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PubMed1975

Effect of ascorbic acid on ACTH-induced cyclic AMP formation and steroidogenesis in isolated adrenal cells of vitamin E-deficient rats.

Isolated adrenal cells from Vitamin E-deficient and control rats were prepared by a trypsin digestion method. Cyclic adenosine 3',5'-monophosphate (cyclic AMP) formation was studied in response to adrenocorticotropin (ACTH) in the presence and absence of ascorbate by measuring the conversion of prelabeled adenosine 5'-triphosphate [14C]ATP to cyclic [14C]AMP. Ascorbate (0.5 mM) inhibited ACTH-induced cyclic [14C]AMP formation in adrenal cells isolated from Vitamin E-deficient rats but had no effect in the control cells. The inhibitory effect of ascorbate on ACTH-induced cyclic AMP formation in Vitamin E-deficient rats decreased as the concentration of ACTH increased. In Vitamin E-deficient rats ascorbate inhibited ACTH-induced cyclic [14C]AMP formation after 30 min of incubation. There was no further significant accumulation of cyclic [14C]AMP at 60 min or 120 min although in the absence of ascorbate cyclic [14C]AMP continued to be formed. The in vitro addition of alpha-tocopherol reduced the inhibition of ACTH-induced cyclic [14C]AMP formation by ascorbate in Vitamin E-deficient rats. These studies suggest that alpha-tocopherol and ascorbate may affect ACTH-induced cyclic AMP formation through interaction with the membrane-bound enzyme adenylate cyclase.

Adrenal Glands↗
PubMed1976

Effect of hexoses and mannoheptulose on cyclic AMP accumulation and insulin secretion in rat pancreatic islets.

The effects of various sugars on the simultaneous release of insulin and accumulation of cyclic AMP were studied in collagenase isolated rat pancreatic islets. D-Glucose stimulated the formation of cyclic AMP at 3 and 60 min of incubation, whether measured by a label incorporation technique, or by the protein kinase binding assay of Gilman. Only D-glucose and D-mannose were able to stimulate insulin release and cyclic [3H]AMP accumulation in the absence of other substrate. D-fructose had a stimulatory effect in the presence of 3.3 mM D-glucose only at a high concentration (33.8 mM), and enhanced the effects of 8.3 mM glucose when added at the concentration of 8.3 mM. D-Galactose was effective only together with 8.3 mM D-glucose. The order of potency of these hexoses, both regarding insulin secretion and cyclic [3H]AMP accumulation, was glucose-mannose-fructose-galactose. L-Glucose and 3-O-methylglucose had no effects at 60 min when incubated together with 8.3 mM D-glucose, whereas at 3 min, 3-O-methylglucose induced a small stimulation of the cyclic [3H]AMP response. D-mannoheptulose and D-glucosamine inhibited the insulin and cyclic [3H]AMP responses to 27.7 mM glucose. Mannoheptulose suppressed completely the glucose effect on cyclic nucleotide accumulation within 90 s. Although under all incubation conditions, the threshold stimulatory or inhibitory concentration of a given agent was identical for insulin release and cyclic [3H]AMP accumulation, these two variables showed quantitative differences in incubations of 60 min, the magnitude of the changes in insulin secretion being larger than that for the cyclic nucleotide. It is suggested that modulation of islet cyclic AMP level is an important step in the transmission of the effect of various sugars on insulin release; however, glucose and possibly other sugars may also enhance insulin release by additional mechanisms not involving the adenylate cyclase-cyclic AMP system of the beta-cell.

Animals↗
PubMed1980

Cyclic AMP in the cell cycle of Physarum polycephalum.

Cyclic AMP, [3H]thymidine incorporation, and DNA content were measured in the cell cycle of Physarum polycephalum. A sensitive radioimmunoassay was employed to assay cyclic AMP so that plasmodia could be assayed individually. In contrast to previously published results (Lovely, J.R. and Threlfall, R.J. (1976) Biochem. Biophys. Res. Commun. 71, 789-795), no pre-mitotic peak of cyclic AMP was detected. In seven experiments levels of cyclic AMP showed only small changes in individual experiments and ranged from 1-6 pmol/mg protein in different experiments. When plasmodia in the immediate premitotic period were collected on the basis of nuclear mitotic morphology, no evidence of a peak of cyclic AMP was found. Light was found to increase plasmodial cyclic AMP in a rapid, transient fashion. However, the brief exposure of cell cycle samples to light during collection did not induce any apparent cell cycle specific peaks of cyclic AMP. Although the occurrence of extremely rapid transient peaks of cyclic AMP in the cell cycle cannot be ruled out, it appears likely that the P. polycephalum cell cycle can proceed normally without major changes in cyclic AMP.

