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PubMed1987

NMR studies of the AMP-binding site and mechanism of adenylate kinase.

NMR has previously been used to determine the conformation of enzyme-bound MgATP and to locate the MgATP-binding site on adenylate kinase [Fry, D. C., Kuby, S. A., & Mildvan, A. S. (1985) Biochemistry 24, 4680-4694]. To determine the conformation and location of the other substrate, AMP, distances have been measured from Cr3+AMPPCP, a linear competitive inhibitor with respect to MgATP, to six protons and to the phosphorus atom of AMP on adenylate kinase, with the paramagnetic probe-T1 method. Time-dependent nuclear Overhauser effects (NOEs) have been used to measure five interproton distances on enzyme-bound AMP. These distances were used to determine the conformation of bound AMP in addition to its position with respect to metal-ATP. Enzyme-bound AMP exhibits a high anti-glycosyl torsional angle (chi = 110 +/- 10 degrees), a 3'-endo,2'-exo ribose pucker (delta = 105 +/- 10 degrees), and gauche-trans orientations about the C4'-C5' bond (gamma = 180 +/- 10 degrees) and the C5'-O5' bond (beta = 170 +/- 20 degrees). The distance from Cr3+ to the phosphorus of AMP is 5.9 +/- 0.3 A, indicating a reaction coordinate distance of approximately 3 A, which is consistent with an associative SN2 mechanism for the phosphoryl transfer. Ten intermolecular NOEs, from protons of the enzyme to those of AMP, were detected, indicating the proximity of at least three hydrophobic amino acids to bound AMP. These constraints, together with the conformation of AMP and the intersubstrate distances, were used to position AMP into the X-ray structure of adenylate kinase. The AMP binding site is found to be near (less than or equal to 4 A from) Leu-116, Arg-171, Val-173, Val-182, and Leu-190; all of these residues have been found to be invariant in muscle-type rabbit, calf, human, porcine [Kuby, S. A., Palmieri, R. H., Frischat, A., Fischer, A. H., Wu, L. H., Maland, L., & Manship, M. (1984) Biochemistry 23, 2393-2399], and chicken adenylate kinase [Kishi, F., Maruyama, M., Tanizawa, Y., & Nakazawa, A. (1986) J. Biol. Chem. 261, 2942-2945].

Adenosine Monophosphate↗
PubMed1997

Importance of the dimer-dimer interface for allosteric signal transduction and AMP cooperativity of pig kidney fructose-1,6-bisphosphatase. Site-specific mutagenesis studies of Glu-192 and Asp-187 residues on the 190's loop.

The role of the 190's loop of fructose-1,6-bisphosphatase (Fru-1, 6-P2ase) in the allosteric regulation of Fru-1,6-P2ase has been investigated through kinetic studies on three mutant enzymes, Glu-192 --> Ala, Glu-192 --> Gln, and Asp-187 --> Ala. AMP is an allosteric inhibitor, which binds to the regulatory sites and induces the R- to T-state transition; for wild-type Fru-1,6-P2ase AMP inhibition is cooperative with a Hill coefficient of 2.0. The replacement of Asp-187, which forms an interaction across the C1:C2 monomer-monomer interface, with alanine did not change the catalytic efficiency, and it had no effect on the cooperativity of AMP inhibition; however, the apparent dissociation constant for AMP increased more than 4-fold as compared to the value for the wild-type enzyme. The replacement of Glu-192, which forms interactions across the C1:C4 dimer-dimer interface, with Ala and Gln lowered kcat from 21 s-1 for wild-type enzyme to 15 s-1 and 13 s-1, respectively, for the mutant enzymes, while their respective Km values were not changed. However, these replacements did have dramatic effects on AMP inhibition; first, cooperative AMP inhibition was lost; second, the AMP inhibition was biphasic, which can be interpreted as due to AMP binding to two classes of binding sites. The high affinity class of sites corresponds to the regulatory sites, while the low affinity class of sites may be the active sites. The results reported here, combined with the structural and kinetic results from the Lys-42 --> Ala enzyme, strongly suggest that the C1:C4 dimer-dimer interface, rather than the C1:C2 monomer-monomer interface, is critical for the propagation of the allosteric signal between the AMP sites on different subunits; in addition, cooperative AMP inhibition is essential for the enzyme to be fully inhibited by the binding of AMP to the allosteric site.

