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At least 19 recordsLinked to original sources

PubMed1984

[cAMP-dependent protein kinase from pigeon breast muscle. Isolation of regulatory subunits by affinity chromatography and study of the topography of the cAMP binding site using cAMP analogs].

The cAMP-dependent protein kinase from the soluble fraction of pigeon breast muscle is represented by two forms, PK I and PK II. The ratio of the phosphotransferase activity of the two forms is 35-40% and 60-65% for PK I and PK II, respectively. The regulatory subunit of PK I was isolated in a homogeneous state by affinity chromatography on 8-(2-oxoethylthio)-cAMP immobilized on epoxy-activated Sepharose 4B. The molecular weight of the regulatory subunit of PK I as determined by SDS polyacrylamide gel electrophoresis is 45 000. The specific cAMP-binding activity is equal to 16 nmol of [3H]cAMP per mg of protein. The apparent dissociation constant (Kd') for cAMP equals to 380 nM. The preparation of the regulatory subunit of PK II obtained by affinity chromatography on the same adsorbent is made up of polypeptides with Mr 56 000, 39 000, 29 000, 17 000 and 11 000. The preparation possesses a cAMP-binding activity of 22 nmol of [3H]cAMP per mg of protein. The interaction of several analogs of cAMP containing substituents at different positions of the nucleotide molecule with the regulatory subunit of PK I was studied. Practically all the analogs with substituents at positions 8 and 6 of the adenine ring in the cAMP molecule had the affinity which was 2-9 times less than that of cAMP. The only exceptions were 8-carboxymethylamino- and 8-(2-oxyethyl)-amino-cAMP whose binding to the regulatory subunit was 100 and 53 times lower than that of cAMP. The substitutions in position N-1 of the cAMP molecule leads to a 30-50-fold decrease of the analogs affinity. beta-Bromoethyl ester of cAMP does not reveal the ability to bind to the regulatory subunit. The carboxymethyl ester of cAMP possesses the affinity for the cAMP-binding site that is 35 times less than that of cAMP. Modification of the 2'-hydroxyl of ribose (as in the case of 2'-amino-2'-deoxy-8-hydroxy-cAMP, 2'-deoxy-cAMP and 2'-O-acrylyl-cAMP) decreases the affinity of these compounds 125-, 313- and 126-fold as compared with cAMP. It was assumed that the cAMP molecule is bound to the regulatory subunit in the syn-conformation. A structural model of the cAMP-binding site in the regulatory subunit of cAMP-dependent protein kinase I from pigeon breast muscle is proposed.

Animals↗

PubMed1995

Novel (Rp)-cAMPS analogs as tools for inhibition of cAMP-kinase in cell culture. Basal cAMP-kinase activity modulates interleukin-1 beta action.

Novel (Rp)-cAMPS analogs differed widely in ability to antagonize cAMP activation of pure cAMP-dependent protein kinase I and II and to antagonize actions of cAMP on gene expression, shape change, apoptosis, DNA replication, and protein phosphorylation in intact cells. These differences were related to different abilities of the analogs to stabilize the holoenzyme form relative to the dissociated form of cAMP kinase type I and II. (Rp)-8-Br-cAMPS and (Rp)-8-Cl-cAMPS were the most potent cAMP antagonists for isolated type I kinase and for cells expressing mostly type I kinase, like IPC-81 leukemia cells, fibroblasts transfected with type I regulatory subunit (RI), and primary hepatocytes. It is proposed that (Rp)-8-Br-cAMPS or (Rp)-8-Cl-cAMPS should replace (Rp)-cAMPS as the first line cAMP antagonist, particularly for studies in cells expressing predominantly type I kinase. The phosphorylation of endogenous hepatocyte proteins was affected oppositely by (Rp)-8-Br-cAMPS and increased cAMP, indicating that (Rp)-8-Br-cAMPS inhibited basal cAMP-kinase activity. The inhibition of basal kinase activity was accompanied by enhanced DNA replication, an effect which could be reproduced by microinjected mutant cAMP-subresponsive RI. It is concluded that the basal cAMP-kinase activity exerts a tonic inhibition of hepatocyte replication. (Rp)-8-Br-cAMPS and microinjected RI also desensitized hepatocytes toward inhibition of DNA synthesis by interleukin-1 beta. This indicates that basal cAMP-kinase activity can have a permissive role for the action of another (interleukin-1 beta) signaling pathway.