Cell Cycle↗
PubMed1980

Evidence for a cyclic AMP system highly sensitive to secretin in gastric glands isolated from the rat fundus and antrum.

The effects of secretin and vasointestinal peptide (VIP) on the production of cyclic AMP have been studied in gastric glands isolated by means of EDTA from rat fundic and antral mucosa. (1) In gastric fundus, secretin and VIP caused a time- and temperature-dependent stimulation of cyclic AMP production that was maximal when the test agents were incubated for 60 min at 20 degrees C in the presence of 0.5 mM 3-isobutyl-1-methylxanthine as a phosphodiesterase inhibitor. The dose-response curve was monophasic for both peptides, the production of cyclic AMP being sensitive to 10(-10) M secretin and to 5 . 10(-8) M VIP. Half-maximal stimulation was obtained with 2.9 10(-9) M secretin or 2 . 10(-7) M VIP and the maximal stimulation represented a 21-fold and a 19-fold increase above control for secretin and VIP, respectively. Histamine also stimulated cyclic AMP production, with a Km of about 5 . 10(-4) M. No additive effect on cyclic AMP production was oberved when secretin and VIP were simultaneously added at maximally active concentrations, while an additive effect was observed when secretin and histamine were added together. (2) In gastric antrum, the characteristics of the secretin- and VIP-stimulated cyclic AMP production were similar to those observed in gastric fundus. Histamine nevertheless failed to stimulate the formation of cyclic AMP in antral mucosa. (3) These data demonstrate the existence of a cyclic AMP system highly sensitive to secretin in gastric glands isolated from the rat fundus and antrum and suggest that VIP operates through this system. (4) It is proposed that the pepsinogen- and/or mucous-secreting cells are implicated in the regulation of cyclic AMP production by secretin in gastric glands of the rat.

Animals↗
PubMed1993

Cyclic AMP and long-term potentiation in the CA1 region of rat hippocampus.

Bath application of dibutyryl cyclic AMP, 8-bromo cyclic AMP or forskolin to hippocampal slices caused a short-lasting decrease, followed after washout by a long-lasting increase in the amplitude of population spikes recorded in area CA1 in response to 0.05 Hz stimulation of the Schaffer collateral/commissural pathway. Population spike depression lasted only as long as the cyclic AMP analogues were present in the bath, while the potentiation lasted for at least 2 h after they were washed out. Population excitatory postsynaptic potential amplitude was depressed while dibutyryl cyclic AMP was present in the bath, but after washout it was not significantly different from baseline amplitude. The population spike depression caused by dibutyryl cyclic AMP was prevented by the adenosine antagonist 8-p-sulfophenyltheophylline, but the long-lasting potentiation was not. Thus the transient depression induced by dibutyryl cyclic AMP was probably caused by an action of the drug or its breakdown products as adenosine agonists. The long-lasting potentiation is caused by a different mechanism. Occlusion experiments were performed to examine the relationship of the long-lasting population spike potentiation caused by dibutyryl cyclic AMP to the population spike long-term potentiation caused by high-frequency stimulation. High-frequency stimulation delivered after the population spike potentiation caused by dibutyryl cyclic AMP was established did cause a further potentiation, but only up to the level caused by high-frequency stimulation alone. Dibutyryl cyclic AMP administered after high frequency stimulation-induced long-term potentiation was established had no further potentiating effect.(ABSTRACT TRUNCATED AT 250 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2003

Apolipoprotein (a) mediates the lipoprotein (a)-induced biphasic shift in human platelet cyclic AMP.