Adenosine Monophosphate↗
PubMed2004

Adenosine A1/A2a receptor agonist AMP-579 induces acute and delayed preconditioning against in vivo myocardial stunning.

The purpose of this study was to determine whether the adenosine A1/A2a receptor agonist AMP-579 induces acute and delayed preconditioning against in vivo myocardial stunning. Regional stunning was produced by 15 min of coronary artery occlusion and 3 h of reperfusion (RP) in anesthetized open-chest pigs. In acute protection studies, animals were pretreated with saline, low-dose AMP-579 (15 microg/kg iv bolus 10 min before ischemia), or high-dose AMP-579 (50 microg/kg iv at 14 microg/kg bolus + 1.2 microg.kg(-1).min(-1) for 30 min before coronary occlusion). The delayed preconditioning effects of AMP-579 were evaluated 24 h after administration of saline vehicle or high-dose AMP-579 (50 microg/kg iv). Load-insensitive contractility was assessed by measuring regional preload recruitable stroke work (PRSW) and PRSW area. Acute preconditioning with AMP-579 dose dependently improved regional PRSW: 129 +/- 5 and 100 +/- 2% in high- and low-dose AMP-579 groups, respectively, and 78 +/- 5% in the control group at 3 h of RP. Administration of the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (0.7 mg/kg) blocked the acute protective effect of high-dose AMP-579, indicating that these effects are mediated through A1 receptor activation. Delayed preconditioning with AMP-579 significantly increased recovery of PRSW area: 64 +/- 5 vs. 33 +/- 5% in control at 3 h of RP. In isolated perfused rat heart studies, kinetics of the onset and washout of AMP-579 A1 and A2a receptor-mediated effects were distinct compared with those of other adenosine receptor agonists. The unique nature of the adenosine agonist AMP-579 may play a role in its ability to induce delayed preconditioning against in vivo myocardial stunning.

Acute Disease↗
PubMed1989

Characterization of mitotic motors by their relative sensitivity to AMP-PNP.

The relative sensitivities of the motors for mitotic chromosome movements and saltatory motion were compared using a nonhydrolyzable analog of ATP, AMP-PNP. K+AMP-PNP was microinjected into PtKl cells at the time of nuclear envelope disassembly or at anaphase onset. To produce a dose-response curve for the effect of AMP-PNP on the rate of movement, the intracellular concentration of AMP-PNP in individual cells was measured. The volume injected into each cell was determined by adding dextrans labeled with Lucifer Yellow to the injection buffer, measuring the injected cell's fluorescence intensity, and then comparing the value with the fluorescence intensity of known volumes of Lucifer Yellow dextran solution. AMP-PNP produced a 50% inhibition of spindle elongation at 0.2 mM, of saltatory motion at 0.8 mM, and of chromosome movement at 8.6 mM. Prometaphase chromosome movement and anaphase chromosome-to-pole movement were similarly inhibited by AMP-PNP. Equivalent volumes of injection buffer containing 1% Lucifer Yellow dextran had no effect on chromosome movement, spindle elongation or saltatory motion. Although AMP-PNP occasionally produced shorter anaphase spindles, tubulin immunofluorescence revealed the presence of abundant spindle microtubules. Metaphase cells treated with very high cell concentrations of AMP-PNP had spindles with unusually long astral microtubules; thus microtubules are stabilized rather than broken down by AMP-PNP. In conclusion, spindle elongation is four times more sensitive than saltatory motion to AMP-PNP and 40 times more sensitive than chromosome movement. When these sensitivities to AMP-PNP are considered with the results from other studies, it can be concluded that the molecular motors for spindle elongation, chromosome movement and saltatory motion are different.

Adenosine Triphosphate↗
PubMed2006

Identification of AMP N1-oxide in royal jelly as a component neurotrophic toward cultured rat pheochromocytoma PC12 cells.

An extract of royal jelly (RJ) induced processes from cultured rat pheochromocytoma PC12 cells. Active components were isolated, and identified as adenosine monophosphate (AMP) and AMP N1-oxide. AMP N1-oxide was more than 20 times as active as AMP, judging from the minimal concentration to elicit activity. AMP N1-oxide was thought to be responsible for about half of the process-forming activity of whole RJ. Chemically-synthesized AMP N1-oxide was active similarly to the molecule purified from RJ, confirming AMP N1-oxide as the active entity. AMP N1-oxide also suppressed proliferation of PC12 cells and stimulated expression of neurofilament M, a specific protein of mature neurons, demonstrating the stimulatory activity of AMP N1-oxide to induce neuronal differentiation of PC12 cells. Pharmacological experiments suggested that AMP N1-oxide actions are mediated by adenyl cyclase-coupled adenosine receptors, including A2A. Thus AMP N1-oxide is a key molecule that characterizes RJ, and is not found in natural products other than RJ.