3T3 Cells↗

PubMed1999

Regulation of adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in UMR-106 osteoblast-like cells: role of cAMP-phosphodiesterase and cAMP efflux.

The present study aimed to define the role of adenosine 3',5'-cyclic monophosphate (cAMP)-phosphodiesterase (PDE) activity and the possible involvement of cAMP efflux on parathyroid hormone (PTH)-stimulated intracellular cAMP accumulation in cultured osteoblast-like UMR-106 cells. Treatment of the cells with 10 nM PTH (1-84) rapidly increased the level of intracellular cAMP. PTH stimulation also increased the cAMP efflux rate. The efflux of cAMP could only account for a minor part of the decrease in intracellular cAMP. Six peaks of cAMP-hydrolyzing PDE activity were separated by Q-Sepharose chromatography. The first peak to elute was stimulated by Ca2+/calmodulin and provided less than 2% of the total eluted cAMP-PDE activity. The second peak, providing less than 4% of the cAMP-PDE activity, was stimulated 3-fold by 4 microM cyclic GMP (cGMP) and was sensitive to the PDE2 isoenzyme-selective inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA). The third peak, providing less than 10% of the cAMP-PDE activity, was insensitive to rolipram, EHNA, Ca2+/calmodulin, and cGMP. Peaks 4, 5 and 6 were sensitive to rolipram (IC50 < 0.1 microM) and provided approximately 85% of the total cAMP-hydrolyzing activity. It is concluded that cAMP-PDE activity in UMR-106 cells plays a major role in the control of intracellular cAMP accumulation, whereas only moderate amounts of cAMP are extruded from the cells through cAMP efflux. The main cAMP-hydrolyzing PDE isozyme is cAMP-specific/rolipram-sensitive. Ca2+/calmodulin-stimulated PDE, cGMP-stimulated PDE, and presently unidentified cAMP-specific/rolipram-insensitive PDE are also present in UMR-106 cells.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2003

cAMP analog mapping of Epac1 and cAMP kinase. Discriminating analogs demonstrate that Epac and cAMP kinase act synergistically to promote PC-12 cell neurite extension.

Little is known about the relative role of cAMP-dependent protein kinase (cAPK) and guanine exchange factor directly activated by cAMP (Epac) as mediators of cAMP action. We tested cAMP analogs for ability to selectively activate Epac1 or cAPK and discriminate between the binding sites of Epac and of cAPKI and cAPKII. We found that commonly used cAMP analogs, like 8-Br-cAMP and 8-pCPT-cAMP, activate Epac and cAPK equally as well as cAMP, i.e. were full agonists. In contrast, 6-modified cAMP analogs, like N6-benzoyl-cAMP, were inefficient Epac activators and full cAPK activators. Analogs modified in the 2'-position of the ribose induced stronger Epac1 activation than cAMP but were only partial agonists for cAPK. 2'-O-Alkyl substitution of cAMP improved Epac/cAPK binding selectivity 10-100-fold. Phenylthio substituents in position 8, particularly with MeO- or Cl- in p-position, enhanced the Epac/cAPK selectivity even more. The combination of 8-pCPT- and 2'-O-methyl substitutions improved the Epac/cAPK binding selectivity about three orders of magnitude. The cAPK selectivity of 6-substituted cAMP analogs, the preferential inhibition of cAPK by moderate concentrations of Rp-cAMPS analogs, and the Epac selectivity of 8-pCPT-2'-O-methyl-cAMP was also demonstrated in intact cells. Using these compounds to selectively modulate Epac and cAPK in PC-12 cells, we observed that analogs selectively activating Epac synergized strongly with cAPK specific analogs to induce neurite outgrowth. We therefore conclude that cAMP-induced neurite outgrowth is mediated by both Epac and cAPK.

Cyclic AMP↗

PubMed1995

(RP)-cAMPS inhibits the cAMP-dependent protein kinase by blocking the cAMP-induced conformational transition.