Lipoproteins are known to influence platelet cyclic adenosine monophosphate (c-AMP) levels. Lipoprotein (a) (Lp(a))'s impact on platelet c-AMP levels has never been assessed. Increasing levels of purified human Lp(a) (1-100) mg/dl were incubated with washed human platelets. Lp(a) concentrations of 1-25 mg/dl resulted an initial statistically significant increase of platelet c-AMP above basal levels and decreased collagen-stimulated platelet aggregation levels. Higher concentrations progressively returned the platelet c-AMP concentrations to basal levels accompanied by further decreases in platelet aggregation. Increasing concentrations of purified apolipoprotein (a) (apo(a)) also resulted in a similar biphasic c-AMP response while Lp(a) without apo(a) was without impact. One antibody directed against apo(a) in intact Lp(a) removed the biphasic c-AMP pattern and eliminated Lp(a) platelet aggregation. Antibodies directed against apo B in intact Lp(a) gave results similar to intact Lp(a) in terms of the biphasic response of c-AMP upon platelet exposure to increasing levels of Lp(a). It is concluded that apo(a) mediates the Lp(a)-induced biphasic response in platelet c-AMP as the result of platelet exposure to increasing levels of Lp(a). The biphasic response in c-AMP assists in platelet aggregation decreases up to a concentration of 25 mg/dl Lp(a), such assistance being lost at higher Lp(a) concentrations.

Antibodies, Monoclonal↗
PubMed1999

Modulation of rhythmic melatonin synthesis in Xenopus retinal photoreceptors by cyclic AMP.

Cyclic AMP regulates melatonin synthesis in vertebrate photoreceptor cells. In the present study, we investigated whether the circadian rhythm of melatonin synthesis in Xenopus retinal photoreceptor layers is driven by rhythmic changes in cyclic AMP. When the photoreceptor layers were continuously treated with 8-(4-chlorophenylthio)-cyclic AMP (8-CPT-cAMP) at a saturating concentration (1 mM), melatonin release was increased at all times of the day, but robust melatonin rhythms were maintained for 2 days in constant darkness (DD). We also measured cyclic AMP efflux and melatonin release simultaneously from photoreceptor layers that were continuously treated with forskolin and/or 3-isobutyl-1-methylxanthine (IBMX) in light-dark (LD) and DD. Circadian rhythmicity was observed in melatonin release, but not in cyclic AMP efflux, suggesting that changes of melatonin levels are not always caused by the changes of the cyclic AMP levels. In addition, the simultaneous treatment of forskolin and IBMX appeared to saturate sensitivity of melatonin synthesis to cyclic AMP, but this treatment did not abolish melatonin rhythms. These results suggest that circadian rhythms of melatonin can be driven without rhythmic changes of cyclic AMP, and that cyclic AMP regulates melatonin in parallel with the output pathways from the circadian oscillator.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Total iridectomy does not alter outflow facility responses to cyclic AMP in cynomolgus monkeys.

Total outflow facility was measured by two-level constant pressure anterior chamber perfusion in surgically untouched and totally iridectomized cynomolgus monkey eyes receiving bolus intracameral infusions of cyclic AMP, dibutyryl cyclic AMP, or 5'AMP. Cyclic AMP doses of 50 and 100 micrograms (corresponding to initial intracameral concentrations of 1.5- and 3 mM) increased facility by approximately 20 and 40% respectively in both surgically untouched and aniridic eyes. Higher doses produced no greater effect, and lower doses were ineffective. Dibutyryl cyclic AMP and 5'AMP were ineffective in doses up to 100 micrograms (2- and 3 mM respectively). These findings indicate that the iris plays no mechanical role in mediating the facility response to cyclic AMP, and that the iridectomy procedure per se does not compromise the ability of the outflow pathways to respond to cyclic AMP. Since untouched and aniridic eyes exhibit similar facility responses to epinephrine and norepinephrine, the present findings also suggest that the iris is not the source of cyclic AMP mediating facility responses to catecholamines.

Adenosine Monophosphate↗
PubMed2003

Cyclic AMP inhibits production of interleukin-6 and migration in human vascular smooth muscle cells.