Adenosine Monophosphate↗
PubMed1975

Interactions of ethanol with cyclic AMP.

Ethanol and other alcohols stimulate adenylate cyclase activity in various tissues and potentiate its stimulation by some hormones. This effect, however, usually requires a high alcohol concentration. In some cases, an unknown substance, different from cyclic AMP, was formed from ATP in the presence of an alcohol and mimicked stimulation of adenylate cyclase. Ethanol inhibits phosphodiesterase activity in some tissues. In the brain, only the low affinity enzyme of pons-medulla region is inhibited. ATP levels and ATPase activities are affected by ethanol treatment and this can lead to secondary changes of the cyclic AMP levels. Cyclic AMP levels in the brain and liver are decreased by acute ethanol administration while levels in other organs are unchanged. High doses of ethanol inhibit the postdecapitation-induced rise of cyclic AMP level in the brain while low ethanol doses potentiate the postdecapitation rise of cyclic AMP in the lower brain stem. Chronic ethanol administration increases basal adenylate cyclase activity and cyclic AMP levels, and decreases stimulation of adenylate cyclase by norepinephrine in the brain. In contrast, the stimulation of cyclic AMP formation by norepinephrine and other biogenic amines is increased in the brain of ethanol-withdrawn animals. Chronic administration of ethanol affects also cyclic AMP levels and cyclic AMP formation in some peripheral organs. Cyclic AMP might be involved in ethanol-induced fatty liver, since it activates hepatic lipase and might also participate in the fatty acid oxidation.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1981

Calcium-mediated enhancement of the cyclic AMP response in cultured bone cells.

We have examined the influence of extracellular Ca2+ on cyclic AMP metabolism in an osteoblast-enriched population of bone cells isolated from the calvaria of rat fetuses. The cyclic AMP response to stimulators of cyclic AMP formation (PTH and PGE2), but not basal cyclic AMP levels, increased progressively as the extracellular Ca2+ concentration was raised from 0.2 to 4.0 mM. The response to changes in extracellular Ca2+ were rapid (within 3.5 min), and the level of responsivity that characterized each Ca2+ concentration persisted for at least 6 h when the Ca2+ concentration was kept constant. The effect of Ca2+ spanned the entire time course of PTH action, was not accompanied by altered excretion of cyclic AMP from the cells, and was evident at low as well as at high hormone concentrations. Ca2+ augmented the action of PTH in the presence as well as in the absence of cyclic AMP phosphodiesterase inhibitors, and failed to decrease cyclic AMP phosphodiesterase activity in the short term. Mn2+ and, to a smaller degree, Ba2+ substituted for Ca2+ in promoting the cyclic AMP response to PTH. Verapamil, an inhibitor of Ca2+ penetration, blunted the Ca2+-mediated increments in the cyclic AMP response, and the divalent cation ionophore A23187 enhanced these increments. These results indicate that Ca2+ and other cations are positive effectors of the stimulated cyclic AMP response in isolated bone cells. Accumulation into an as yet unknown cellular compartment may be required for the cation effect. The data are most consistent with enhancement of adenylate cyclase reactivity as the mode of cation action.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1982

Purification, characterization and production of rabbit antibodies to rat liver particulate, high-affinity, cyclic AMP phosphodiesterase.