(RP)-cAMPS is known to inhibit competitively the cAMP-induced activation of cAMP-dependent protein kinase (PKA). The molecular nature of this inhibition, however, is unknown. By monitoring the intrinsic tryptophan fluorescence of recombinant type I regulatory subunit of PKA under unfolding conditions, a free energy value (delta GDH2O) of 8.23 +/- 0.22 kcal/mol was calculated. The cAMP-free form of the regulatory subunit was less stable with delta GDH2O = 6.04 +/- 0.05 kcal/mol. Native stability was recovered by treatment of the cAMP-free protein with either cAMP or (SP)-cAMPS but not with (RP)-cAMPS. Thus, (RP)-cAMPS binding to the regulatory subunit keeps the protein in a locked conformation, unable to release the catalytic subunit. This finding was further supported by demonstrating that holoenzyme formation was greatly accelerated only when bound cAMP was replaced with (RP)-cAMPS but not with cAMP or (SP)-cAMPS.

Arginine↗

PubMed1995

Cyclic adenosine monophosphate (cAMP) analogs 8-Cl- and 8-NH2-cAMP induce cell death independently of cAMP kinase-mediated inhibition of the G1/S transition in mammary carcinoma cells (MCF-7).

Human mammary carcinoma cells (MCF-7) were arrested in late G1-phase after treatment with agents (forskolin, interleukin-1 beta 3-isobutyl-1-methylxanthine) that increased the endogenous concentrations of cAMP. The effect of elevated cAMP was mimicked by microinjected catalytic (C alpha) cAMP-dependent protein kinase (cAK) subunit and reversed by the injection of a dominant negative cAK regulatory mutant (RID199). Further evidence that activation of cAK induced growth arrest was provided by the use of pairs of stable cAMP analogs known to synergistically activate isolated cAK isozymes. Furthermore, the effect of cAMP was not potentiated by serine/threonine phosphatase inhibitors that profoundly restricted MCF-7 growth. Some 8-substituted cAMP analogs, e.g. 8-Cl-cAMP and 8-NH2-cAMP, induced cell death rather than reversible inhibition of growth. Their effect was not synergized with complementary cAMP analogs. Furthermore, their potency was decreased rather than increased in the presence of an inhibitor of degradation (3-isobutyl-1-methylxanthine). Finally, their effect could be mimicked by degradation products unable to activate cAK. We concluded that 8-Cl-cAMP (and 8-NH2-cAMP) induced irreversible growth arrest by a mechanism not involving cAK, whereas activation of cAK resulted in a transient and fully reversible inhibition of cell proliferation.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2003

Interaction of CRP L124 with cAMP affects CRP cAMP binding constants, cAMP binding cooperativity, and CRP allostery.

A cyclic nucleotide-binding pocket of the CRP dimer is composed of amino acid residues contributed by both subunits. Leucine (L) 124 of one subunit packs against the adenine ring of cAMP bound to the opposing subunit. We have undertaken a study designed to evaluate the role of L124 in CRP allostery. Wild-type (WT) apo-CRP is a 47 kDa protease-resistant dimer composed of identical subunits that exhibits a biphasic isotherm in cAMP titration studies. The WT CRP-cAMP complex is a protease-sensitive dimer degraded by protease to a dimer core that ranges between 26.5 and 30.5 kDa. Substitution of L124 with isoleucine (I), valine (V), cysteine (C), or alanine (A) generated a series of CRP variants that exhibited unique differences in apo-CRP resistance to protease, the mass of the core fragments generated in protease digestion reactions, cAMP-mediated allostery, and CRP-cAMP complex functionality. Differences in the affinity of the position 124 CRP variants for cAMP were observed. The binding constants that drive the formation of the WT and L124I CRP-cAMP complexes deviated by not more than a factor of 1.5. In contrast, the L124V, L124A, and L124C forms of CRP exhibited both a decreased K(cAMP1)(app) and an increased K(cAMP2)(app) to produce 2.4-, 55-, and 204-fold reductions, respectively, in the difference between these two parameters compared to that observed for WT CRP. The data indicate that the van der Waals volume and/or the hyrophobicity of the L124 side chain are important determinants of CRP cAMP binding properties and affect, either directly or indirectly, cAMP-mediated conformation changes in CRP.

Allosteric Site↗

PubMed1998

Dissecting cAMP binding domain A in the RIalpha subunit of cAMP-dependent protein kinase. Distinct subsites for recognition of cAMP and the catalytic subunit.