BACKGROUND: Gene expression induced by tumor necrosis factor-alpha (TNF-alpha) is involved in the regulation of vascular smooth muscle cell (VSMC) proliferation and migration, two events critical to formation of stenotic vascular lesions. In some systems, elevating adenosine 3',5'-cyclic monophosphate (cyclic AMP) inhibits TNF-alpha induced gene transcription. We recently demonstrated that interleukin-6 (IL-6) was chemotactic to VSMC. Therefore, we tested the hypothesis that elevating cyclic AMP would inhibit TNF-alpha-mediated IL-6 expression and VSMC migration. MATERIALS AND METHODS: VSMC were cultured from saphenous vein remaining after coronary artery bypass grafting. Migration of VSMC through a porous membrane was determined. Intracellular cyclic AMP was elevated by exposing the cells to forskolin or 8-Br-cyclic AMP and was measured by radioimmunoassay. IL-6 was measured by enzyme-linked immunosorbent assay. RESULTS: TNF-alpha induced migration of VSMC in a concentration-dependent manner. Incubation of cells with forskolin significantly increased cyclic AMP. Co-incubation of cells with TNF-alpha in combination with 8-Br-cyclic AMP or forskolin inhibited migration by approximately 25 and 70%, respectively. Incubation with TNF-alpha increased release of IL-6 from VSMC 18-fold over basal. This stimulated release was inhibited by either 8-Br-cyclic AMP or forskolin. In cells stimulated with TNF-alpha, addition of an antibody to IL-6 reduced migration by 25%. CONCLUSIONS: These data show that IL-6 produced by VSMC contributes to cell migration induced by TNF-alpha. Further, elevating cyclic AMP inhibited TNF-alpha-induced release of IL-6, and migration of VSMC. These results are consistent with the notion that mechanisms that increase intracellular cyclic AMP, such as activation of beta-adrenergic receptors on VSMC, act as a brake on cell migration.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1999

Cyclic AMP metabolism by swine adipocyte microsomal and plasma membranes.

Extracellular cyclic AMP is source of extracellular adenosine in brain and kidney. Whether this occurs in adipose tissue is unknown. The present study evaluated the capacity of swine adipocyte plasma membranes to metabolize cyclic AMP to AMP and adenosine, via phosphodiesterase (PDE) and 5'-nucleotidase (5'-NT), respectively. Plasma membranes (PM) and microsomal membranes (MM) were isolated from over-the-shoulder subcutaneous adipose tissue of 3 month-old male miniature swine. The purity of the membrane fractions was determined and PDE and 5'-NT activities in PM and MM fractions were corrected for cross-contamination. The maximal activity of MM-PDE was 7-fold greater than that of PM-PDE. MM-PDE was 100% inhibited by 5 microM cilostamide, while PM-PDE was unaffected by this PDE3B inhibitor. Inhibitors of PDE1, PDE2, PDE4 and PDE5 also failed to inhibit PM-PDE. However, 1 mM DPSPX inhibited PM-PDE activity by 72%. When PM were incubated with 0.8 microM cyclic AMP for 20 min, AMP accumulation was four times that of adenosine. These data demonstrate that cyclic AMP can be converted to AMP and adenosine by the PM-bound enzymes 5'-NT and PDE, and suggest that the PM-PDE responsible for extracellular cyclic AMP metabolism to AMP is distinct from the intracellular MM-PDE.

5'-Nucleotidase↗
PubMed1989

Characterization of AMD, the AMP deaminase gene in yeast. Production of amd strain, cloning, nucleotide sequence, and properties of the protein.

The structural gene for AMP deaminase (AMD) from Saccharomyces cerevisiae has been cloned and characterized. A yeast strain deficient in AMP deaminase activity was produced and shown to be deficient in AMP deaminase protein by Western blot analysis. The gene for AMP deaminase was located in a lambda gt11 library of yeast genomic DNA, and a DNA fragment from the lambda gt11 clone was used to locate homologous DNA in a yeast genomic library in the centromeric plasmid YCp50, a yeast-Escherichia coli shuttle vector. One plasmid was selected for its ability to restore AMP catalytic activity to the deficient strain. Yeast deficient in AMP deaminase or those overproducing the enzyme grow at near normal rates. The open reading frame corresponding to AMD codes for a protein of 810 amino acids, molecular weight 93,286. The yeast AMD transcript is 3.0 +/- 0.2 kb, and the transcriptional initiation sites have been identified. Western blot analysis of extracts prepared from actively growing yeast indicates a major band at approximately 96,000 molecular weight with several bands at lower molecular weight, including 83,000. When the AMD gene is expressed in E. coli, the large Mr form of AMP deaminase is produced. These results show that the purified enzyme (Mr = 83,000) is a truncated form of the full-length translation product. No adenine nucleotide binding sites were located based on the consensus sequence from other nucleotide binding proteins. No overall homology was found between yeast AMP deaminase and E. coli AMP nucleosidase. Although their metabolic roles and regulatory mechanisms are similar, these enzymes have arisen from separate ancestral proteins.

AMP Deaminase↗
PubMed1997

Redox modulation of the response of NADH oxidase activity of rat liver plasma membranes to cyclic AMP plus ATP.

