layer is characterized by a larger contractile responsiveness to phenylephrine and a lower relaxant response to compounds influencing the cyclic AMP-system than those of the medial layer.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1980

Mannose utilization in Escherichia coli requires cyclic AMP but not an exogenous inducer.

It has been clarified whether the utilization of mannose by Escherichia coli requires adenosine 3',5'-cyclic monophosphate (cyclic AMP). Using an adenylyl cyclase deficient mutant (CA8306B) and a cyclic AMP receptor protein (CRP) deficient mutant (5333B) we have shown that the utilization of mannose is dependent on the cyclic AMP - CRP complex. 2-Deoxyglucose (DG) is a nonmetabolizable glucose analog specific for the phosphotransferase system (PTS) which transports mannose (termed here PTSM). Growth of CA8306B on glycerol is unaffected by addition of the analog, whereas growth of the strain on glycerol plus cyclic AMP ceases immediately upon addition of DG. These results suggest that the formation of PTSM is dependent on cyclic AMP. In addition, CA8306B grown on glycerol plus cyclic AMP can immediately utilize mannose when transferred to a medium containing mannose as a sole carbon source, whereas the same strain grown on glycerol without cyclic AMP cannot utilize mannose when so transferred. The results suggest that the formation of PTSM does not require an exogenous inducer.

Cyclic AMP↗
PubMed1975

Uncoupling of lipolysis from cyclic AMP by procaine: a tool for studying the mechanism of action of antilipolytic agents.

The initial rate of net glycerol release in norepinephrine-stimulated adipose tissue fragments was inhibited (40-78%) by procaine-HCl (1-5mM), whereas basal (unstimulated) lipolysis was unaffected. A dose-related inhibition of norepinephrine-induced lipolysis by procaine-HCl (0.1-1 mM) also occurred in adipocytes. Procaine-induced antilipolysis was associated with an augmented rather than a reduced hormone-stimulated increment in intracellular cyclic AMP. The dissociation of lipolysis from cyclic AMP accumulation has been termed the uncoupling effect of procaine. This effect of procaine was employed to define the precise mechanism of action of the antilipolytic drug clofibrate (Atromid-S) which inhibits lipolysis by reducing cyclic AMP. A reduction in cyclic AMP by clofibrate was demonstrated in norepinephrine-stimulated cells exposed to procaine (uncoupled system). Thus, the inhibitory effect of clofibrate on cyclic AMP could not be attributed to accumulation of products of lipolysis. Because neither procaine-HCl nor clofibrate had any effect on the low Km 3':5'-cyclic-AMP phosphodiesterase (EC 3.1.4.17) activity in hormone stimulated cells, the clofibrate-induced reduction in cyclic AMP was attributed to its direct action on adipocyte adenylate cyclase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1984

Dibutyryl cyclic AMP increases phosphodiesterase activity in the rat heart.

The influence of increasing the in vivo concentration of cyclic AMP on the activity of cyclic nucleotide phosphodiesterase (PDE) in rat heart was investigated. One, three, and five hourly injections of 5.0 mg dibutyryl (Bt2) cyclic AMP significantly increased the activity of PDE in the supernatant fraction of rat heart using 1.0 microM cyclic AMP as the assay substrate concentration. When 100 microM cyclic AMP was used in the assay reaction, increases in enzymes activity were seen following five and eight nucleotide injections. The nucleotide-induced increase in PDE activity was dose dependent. When the five-injection protocol was used, PDE activity remained elevated for at least 4 h, while activity had returned to control levels within this time when two hourly injections were used. The nucleotide stimulation of PDE activity was blocked by cycloheximide. Five hourly infections of Bt2 cyclic AMP increased PDE activity in the liver and fast-twitch red muscle. A reduction in PDE activity in fast-twitch white muscle was seen following nucleotide injections. These findings are consistent with the hypothesis that prolonged elevations in the intracellular concentration of cyclic AMP cause an elevation in myocardial PDE activity. The increased activity seems to be the result of protein synthesis. These data suggest that cyclic AMP contributes significantly in regulating its own metabolism in the rat heart.

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

Cyclic AMP-adenosine pathway induces nitric oxide synthesis in aortic smooth muscle cells.