The two gene-duplicated cAMP binding domains in the regulatory subunits of cAMP dependent protein kinase are each comprised of an A helix, an eight-stranded beta-barrel, and a B and C helix (1). The A domain is required for high affinity binding to C, while the B domain regulates access to the A domain. Using a combination of a yeast two-hybrid screen coupled with deletion analysis, cAMP binding domain A of RI was dissected into two structurally and functionally distinct subsites, one that binds cAMP and another that binds the C subunit. The minimum stable subdomain required for binding to C in the 1-3 micromolar range is composed of residues 94-169, while residues 236-244, mapped to the C helix of cAMP binding domain A, were defined as a second surface necessary for high affinity (5-10 nanomolar) binding to C. This portion of the C helix, due to its position directly between the two subsites, serves as a molecular switch for either a cAMP-bound conformation or a C-bound conformation and can thus modulate interactions of cAMP binding domain A with cAMP, with C, and with cAMP binding domain B.

Amino Acid Sequence↗

PubMed1996

8-bromo-cAMP and 8-CPT-cAMP increase the density of beta-adrenoceptors in hepatocytes by a mechanism not mimicking the effect of cAMP.

Addition of 8-bromo-adenosine 3',5'-cyclic monophosphate (8-bromo-cAMP) or 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (8-CPT-cAMP) to hepatocytes at the time of plating enhanced the acquisition of beta-adrenoceptors that occurs spontaneously upon culturing as primary monolayers. This effect was partially suppressed by the phosphodiesterase inhibitor isobutyl methylxanthine, and was mimicked by 8-bromo-AMP, 8-bromo-adenosine, and the adenosine kinase inhibitor 5'-amino-5'-deoxyadenosine. Agents that elevated the intracellular level of cAMP, such as glucagon and forskolin, and Sp-8-bromo-adenosine 3',5'-monophosphorothioate (Sp-8-bromo-cAMPS), a cAMP analogue that is resistant towards metabolic breakdown, did not significantly enhance beta-adrenoceptor expression when used alone, but glucagon enhanced the effect of 8-bromo-adenosine. 8-bromo-cAMP and 8-bromo-adenosine decreased cellular ATP-levels. These observations suggest that the enhanced beta-adrenoceptor acquisition was mediated mainly through the action of metabolites of 8-bromo-cAMP and 8-CPT-cAMP, although there may be a cAMP-mediated component in the effect. Several mechanisms, including depletion of ATP, are probably involved, and might affect beta-adrenoceptor degradation.

1-Methyl-3-isobutylxanthine↗

PubMed1984

Desensitization of the cAMP system in mouse Leydig cells by hCG, cholera toxin, dibutyryl cAMP and cAMP: localization of the 'lesion' to the guanine nucleotide regulatory protein-adenylate cyclase complex.

The mechanism of hCG-induced desensitization of the cAMP system was studied in Percoll-purified mouse Leydig cells. Pretreatment of Leydig cells with hCG resulted in a time- and dose-dependent decrease in the capacity of hCG-induced cAMP formation. Maximal desensitization (approximately 90%) was induced by only partial prior stimulation. Desensitization, however, was not observed without a prior increase in cAMP or testosterone production. Pretreatment of the cells with N6,O2'-dibutyryl cAMP (DBcAMP) also induced a dose- and time-dependent densensitization. cAMP was only effective in the presence of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX). Cholera toxin desensitized the hormone-induced cAMP response as drastically as hCG. Cholera toxin was unable to reverse the refractory state induced by one of the agonists. hCG-induced desensitization was not associated with a loss in [125I]hCG binding or an increase in maximal phosphodiesterase activity, and appeared not to be dependent on protein synthesis. Membranes from hCG, cholera toxin of DBcAMP-desensitized cells showed an impaired adenylate cyclase activity in response to hCG, hCG plus beta-gamma-imidoguanosine 5'-triphosphate (GPPNP) and NaF. In conclusion, hCG-induced desensitization of the adenylate cyclase system in mouse Leydig cells can be mimicked by cholera toxin, DBcAMP and cAMP, indicating a cAMP-mediated process. The site of the 'lesion' has to be localized to the guanine nucleotide regulatory protein-adenylate cyclase complex rather than to its uncoupling from the hormone receptor.

1-Methyl-3-isobutylxanthine↗

PubMed1985

Evidence for the role of cAMP-dependent protein kinase in the down-regulation of hypothalamic HD: reversal of cAMP-(ATP) induced inhibition of HD activity by the 'Walsh' inhibitor of cAMP-dependent protein kinase and by cyclic GMP.

