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At least 253 records · Page 14Linked to original sources
PubMed1990

Thiol reagents (diamide and N-ethylmaleimide) inhibit increase in cAMP in response to glucose and abolish the clonidine-mediated attenuation of glucagon-induced cAMP formation in isolated rat pancreatic islets.

In freshly collagenase-isolated rat pancreatic islets and in islets cultured for 72 hours, the effects of thiol reagents on glucagon (5 micrograms/ml) and/or glucose (16.7 mM)-mediated increases in cAMP formation as well as on clonidine (10 microM)-induced inhibition of these actions were studied. In freshly isolated islets and to a more pronounced degree in islets cultured for 72 hours glucagon (5 micrograms/ml) increased the cAMP content above the basal value. Clonidine (0.1-100 microM) had no significant effect on the basal cAMP formation, but inhibited the glucagon-mediated effect. The thiol reagents diamide (10-100 microM) and NEM affected neither the basal nor the glucagon-mediated effect, but abolished the inhibitory action of clonidine on cAMP formation. In freshly isolated islets, high glucose concentrations (8.3-16.7 mM) increased the cAMP formation. Diamide (100 microM) and NEM (100 microM) attenuated the stimulatory effect of 16.7 mM glucose. It is suggested that these selective effects of the thiol reagents on glucagon-mediated increase in cAMP formation in the presence of substimulatory concentration of glucose may be due to the differences in the sensitivity of the sulfhydryl groups of the G-proteins to thiol reagents i.e. Gi or proteins closely related to Gi being more sensitive than Gs. The data further suggest that glucose acts on the cAMP cascade at a step distinct from Rs. Since both glucose and glucagon effects were influenced by the addition of clonidine, it is possible to interpret the data as indicating that the effects of both stimulators eventually converge at some common step in the adenylate cyclase cascade.

Animals↗
PubMed1990

Type II regulatory subunit of protein kinase restores cAMP-dependent transcription in a cAMP-unresponsive cell line.

cAMP-dependent protein kinase appears to play a role in cAMP-induced gene expression in mammalian cells. There exist two major types of cAMP-dependent protein kinase, type I and type II, which are distinguished by their regulatory subunits, RI and RII, respectively. We investigated the role of type I and type II protein kinase in the cAMP-induced gene expression by either stable or co-transfection of RI alpha, RII alpha, or RII beta gene in an expression vector together with somatostatin-chloramphenicol acetyltransferase (SS-CAT) fusion gene using a cAMP-unresponsive mutant pheochromocytoma cell line (A126-1B2). Introduction of the RII beta gene restored the capability of these cells to induce the SS-CAT gene expression in response to forskolin stimulus and induced a changed morphology which resembled that of wild type. The RII alpha gene also induced SS-CAT gene expression but to a lesser degree than that achieved by the RII beta gene, whereas the RI alpha gene had no effect. The induction of SS-CAT gene expression by the RII beta gene was specifically blocked by the 21-mer RII beta antisense oligodeoxynucleotide. These results show for the first time that type II but not type I regulatory subunit of cAMP-dependent protein kinase is essential for a cAMP-induced gene transcription.

Animals↗
PubMedDate not supplied

Human platelet aggregation and cAMP system--cAMP level, adenyl cyclase, phosphodiesterase.

The possibility of a direct and casual relationship between various parameters of the adenosine 3',5'-cyclic monophosphate (cAMP) system, e.g., the level of cAMP, adenyl cyclase activity and phosphodiesterase activity, and platelet aggregation was studied by measuring the effects of various environmental conditions, as well as metabolic inhibitors, aggregating agents and aggregation inhibitors upon these parameters. A competitive binding technique using 3H labeled cAMP was used to determine the level of cAMP in intact platelets, and a high performance liquid chromatographic method was developed to measure the adenyl cyclase and phosphodiesterase activities in the platelet membrane fraction. Although the availability of substrate adenosine triphosphate (ATP) correlated well with the amount of cAMP produced, and in turn the availability of cAMP seemed to have a direct effect upon the reversibility of shape changes induced by the various stimuli, the only effect upon aggregation concerned the extent to which it occurred. No direct correlation of the level of cAMP with either the actual inhibition or activation of the aggregation mechanism was observed.

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

cAMP-dependent transcription of the human CYP21B (P-450C21) gene requires a cis-regulatory element distinct from the consensus cAMP-regulatory element.