The cyclic nucleotide phosphodiesterase (EC 3.4.16) activities of a rat liver particulate fraction were analyzed after solubilization by detergent or by freeze-thawing. Analysis of the two extracts by DEAE-cellulose chromatography revealed that they contain different complements of phosphodiesterase activities. The detergent-solubilized extract contained a cyclic GMP phosphodiesterase, a low affinity cyclic nucleotide phosphodiesterase whose hydrolysis of cyclic AMP was activated by cyclic GMP and a high affinity cyclic AMP phosphodiesterase. The freeze-thaw extract contained a cyclic GMP phosphodiesterase and two high affinity cyclic AMP phosphodiesterase, but no low affinity cyclic nucleotide phosphodiesterase. The cyclic AMP phosphodiesterase activities from the freeze-thaw extract and from the detergent extract all had negatively cooperative kinetics. One of the cyclic AMP phosphodiesterases from the freeze-thaw extract (form A) was insensitive to inhibition by cyclic GMP; the other freeze-thaw solubilized cyclic AMP phosphodiesterase (form B) and the detergent-solubilized cyclic AMP phosphodiesterase were strongly inhibited by cyclic GMP. The B enzyme appeared to be converted into the A enzyme when the particulate fraction was stored for prolonged periods at -20 degrees C. The B form was purified extensively, using DEAE-cellulose, a guanine-Sepharose column and gel filtration. The enzyme retained its negatively cooperative kinetics and high affinity for both cyclic AMP and cyclic GMP throughout the purification, although catalytic activity was always much greater for cyclic AMP. Rabbit antiserum was raised against the purified B enzyme and tested via a precipitin reaction against other forms of phosphodiesterase. The antiserum cross-reacted with the A enzyme and the detergent-solubilized cyclic AMP phosphodiesterase from rat liver. It did not react with the calmodulin-activated cyclic GMP phosphodiesterase of rat brain, the soluble low affinity cyclic nucleotide phosphodiesterase of rat liver or a commercial phosphodiesterase preparation from bovine heart. These results suggest a possible interrelationship between the high affinity cyclic nucleotide phosphodiesterase of rat liver.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1999

Isolation and regulation of piglet cardiac AMP deaminase.

The properties of piglet cardiac AMP deaminase were determined and its regulation by pH, phosphate, nucleotides and phosphorylation is described. AMP deaminase purified from the ventricles of newborn piglet hearts displayed hyperbolic kinetics with a Km of 2 mM for 5'-AMP. The enzyme had a pH optimum of 7.0 and was strongly inhibited by inorganic phosphate. ATP decreased the Km of the native enzyme 3-fold, but did not significantly block the inhibitory effects of phosphate. Kinetic parameters were not significantly altered in the presence of adenosine, cyclic AMP and NAD+, whereas, the Km was decreased by 50% in the presence of NADH. Piglet cardiac AMP deaminase was phosphorylated by protein kinase C, resulting in a 2-fold increase in Vmax with no change in Km. However, incubation with cAMP-dependent protein kinase did not affect enzyme kinetics. The 80-85 kD protein subunit of piglet cardiac AMP deaminase immunoreacted with antisera raised against human erythrocyte AMP deaminase, rabbit heart AMP deaminase and human recombinant AMP deaminase 3 (isoform E). These results are discussed in relation to in situ AMP deaminase activity in neonatal piglet heart myocytes.

AMP Deaminase↗
PubMed1999

Differential inhibitory mechanism of cyclic AMP on TNF-alpha and IL-12 synthesis by macrophages exposed to microbial stimuli.

Microbial stimuli such as bacterial lipopolysaccharide (LPS) or glycosylphosphatidylinositol-mucins derived from Trypanosoma cruzi trypomastigotes (tGPI-mucins) are effective stimulators of the synthesis of cytokines by macrophages. Here, we evaluated the ability of cyclic AMP mimetic or elevating agents to modulate TNF-alpha and IL-12 synthesis by murine inflammatory macrophages. Cholera Toxin (ChTx) inhibited tGPI-mucins (2.5 nM) or LPS (100 ng ml(-1)) induced TNF-alpha and IL-12(p40) synthesis in a concentration-dependent manner. Similarly, the cyclic AMP mimetics, 8-bromo cyclic AMP or dibutyryl cyclic AMP, or prostaglandin (PG) E2 inhibited the synthesis of both cytokines by macrophages exposed to microbial stimuli. The protein kinase A inhibitor H-89 partially reversed the inhibitory effects of dibutyryl cyclic AMP and PGE2 on both IL-12(p40) and TNF-alpha synthesis. Pretreatment of macrophages with dibutyryl cyclic AMP or ChTx augmented the synthesis of IL-10 triggered by microbial products. Elevation of cyclic AMP inhibited the synthesis of TNF-alpha, but not IL-12(p40), by inflammatory macrophages from IL-10 knockout mice. Kinetic studies showed that synthesis of both TNF-alpha and IL-10 peaked at 8 h and IL-12 at 24 h after stimulation with microbial stimuli. Together, our findings favour the hypothesis that the cyclic AMP inhibitory activity on IL-12(p40) but not on TNF-alpha synthesis is dependent on de novo protein synthesis, most likely involving IL-10, by macrophages stimulated with microbial products. Accordingly, dibutyryl cyclic AMP inhibited IL-12(p40) synthesis only when added before or at the same time of the stimuli. In contrast, the effect of this cyclic AMP analogue on TNF-alpha synthesis was protracted and observed even 2 h after the addition of the stimuli.