Under the total blockade of PDE1 and the presence of endogeneous ATP and MgCl2, the inhibitory effect of cAMP on HD activity could be demonstrated as low as 8.7 X 10(-8) M concentration in a 20,000 g supernatant of a sustained homogenate of rat hypothalamus. A total reverse of this action and also a partial release of the cAMP-induced inhibition of HD, occurred at higher concentrations of cAMP, and ATP could be achieved by an endogeneous inhibitor of cAMP-dependent protein kinase or by cyclic GMP. The reversal of cAMP action by PKI seems to serve a strong evidence for the role of cAMP-dependent protein kinase (EC 2.7.37: ATP-protein phosphotransferase) in this action and emphasized the involvement of a direct or an indirect phosphorylation in the regulation of HD activity. The stimulatory effect of cyclic GMP on cAMP-induced inhibition of HD or its 'direct' effect on histamine formation is asserted, probably through the activation of PDE, or through independent stimulatory machinery, coupled to the cyclic GMP system.

Adenosine Triphosphate↗

PubMed1990

Post-transcriptional regulation of cAMP-dependent protein kinase activity by cAMP in GH3 pituitary tumor cells. Evidence for increased degradation of catalytic subunit in the presence of cAMP.

The effects of cyclic AMP treatment on total cAMP-dependent protein kinase activity in GH3 pituitary tumor cells have been studied. Incubation of cells for 24 h with 1 microM forskolin resulted in a 50% decrease in total cAMP-dependent protein kinase activity which was reversible upon removal of forskolin from culture media. A similar response was observed in GH3 cells treated with 5 ng/ml cholera toxin and 0.5 mM dibutyryl cAMP but not 0.5 mM dibutyryl cGMP. Northern blot analysis demonstrated that the steady-state level of the mRNA for each of the six kinase subunit isoforms studied was not detectably altered after treatment with 1 microM forskolin for 24 h. The concentration of catalytic subunit was also assessed by binding studies using a radiolabeled heat-stable protein kinase inhibitor. Treatment of GH3 cells with 1 microM forskolin for 24 h reduced protein kinase inhibitor binding activity by 50%, consistent with the observed forskolin-induced decrease in total kinase activity. Analysis of endogenous heat-stable protein kinase inhibitor activity in GH3 cell extracts showed no significant difference between forskolin-treated cells and cells maintained under control conditions. To assess possible effects on catalytic subunit degradation, pulse-chase experiments were performed and radiolabeled catalytic subunit was isolated by affinity chromatography. The results demonstrated that treatment of cells with chlorophenylthio-cAMP detectably increased the apparent degradation of radiolabeled catalytic subunit. The increased degradation of the catalytic subunit was sufficient to account for the observed decreases in kinase activity. These results suggest that relatively long term cAMP treatment can alter total cAMP-dependent protein kinase activity through effects to alter the degradation of the catalytic subunit of the enzyme.

Animals↗

PubMed1982

Interaction of cAMP derivatives with the 'stable' cAMP-binding site in the cAMP-dependent protein kinase type I.

cAMP binding to the 'stable' cAMP-binding sites in the regulatory subunit of the cAMP-dependent protein kinase type I was investigated using a set of 18 selected derivatives. All the tested analogues were competitive with [3H]cAMP and inhibitor constants from 12 nM to 20 microM with the free regulatory subunit were determined. The cAMP molecule seemed to be bound by these specific hydrogen bonds to the 5' and 3' oxygen, the 2' hydroxyl, and an ion pair interaction between the negative charge in equatorial position and a positively charged amino acid side chain. The adenine base is rather unspecifically bound with no hydrogen bonds involved. This binding specificity of the 'stable' site is similar to the requirement for dissociation as determined by the activation of the kinase by a respective analogue. This indicates that occupation of the 'stable' sites leads to activation of the protein kinase. The presence of the catalytic subunit reduced the affinity of most analogues. The binding of one derivative with the negative charge fixed in the axial position is not influenced by the addition of the catalytic subunit and ATP. A plausible model for a conformational change during the activation process in the 'stable' site is discussed.