The main purpose of this investigation was to evaluate whether the cyclic AMP-adenosine pathway, ie, the conversion of cAMP to AMP and, hence, to adenosine, is involved in the regulation of nitric oxide (NO) synthesis by vascular smooth muscle cells (SMCs). Treatment of confluent monolayers of SMCs with adenosine, 2-chloroadenosine (stable analog of adenosine), and agents that elevate endogenous (SMC-derived) adenosine (EHNA and iodotubericidin) increased nitrite/nitrate (stable metabolites of NO) levels in the medium and enhanced the conversion of 3H-L-arginine to 3H-L-citrulline by cytosolic extracts obtained from the pretreated SMCs. The stimulatory effects of adenosine were not mimicked by low (1 to 100 nmol/L) concentrations of CGS21680, an A2A receptor agonist, or CPA, a selective A1 receptor agonist. The stimulatory effects of 2-chloroadenosine and EHNA plus iodotubericidin were significantly inhibited by KF17837, a selective A2 receptor antagonist, and by DPSPX, an A1/A2 receptor antagonist, but not by DPCPX, a selective A1 receptor antagonist. DDA (adenylyl cyclase inhibitor) and Rp-cyclic AMP (protein kinase A inhibitor) did not block the effects of adenosine on NO synthesis. Incubation of SMCs with exogenous cyclic AMP, at concentrations previously shown to elevate levels ofadenosine in the medium, also increased nitrite/nitrate levels and 3H-L-citrulline formation, and the effects of cyclic AMP on NO synthesis were blocked by DPSPX and KF17837, but not by DPCPX. These findings provide evidence that exogenous and SMC-derived adenosine induce NO synthesis via A2B receptors linked to a pathway not involving adenylyl cyclase/protein kinase A. Moreover, extracellular cyclic AMP induces NO synthesis via conversion to adenosine and activation of A2B adenosine receptors. The cyclic AMP-adenosine pathway may be importantly involved in the vascular production of NO.

2-Chloroadenosine↗
PubMed1975

Effect of acute metabolic acidosis on vasopressin-dependent cyclic AMP in rat kidney.

Acidic media have been reported to inhibit the hydro-osmotic effect of vasopressin in toad bladders, probably through inhibition of the cyclic AMP system. However, the mechanism of inhibition of the cyclic AMP system is controversial. Therefore, that inhibitory mechanism was further investigated in rat kidneys. The antidiuretic response to vasopression was significantly inhibited in animals with metabolic acidosis. The inhibition of the antidiuretic response was associated with a smaller than normal increase of urinary excretion of cyclic AMP after the iv injection of vasopressin. In in vitro experiments, both the increase of cyclic AMP concentration in renal medullary slices and the activation of adenylate cyclase in medulla by vasopressin were significantly less in acidic than in control media. These findings suggest that medabolic acidosis inhibits the antidiuretic effect of vasopressin by inhibiting the vasopressin-dependent cyclic AMP system in the kidney. Acidic media also inhibited cyclic AMP-phosphodiesterase. These dual effects of acidosis on adenylate cyclase and cyclic AMP-phosphodiesterase may explain the conflicting findings observed in the experiments on toad bladders.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1975

Effects of lithium on vasopressin-dependent cyclic AMP in rat renal medulla.

A vasopressin resistant urinary concentrating defect has been described in patients receiving lithium salt for affective disorders. For the pathogenic mechanism of the concentrating defect it has been postulated that lithium inhibits the vasopressin-dependent cyclic AMP system. However, the results of indirect studies on the lithium effect are equivocal. Therefore, the effect of lithium specifically on the vasopressin-dependent cyclic AMP system was investigated in rat renal medulla. The increase of cyclic AMP concentration by vasopressin was inhibited by lithium. But lithium had no effect on the PTH-dependent cyclic AMP concentration in renal cortical slices. Regardless of magnesium concentrations from 0-10 mM in the incubation media, 10 mM lithium had no moeasurable effect on the vasopressin-dependent adenylate cyclase of rat renal medulla. However, 10 mM lithium augmented the cyclic AMP-phosphodiesterase activity in renal medulla in the high Km system. These results suggest that lithium inhibits the vasopressin-dependent cyclic AMP concentration in renal medulla via the augmentation of its catabolism, rather than via the inhibition of cyclic AMP generation.

3',5'-Cyclic-AMP Phosphodiesterases↗