NADH oxidase activity of rat liver plasma membranes was inhibited by low concentrations (1-100 nM) of ATP. The inhibition was amplified by addition of nanomolar concentrations (0.1-10) of cyclic AMP. The inhibition was complex and related to a marked increase in the Km for NADH at high NADH concentrations together with a concomitant decrease in the Vmax. In the absence of added or residual ATP, cyclic AMP was without effect. The response of cyclic AMP + ATP was inhibited by low concentrations of the selective inhibitor of cyclic AMP-dependent protein kinase, H-89 but not by staurosporin. The Vmax but not the Km was modified by treating the plasma membranes with a mild oxidizing agent, N-chlorosuccinamide, or with the reducing agent, dithiothreitol. In the presence of dithiothreitol, the Vmax was reduced by cyclic AMP + ATP. In contrast, in the presence of N-chlorosuccinamide, the Vmax was increased by cyclic AMP + ATP relative to cyclic AMP + ATP alone. Thus, the effect of cyclic AMP + ATP on the Vmax could be either an increase or a decrease depending on whether the membranes were oxidized or reduced. The results demonstrate regulation of NADH oxidase activity of rat liver plasma membranes through cyclic AMP-mediated phosphorylation by membrane-located protein kinase activities where the final response is dependent on the oxidation-reduction status of the plasma membranes.

Adenosine Triphosphate↗
PubMed2004

AMP-deaminase from normal and cirrhotic human liver: a comparative study.

AMP-deaminase (EC 3.5.4.6) is an enzyme of nucleotide breakdown involved in regulation of adenine nucleotide pool in mammalian cells. Reaction catalysed by AMP-deaminase constitutes a rate-limiting step in adenine nucleotide catabolism in liver. In this study kinetic and regulatory properties of AMP-deaminase purified from normal and cirrhotic human liver were investigated. In comparison to AMP-deaminase extracted from the normal human liver, AMP-deaminase extracted from the cirrhotic liver was less sensitive towards substrate analogues, and only a very limited response towards pH and adenylate energy charge changes tested for enzyme isolated from this tissue source had been observed. At physiological pH 7.0, in the absence and in the presence of important allosteric effectors (ATP, ADP, GTP and orthophosphate), AMP-deaminases from the two sources studied manifested different regulatory profiles, with half-saturation constant (S0.5) values being distinctly higher for the enzyme extracted from the pathological organ. In contrast to AMP-deaminase isolated from the normal, healthy liver, where presence of relatively large (68 kDa) protein fragment was also detected, only smaller protein fragments were identified, while SDS-PAG electrophoresis of AMP-deaminase isolated from the cirrhotic liver was performed. The obtained results indicate clearly that advanced proteolytic processes occurring in the cirrhotic liver may affect structural integrity of AMP-deaminase studied, making enzyme less active and less sensitive to regulatory action of important allosteric effectors.

AMP Deaminase↗
PubMed2000

Nitric oxide-dependent vasodilatation of rabbit femoral artery by beta(2)-adrenergic stimulation or cyclic AMP elevation in vivo.

Some studies suggest that beta-adrenoceptor-mediated vasorelaxation is in part mediated through nitric oxide (NO) release. We wished to determine the contribution of the L-arginine / NO system to vasodilatation in response to beta-adrenoceptor stimulation with isoprenaline or cyclic adenosine-3',5'-monophosphate (cyclic AMP) elevation with forskolin and dibutyryl cyclic AMP in vivo, using a rabbit femoral artery constant perfusion model. Baseline femoral artery pressure was similar in rabbits receiving isoprenaline, forskolin or dibutyryl cyclic AMP. Isoprenaline, forskolin and dibutyryl cyclic AMP each decreased femoral artery pressure in a dose-dependent manner. The doses (mol kg(-1)) of isoprenaline, forskolin and dibutyryl cyclic AMP which decreased pressure by 10% from baseline, expressed as a negative logarithm (-log ED(10)) were: 10.0+/-0.2, 9.5+/-0.1 and 4.9+/-0.1 respectively (P<0.0001 for each). Use of beta-adrenoceptor subtype-selective antagonists showed that the vascular response to isoprenaline was purely due to stimulation of the beta(2)-adrenoceptor subtype. Injection of 1 micromol kg(-1) N(G)-nitro-L-arginine methyl ester (L-NAME) did not alter baseline pressure. However, it abolished the pressure response to isoprenaline (P<0.0001), and significantly attenuated the pressure responses to forskolin and dibutyryl cyclic AMP: -log ED(10) values for forskolin and dibutyryl cyclic AMP, in the presence of L-NAME, were 7.9+/-0.1 and 3.5+/-0.3 respectively (P<0.0001 for each, as compared with values in the absence of L-NAME). These results indicate that beta(2)-adrenergic stimulation and cylic AMP elevation activate the L-arginine/NO system in rabbit femoral artery in vivo, and that NO generation contributes importantly to the changes in vascular tone induced by agents which modulate beta-adrenoceptors or cyclic AMP.