Animals↗
PubMed1981

Cyclic AMP phosphodiesterase activity during differentiation of rabbit erythroid bone marrow cells.

Changes in the activity of cyclic AMP phosphodiesterase during differentiation of rabbit bone marrow erythroid cells were investigated. The cells were separated by velocity sedimentation at unit gravity into six fractions corresponding to different stages of development: proerythroblasts, basophilic cells, polychromatic cells, early orthochromatic and late orthochromatic cells and reticulocytes. Cyclic AMP phosphodiesterase was found to be very active in the most immature cells, the proerythroblasts, which also have the highest content of cyclic AMP. After differentiation into basophilic erythroblasts, a 4-fold decrease in cyclic AMP phosphodiesterase activity was observed. In these cells the amount of cyclic AMP was about 80% lower than that in proerythroblasts. In polychromatic cells a further drop in phosphodiesterase activity occurred. After the final cell division the enzyme activity was very low and the levels of cyclic AMP in the early and late orthochromatic cells remained constant. Kinetic studies demonstrated a heterogeneity of erythroid cell cyclic AMP phosphodiesterase: high affinity, low-Km (5.5 X 10(-6) M) and low affinity, high-Km (0.1 X 10(-3) M) enzymes were found. The phosphodiesterase activity was dependent on the presence of Mg2+ and was activated by Ca2+ at low Mg2+ concentrations (1 mM). The changes in cyclic AMP phosphodiesterase activity during differentiation and maturation of erythroid cells suggest the possible importance of this enzyme in the physiological control of cyclic AMP concentrations in developing erythroblasts. The loss of cyclic AMP phosphodiesterase activity after cessation of cell division supports the concept of the significance of the final cell division in erythroblast differentiation.

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

Role of phosphodiesterases III and IV in the modulation of vascular cyclic AMP content by the NO/cyclic GMP pathway.

1. The effect on cyclic nucleotide contents of selective inhibitors of cyclic nucleotide phosphodiesterase (PDE) isoforms III and IV (respectively SK&F 94120 and rolipram) and their interactions with endothelium and NO have been studied in rat aorta in the presence of indomethacin (10 microM). The participation of NO was assessed by using either NG-nitro-L-arginine methyl ester (L-NAME) (NO synthase inhibitor: 30 microM) or 3-morpholinosydnonimine (SIN-1, NO donor: 10 microM with SOD 100 units ml-1). 2. The presence of endothelium significantly increased both adenosine 3':5'-cyclic monophosphate (cyclic AMP, 1.7 fold) and guanosine 3':5'-cyclic monophosphate (cyclic GMP, 2.2 fold) contents. Cyclic GMP was largely affected by L-NAME or SIN-1 treatment, this was not the case for cyclic AMP suggesting that the presence of endothelium modified cyclic AMP content in aorta independently of the NO production. 3. In the presence or absence of endothelium, neither SK&F 94120 nor rolipram, alone or combined, significantly modified cyclic GMP content. 4. The PDE III inhibitor significantly affected cyclic AMP content only in non treated aorta without endothelium. In contrast, the PDE IV inhibitor increased cyclic AMP in all conditions. These increases were generally about 2 fold but markedly higher in aorta treated with SIN-1 and superoxide dismutase (SOD, 6 fold). Association of a low concentration of the PDE III inhibitor (5 microM) with the PDE IV inhibitor (30 microM) potentiated the effect of the PDE IV inhibitor on cyclic AMP content, except for aorta without endothelium treated with SIN-1 plus SOD. 5. These data indicate that the presence of the endothelium could increase cyclic AMP content independently of NO and prostacyclin (PGI2) production. Furthermore, an increase in cyclic GMP content (modulated by NO production) could enhance the cyclic AMP accumulation induced by the PDE IV inhibitor. This result supports the hypothesis that PDE III inhibition by endogenous cyclic GMP may potentiate the effect of PDE IV inhibition on cyclic AMP content. Taken together with our previous studies on relaxation, these results suggest that the NO/cyclic GMP pathway could induce PDE IV-dependent regulation of cyclic AMP via PDE III inhibition.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1982

Cyclic AMP may not be involved in catabolite repression in Saccharomyes cerevisiae: evidence from mutants capable of utilizing it as an adenine source.