Adenosine Triphosphate↗

PubMed1982

Activation of protein kinase isoenzymes under near physiological conditions. Evidence that both types (A and B) of cAMP binding sites are involved in the activation of protein kinase by cAMP and 8-N3-cAMP.

cAMP-dependent protein kinase I and II (cAKI and cAKII) were incubated under near physiological conditions in the presence of various concentrations of 8-N3-c[3H]AMP or c[3H]AMP. Both types (A and B) of cyclic nucleotide binding sites of cAKI or cAKII were occupied to a similar extent and the degree of their occupation correlated with the degree of kinase activation. cAKI and cAKII bound cAMP in an apparent positively cooperative manner in the presence of Mg2+, ATP. 8-N3-c[3H]AMP dissociated several orders of magnitude faster from site A than site B of the regulatory moiety of cAKII, and was photo-incorporated only when bound to site B.

Azides↗

PubMed1983

Adenosine 3',5'-monophosphate (cAMP)-binding protein and cAMP-dependent protein kinase in human placenta.

cAMP modulates estrogen, hCG, and lactate syntheses by human placenta, cAMP presumably exerts its major intracellular effect by binding to cAMP-dependent protein kinase (cAMP-PK), which, in turn, phosphorylates regulatory proteins within the target cell. cAMP binding and cAMP-PK have not been previously identified in placenta. [3H]cAMP binding to crude cytosol fractions of term placenta was rapid, saturable, and reversible. Scatchard analyses of saturation experiments of [3H]cAMP binding to placental cytosol were linear (Kd = 1.13 +/- 0.11 x 10(-8) M; n = 5). The binding capacity was 1.27 +/- 0.18 pmol/mg protein. Competition for the [3H]cAMP-binding site followed the potency order cAMP much greater than cGMP much greater than (Bu)2cAMP, analogous to cAMP binding to cAMP-PK in other tissues. ADP, ATP, and adenosine did not compete for the [3H]cAMP-binding site. cAMP significantly enhanced phosphorylation of histone protein by placental cytosol (activity ratio, 0.57 +/- 0.04; P less than 0.01). Two peaks of [3H]cAMP binding and coincident cAMP-PK activity were identified by DEAE-cellulose column chromatography of placental cytosol corresponding to classical type I and type II cAMP-PK. While the majority of the cAMP-PK was found in placental cytosol, cAMP-PK was also demonstrated in crude microsomal and microvillous brush border membranes of human placenta after solubilization with Triton X-100 (P less than 0.05). Regulation of placental function by catecholamines and other hormones known to mediate cAMP levels may be accomplished through the phosphorylation of cellular proteins by cAMP-dependent protein kinases.

Binding, Competitive↗

PubMed1996

Rp diastereomeric analogs of cAMP inhibit both cAMP- and cGMP-induced dilation of hamster mesenteric small arteries.

Cross talk between the adenosine (3',5'-cyclic monophosphate) (cAMP) and the guanosine (3',5'-cyclic monophosphate) (cGMP) signalling pathways in vascular smooth muscle may occur such that cAMP may act through cGMP-dependent protein kinase rather than cAMP-dependent protein kinase to induce relaxation of this tissue. Therefore, it was hypothesized that due to this crosstalk, competitive antagonists of cAMP may not show much selectivity in inhibition of cAMP- or cGMP-induced vasodilation. To test this hypothesis, the effects of Rp-diastereomeric phosphorothioate derivatives of cAMP, putative competitive antagonists of cAMP at cAMP-dependent protein kinase, were assessed on vasodilation induced by Sp-phosphorothioate derivatives of cAMP, dibutyryl cAMP, 8-Br cGMP and sodium nitroprusside. Hamster mesenteric arteries (200-400 microns i.d.) were cannulated and pressurized to 75 mm Hg and constricted to approximately 50% of maximum with 1 mumol/l phenylephrine. Vasodilators were then added in cumulative fashion and diameter responses recorded in the absence and presence of (Rp)-adenosine (3',5'-cyclic monophosphorothioate) (Rp cAMPs) or (Rp)-8-(parachlorophenylthio) adenosine (3',5'-cyclic monophosphorothioate) (Rp 8CPT cAMPs). Rp cAMPs (0.1-0.5 mmol/l) inhibited dilations induced by the cAMP agonists, (Sp)-adenosine (3',5'-cyclic monophosphorothioate) (Sp cAMPs) and dibutyryl cAMP, but also inhibited dilations induced by 8-Br cGMP and sodium nitroprusside (p < 0.05 and n > 4 for all). In a more detailed study we found that Rp 8CPT cAMPs against Sp 8CPT cAMPs (3.6 +/- 1.2) was similar to the pA2 for Rp 8CPT cAMPs against 8-Br cGMP (4.1 +/- 1.2) (p > 0.05, d.f. = 37). These data support the hypothesis that both cAMP and cGMP act through a common protein kinase to cause vasodilation and urge caution in the use of Rp-diastereomeric analogs of cyclic nucleotides to dissect out specific signal transduction pathways in blood vessels.