Adrenergic beta-2 Receptor Agonists↗
PubMed1984

Cyclic AMP-dependent protein phosphorylation and insulin secretion in intact islets of Langerhans.

Effects on insulin release, cyclic AMP content and protein phosphorylation of agents modifying cyclic AMP levels have been tested in intact rat islets of Langerhans. Insulin release induced by glucose was potentiated by dibutyryl cyclic AMP, glucagon, cholera toxin and 3-isobutyl-1-methylxanthine (IBMX); the calmodulin antagonist trifluoperazine reversed these potentiatory effects. Inhibition by trifluoperazine of IBMX-potentiated release was, however, confined to concentrations of IBMX below 50 microM; higher concentrations, up to 1 mM, were resistant to inhibition by trifluoperazine. IBMX-potentiated insulin release was also inhibited by 2-deoxyadenosine, an inhibitor of adenylate cyclase. In the absence of glucose, IBMX at concentrations up to 1 mM did not stimulate insulin release and in the presence of 3.3 mM-glucose IBMX was effective only at a concentration of 1 mM; under the latter conditions trifluoperazine again did not inhibit insulin secretion. The maximum effect on insulin release was achieved with 25 microM-IBMX. Islet [cyclic AMP] was increased by IBMX, with the maximum rise occurring with 100 microM-IBMX. The increase in [cyclic AMP] elicited by IBMX was more rapid than that induced by cholera toxin. Trifluoperazine did not significantly affect islet cyclic AMP levels under any of the conditions tested. When islets were incubated with [32P]Pi, radioactivity was incorporated into islet ATP predominantly in the gamma-position. The rate of equilibration of label was dependent on medium Pi and glucose concentration and at optimal concentrations of these 100% equilibration of internal [32P]ATP with external [32P]Pi required a period of 3h. Radioactivity was incorporated into islet protein and, in response to an increase in islet [cyclic AMP], the major effect was on a protein of Mr 15 000 on sodium dodecyl sulphate/polyacrylamide gels. The extent of phosphorylation of the Mr-15 000 protein was correlated with the level of cyclic AMP: phosphorylation in response to IBMX was inhibited by 2-deoxyadenosine but not by trifluoperazine. Fractionation of islets suggested that the Mr-15 000 protein was of nuclear origin: the protein co-migrated with histone H3 on acetic acid/urea/Triton gels. In the islet cytosol a number of proteins were phosphorylated in response to elevation of islet [cyclic AMP]: the major species had Mr values of 18 000, 25 000, 34 000, 38 000 and 48 000. Culture of islets with IBMX increased the rate of [3H]-thymidine incorporation.(ABSTRACT TRUNCATED AT 400 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1985

Basal phosphorylation of cyclic AMP-regulated phosphoproteins in intact S49 mouse lymphoma cells.

Protein phosphorylation in intact S49 mouse lymphoma cells was studied by using high-resolution two-dimensional gel electrophoresis of proteins labelled with [35S]methionine or [32P]Pi. In wild-type cells substrates for cyclic AMP-stimulatable phosphorylation exhibited high basal phosphorylation; in mutant cells deficient in activities of either cyclic AMP-dependent protein kinase or adenylate cyclase, basal phosphorylation of most of these substrates was negligible. Analysis of tryptic phosphopeptides from proteins labelled with [32P]Pi in wild-type cells suggested that identical sites were phosphorylated under conditions of both basal and hormonally elevated concentrations of cyclic AMP. These results argue that most basal phosphorylation is a consequence of partial activation of cyclic AMP-dependent protein kinase and that this activation is attributable to basal concentrations of cyclic AMP. For the intermediate filament protein vimentin, basal phosphorylation was largely at a site distinct from that stimulated by increased cyclic AMP, and basal phosphorylation was not markedly different in mutant and wild-type cells. Vimentin phosphorylated at both sites was not observed. Cyclic AMP treatment resulted in enhanced phosphorylation at the cyclic AMP-specific site and decreased phosphorylation at the cyclic AMP-independent site.