Mutants able to utilize 5'-AMP or cyclic AMP as the adenine source were isolated from an ade6 ade10 double mutant by ethyl methane sulfonate mutagenesis. A single amp1 mutation, primarily selected on 5'-AMP medium, confers the phenotype for utilization of exogenous 5'-AMP as the adenine source. From the ade6 ade10 amp1 triple mutant, a mutant able to utilize cyclic AMP was isolated, and the mutant phenotype was proven to be due to the simultaneous occurrence of triple mutations designated as cam1, cam2, and cam3. The cam3 mutation, but not cam1 or cam2, also confers the phenotype for utilizing 5'-AMP, the same phenotype as the amp1 mutation. All of these mutations are recessive to the respective wild-type counterparts. Cells having the ade6 ade10 amp1 cam1 cam2 cam3 genotype showed significant ability to take up exogenous cyclic AMP, whereas no differences were observed in cyclic AMP phosphodiesterase activity in comparison with that of the original strains used in the mutant isolation. Since glucose severely repressed galactokinase synthesis in the constitutive GAL81 mutant having the ade6 ade10 amp1 cam1 cam2 cam3 genotype, irrespective of the presence or absence of cyclic AMP in the medium, it was suggested that cyclic AMP is not involved in the mechanism of catabolite repression in Saccharomyces cerevisiae. It does, however, have a stimulative effect on the galactokinase synthesis in the GAL81 mutant in the absence of glucose.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1988

Effect of exercise on cardiac cyclic AMP.

The effect of one bout of intense swimming caused significant increases in the cyclic AMP content of fast-twitch white skeletal muscle, liver, and heart. Further investigation of the exercise-induced increase in myocardial cyclic AMP indicates that the nucleotide content remained elevated long after (24 h) termination of exercise. This increase in cyclic AMP was time dependent, with the level increasing gradually throughout the work bout. The increase in cardiac cyclic AMP seemed to be independent of work intensity, provided that work time was of sufficient duration (greater than or equal to 30 min). Increases in cardiac cyclic AMP were also seen when rats were exposed to 2 degrees C for 1-7 d. The increases in cyclic AMP seen following exercise and cold exposure were accompanied by an increase in cardiac cyclic AMP phosphodiesterase (PDE) activity. Our working hypothesis was that prolonged elevations in cyclic AMP produced an induction and/or activation of one or more of the PDE isozymes. When we administered dibutyryl cyclic AMP to rats, cardiac PDE activity was increased. This increase was inhibited by cycloheximide, suggesting that the elevated enzyme activity is mediated by the synthesis of new protein. These data support the concept that cyclic AMP is involved in the regulation of its own metabolism during physiological stress.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1982

Forskolin as an activator of cyclic AMP accumulation and lipolysis in rat adipocytes.

Forskolin increased cyclic AMP accumulation in isolated adipocytes and markedly potentiated the elevation of cyclic AMP due to isoproterenol. In adipocyte membranes, forskolin stimulated adenylate cyclase activity at concentrations of 0.1 microM or greater. Forskolin did not affect the EC50 for activation of adenylate cyclase but did increase the maximal effect of isoproterenol. Neither the soluble nor particulate low-Km cyclic AMP phosphodiesterase activity was affected by forskolin. Low concentrations of forskolin (0.1-1.0 microM), which significantly elevated cyclic AMP levels, did not increase lipolysis, whereas similar increases in cyclic AMP levels due to isoproterenol elevated lipolysis. Forskolin did not inhibit the activation of triacylglycerol lipase by cyclic AMP-dependent protein kinase or the subsequent hydrolysis of triacylglycerol. Higher concentrations of forskolin (10-100 microM) did increase lipolysis. Both the increased cyclic AMP production and lipolysis due to forskolin were inhibited by the antilipolytic agents insulin and N6-(phenylisopropyl)adenosine. Hypothyroidism reduced the ability of forskolin to stimulate cyclic AMP production and lipolysis. These results indicate that forskolin increases cyclic AMP production in adipocytes through an activation of adenylate cyclase. Lipolysis is activated by forskolin but at higher concentrations of total cyclic AMP than for catecholamines.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1993

Cyclic AMP metabolism in intact rat ventricular cardiac myocytes: interaction of carbachol with isoproterenol and 3-isobutyl-1-methylxanthine.