Animals↗

PubMed1984

Effects of the specific cAMP antagonist, (Rp)-adenosine cyclic 3',5'-phosphorothioate, on the cAMP-dependent protein kinase-induced activity of hepatic glycogen phosphorylase and glycogen synthase.

The cAMP-dependent protein kinase-induced effects on phosphorylase and glycogen synthase activities and glucose production were studied in hepatocytes isolated from fed rats in the presence of the diastereomers of adenosine cyclic 3',5'-phosphorothioate, (Sp)-cAMPS and (Rp)-cAMPS. Incubation of hepatocytes with (Sp)-cAMPS or glucagon, both of which lead to cAMP-dependent protein kinase activation, resulted in a concentration-dependent increase in glycogen phosphorylase activity and a decrease in glycogen synthase activity. Incubation of hepatocytes with the cAMP-dependent protein kinase antagonist, (Rp)-cAMPS, in the absence of an agonist, had no significant effect on phosphorylase or glycogen synthase activities. Incubation of hepatocytes with a half-maximally inhibitory concentration of (Rp)-cAMPS shifted the agonist-induced activation curves for phosphorylase and the agonist-induced inhibition curves for glycogen synthase to 5-fold higher concentrations for both (Sp)-cAMPS and glucagon. Phosphorylase activity was very sensitive to the rapid, concentration-dependent inhibition by (Rp)-cAMPS of agonist-induced activation of cAMP-dependent protein kinase. The effects on phosphorylase activity were observable in 30 s and were concentration-dependent with half-maximal inhibition at 10 microM, similar to that observed for cAMP-dependent protein kinase. In contrast, glycogen synthase activity was less sensitive to (Rp)-cAMPS inhibition of agonist-induced activation of cAMP-dependent protein kinase. The effects on glycogen synthase activity lagged behind those on phosphorylase activity and the concentration dependence did not parallel the cAMP-dependent protein kinase effect, but was shifted to higher concentrations of (Rp)-cAMPS with half-maximal inhibition at 60 microM. Glucose (10 to 40 mM) increased the sensitivity of glycogen synthase to (Rp)-cAMPS inhibition of cAMP-dependent protein kinase over a narrow range of agonist concentration, but had no significant effect throughout most of the agonist-induced activation range. Thus, the diastereomers, (Sp)- and (Rp)-cAMPS, influence glycogen metabolism and the glycogenolytic enzymes through their modulation of cAMP-dependent protein kinase levels.

Animals↗

PubMed1983

The effect of folic acid on cAMP-elicited cAMP production in Dictyostelium discoideum.

In Dictyostelium discoideum, folic acid serves as a chemoattractant, accelerates the appearance of several developmentally regulated enzymes, alters the phase of spontaneous light-scattering oscillations, and elicits increases in cGMP and cAMP. The effect of folic acid and pteridine analogs on rates of [3H]cAMP secretion, monitored by rapid perfusion, was studied. At low cell densities folic acid elicited only minimal [3H]cAMP secretion. At high cell densities, folic acid elicited greater increases in cellular cAMP and rates of [3H]cAMP secretion than did maximally effective doses of cAMP. Following pretreatment with 10(-8) M cAMP for 10 min or 10(-6) M cAMP for 2 min, no [3H]cAMP was secreted in response to folic acid. Conversely, pretreatment with folic acid did not impair subsequent responsiveness to 5 X 10(-9) M cAMP. Simultaneous addition of folic acid and cAMP increased the release of [3H]cAMP two- to threefold compared to control stimuli of the same concentration of cAMP. Pretreatment with a mixed stimulus attenuated the response to a subsequent increment in the cAMP concentration only if the higher cAMP stimulus did not include folic acid. The effects of folic acid are mimicked by the pteridine ring alone; analogs substituted at the 2- or 5-position are ineffective; non-reducible analogs are partially active. The observations presented here suggest significant differences in the mechanisms by which cAMP and folic acid stimulate cAMP production. Rather than there being separate receptors for each chemoattractant that interact in the same way with an intracellular pathway, it appears that folic acid and pteridine analogs potentiate the response to cAMP.

Adaptation, Physiological↗