Adenylyl Cyclases↗
PubMed1986

The role of Ca2+ in steroidogenesis in Leydig cells. Stimulation of intracellular free Ca2+ by lutropin (LH), luliberin (LHRH) agonist and cyclic AMP.

The requirements of purified rat Leydig cells for intra- and extra-cellular Ca2+ during steroidogenesis stimulated by LH (lutropin), cyclic AMP analogues and LHRH (luliberin) agonist were investigated. The intracellular Ca2+ concentrations ([Ca2+]i) were measured by using the fluorescent Ca2+ chelator quin-2. The basal [Ca2+]i was found to be 89.4 +/- 16.6 nM (mean +/- S.D., n = 25). LH, 8-bromo cyclic AMP and dibutyryl cyclic AMP increased [Ca2+]i, by 300-500 nM at the highest concentrations of each stimulator, whereas LHRH agonist only increased [Ca2+]i by a maximum of approx. 60 nM. Low concentrations of LH (less than 1 pg/ml) and all concentrations of LHRH agonist increased testosterone without detectable changes in cyclic AMP. With amounts of LH greater than 1 pg/ml, parallel increases in cyclic AMP and [Ca2+]i occurred. The steroidogenic effect of the LHRH agonist was highly dependent on extracellular Ca2+ concentration ([Ca2+]e), whereas LH effects were only decreased by 35% when [Ca2+]e was lowered from 2.5 nM to 1.1 microM. No increase in [Ca2+]i occurred with the LHRH agonist in the low-[Ca2+]e medium, whereas LH (100 ng/ml) gave an increase of 52 nM. It is concluded that [Ca2+]i can be modulated in rat Leydig cells by LH via mechanisms that are both independent of and dependent on cyclic AMP, whereas LHRH-agonist action on [Ca2+]i is independent of cyclic AMP. The evidence obtained suggests that, at sub-maximal rates of testosterone production, Ca2+, rather than cyclic AMP, is the second messenger, whereas for maximum steroidogenesis both Ca2+- and cyclic-AMP-dependent pathways may be involved.

Animals↗
PubMed1987

The interaction between the adenylate cyclase system and insulin-stimulated glucose transport. Evidence for the importance of both cyclic-AMP-dependent and -independent mechanisms.

UNLABELLED: The counter-regulatory effect of adenosine, isoprenaline and selected cyclic AMP analogues on insulin-stimulated 3-O-methylglucose transport and insulin binding were studied in rat fat-cells. Isoprenaline alone had no consistent effect on glucose transport in the presence of maximally effective insulin concentrations. However, it decreased insulin binding by approx. 20% and increased EC50 (concn. giving 50% of maximal stimulation) for insulin from 8 +/- 1 to 17 +/- 2 mu units/ml. Adenosine deaminase (ADA) alone only exerted a slight effect, whereas isoprenaline and ADA in combination consistently decreased the maximal effect of insulin on glucose transport, decreased insulin binding by approx. 30% and markedly decreased insulin-sensitivity (EC50 61 +/- 8 mu units/ml). In cells from pertussis-toxin-treated animals, isoprenaline alone decreased the insulin response by approx. 75%, decreased insulin binding by approx. 45% and caused a marked rightward shift in the dose-response curve for insulin (EC50 103 +/- 34 mu units/ml). The importance of cyclic AMP for these effects was evaluated with the analogue N6-monobutyryl cyclic AMP, which is resistant to hydrolysis by the phosphodiesterase. The importance of phosphodiesterase activation by insulin was studied with 8-bromo cyclic AMP, which is an excellent substrate for this enzyme. N6-Monobutyryl cyclic AMP, in contrast with 8-bromo cyclic AMP, markedly impaired insulin-sensitivity (EC50 approx. 100 mu units/ml). However, the maximal effect of insulin was only slightly attenuated. IN CONCLUSION: (1) beta-adrenergic stimulation and cyclic AMP markedly alter insulin-sensitivity, but not responsiveness, mainly through post-receptor perturbations; (2) when cyclic AMP is increased phosphodiesterase activation by insulin is a critical step to elicit insulin action; (3) adenosine modulates the insulin-antagonistic effect of beta-adrenergic stimulation via Ni (inhibitory nucleotide-binding protein) through both cyclic-AMP-dependent and -independent mechanisms.

3-O-Methylglucose↗