Experiments were carried out to elucidate the characteristics of regulation of cyclic AMP levels in intact myocardial cells. For this purpose, the influence of isoproterenol, a nonselective cyclic nucleotide phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX) and carbachol on cyclic AMP levels was investigated in isolated rat cardiac myocytes. The extent of cyclic AMP accumulation induced by isoproterenol was much less than that produced by IBMX: submaximal concentrations of isoproterenol and IBMX elevated the cyclic AMP level 2.4- and 4.8-fold of the control level, respectively. Both agents in combination increased the cyclic AMP level markedly 48-fold. Carbachol inhibited the cyclic AMP accumulation induced by isoproterenol, IBMX and their combination by 30%, 60% and 80% of the respective response. The extent of inhibition produced by carbachol of the cyclic AMP accumulation induced by IBMX + isoproterenol was smaller than that caused by propranolol, and carbachol produced only a marginal additional inhibitory action to that of propranolol, implying that carbachol does not affect the process of cyclic AMP degradation. The present findings indicate that in intact cardiac myocytes the rate of cyclic AMP degradation catalyzed by PDE may be a crucial process of cyclic AMP turnover. This view is supported by the observations that the inhibitory action of carbachol on the effect of isoproterenol was less than that on the effect of IBMX, and that the inhibitory action of carbachol was markedly enhanced by the simultaneous presence of IBMX.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Effects of prostaglandin E1, isoproterenol and forskolin on cyclic AMP levels and tension in rabbit aortic rings.

The role of cyclic AMP in the control of vascular smooth muscle tone was studied by monitoring the effects of prostaglandin E1 (PGE1), isoproterenol and forskolin on cyclic AMP levels and tension in rabbit aortic rings. PGE1, isoproterenol and forskolin all increased cyclic AMP levels in rabbit aortic rings. Isoproterenol and forskolin relaxed phenylephrine-contracted aortic rings, but PGE1 contracted the rings in the presence or absence of phenylephrine. Isoproterenol relaxed these PGE1-contracted aortic rings without further change in total cyclic AMP levels, which were already elevated by the PGE1 alone. Pretreatment with forskolin potentiated the effects of PGE1 on cyclic AMP levels. PGE1 caused contractions in muscles partially relaxed by forskolin, even though very large increases in cyclic AMP levels (30 fold) were produced by PGE1 in the presence of forskolin. Isoproterenol was able to relax these forskolin-treated, PGE1-contracted muscles with no further increase in cyclic AMP levels. Thus, there does not appear to be a good correlation between total tissue levels of cyclic AMP and tension in these experiments. Our results suggest that, if cyclic AMP is responsible for relaxation of smooth muscle, some form of functional compartmentalization of cyclic AMP must exist in this tissue.

Alprostadil↗
PubMed1992

Dynamics of cyclic-AMP efflux in relation to alpha-MSH secretion from melanotrope cells of Xenopus laevis.

An important factor in regulating secretion from endocrine cells is the cytoplasmic concentration of cyclic-AMP. Many regulatory substances are known to either stimulate or inhibit the production of this second messenger through activation of their receptors. In the present study, we have monitored changes in cyclic-AMP efflux from melanotrope cells of Xenopus laevis in response to established neurochemical regulators of alpha-MSH secretion. In vitro superfusion of neurointermediate lobes allows for a dynamic recording of cyclic-AMP production in relation to hormone secretion. Unlike alpha-MSH secretion, the efflux of cyclic-AMP was not dependent on the concentration of extracellular calcium, indicating that hormone release and cyclic-AMP efflux are mediated by different mechanisms. The phosphodiesterase inhibitor IBMX and the adenylate cyclase activator forskolin stimulated cyclic-AMP efflux, but had no stimulatory effect on alpha-MSH release. This indicates that an increase in cyclic-AMP production in melanotrope cells is not necessarily accompanied by an increase in the rate of alpha-MSH release. Corticotropin-releasing factor stimulated cyclic-AMP efflux with dynamics similar to that induced by the amphibian peptide sauvagine. Dopamine and the GABAB receptor agonist baclofen both inhibited cyclic-AMP efflux and alpha-MSH release, with similar dynamics of inhibition and similar dose-response relationships. It is proposed that an inhibition of cyclic-AMP efflux is coupled to an inhibition of alpha-MSH secretion.

1-Methyl-3-isobutylxanthine↗