By utilizing chimeric genes constructed from 5'-flanking sequences of the human CYP21B (P-450C21) gene and reporter genes (chloramphenicol acetyltransferase or rabbit beta-globin), a 34-nucleotide sequence has been found to be required for cAMP-dependent transcription. This sequence (-129/-96 base pairs) shows no homology to that of the consensus (CRE) cAMP-regulatory element. Gel retardation analysis shows that a protein-DNA complex is formed between this DNA sequence and nuclear proteins from mouse adrenal Y1 tumor cells or bovine adrenal cortical cells or human fetal adrenal tissue and that formation of this complex cannot be competed by DNA containing the consensus CRE sequence. Even though cAMP-enhanced accumulation of P-450C21 mRNA in primary cultures of bovine adrenocortical cells is inhibited by the protein synthesis inhibitor, cycloheximide, reporter gene transcription enhanced by the cAMP-responsive -129/-96-base pair fragment of the human CYP21B gene is not. We conclude that cAMP-dependent transcription of the human P-450C21 gene (CYP21B), an event required for maintenance of optimal steroidogenic capacity in the adrenal cortex, involves a stable transcription factor(s) distinct from the CRE-binding protein. Furthermore the cAMP-dependent cis-regulatory element of the human P-450C21 gene is distinct from those found associated with the other steroid hydroxylase genes, 17 alpha-hydroxylase cytochrome P-450, cholesterol side chain cleavage cytochrome P-450, and 11 beta-hydroxylase cytochrome P-450, suggesting that each of these genes may require its own set of specific transcription factors for cAMP-dependent regulation.

Adrenal Cortex↗
PubMed1987

Epidermal growth factor activates steroid biosynthesis in cultured Leydig tumor cells without affecting the levels of cAMP and potentiates the activation of steroid biosynthesis by choriogonadotropin and cAMP.

The studies presented herein were designed to investigate the effects of mouse epidermal growth factor (mEGF) on steroid biosynthesis in a clonal strain of cultured murine Leydig tumor cells (designated MA-10). We show that in short-term incubations (up to 8 h), mEGF activates steroid biosynthesis without affecting cAMP levels. The maximal activation of steroid biosynthesis by mEGF (about 10-fold) is, however, much lower than the maximal activation detected with human choriogonadotropin (hCG) or cAMP analogues (about 1000-fold). We also show that mEGF has two (opposing) effects on the activation of steroidogenesis by hCG. Initially, it transiently attenuates the increase in intracellular cAMP and steroid biosynthesis provoked by submaximal concentrations of hCG. At later times, however, it potentiates the stimulatory effects of submaximal concentrations of hCG on steroid biosynthesis in a synergistic fashion. Last, we show that mEGF and submaximal concentrations of cAMP analogues also activate steroidogenesis in a synergistic fashion and that the degree of synergism attained with cAMP analogues plus mEGF is much higher than that attained with hCG plus mEGF. Taken together, our results show that mEGF (i) activates steroidogenesis without affecting cAMP levels and (ii) modulates the activation of steroidogenesis by the cAMP second messenger system.

1-Methyl-3-isobutylxanthine↗
PubMed1985

Identification and differential expression of two forms of regulatory subunits (RII) of cAMP-dependent protein kinase II in Friend erythroleukemic cells. Differentiation and 8-bromo-cAMP elicit a large and selective increase in the rate of biosynthesis of only one type of RII.

The concentration of regulatory subunits (R) of type II cAMP-dependent protein kinase increased 4- to 5-fold when Friend erythroleukemic cells were either grown in medium containing 0.5 mM 8-bromo-cAMP and 0.2 mM methylisobutylxanthine or stimulated to differentiate. Two species of RII with apparent Mr values of 54,000 (RII-54) and 52,000 (RII-52) are expressed in Friend cells. Both forms of RII were (a) covalently labeled with 8-N3-[32P]cAMP, (b) phosphorylated by the catalytic subunit of protein kinase II, and (c) complexed by polyclonal anti-RII IgGs. RII-52 and RII-54 were not interconverted by phosphorylation or dephosphorylation. A monoclonal antibody that recognizes an internal site in RII resolved the two cAMP-binding proteins by preferentially binding RII-54. The structural diversity suggested by the monoclonal antibody experiment was further examined by comparing two-dimensional maps of tryptic peptides obtained from metabolically labeled [( 35S]met) RII-52 and RII-54. Groups of 35S-labeled peptides that were either uniquely derived from RII-54 or obtained only from RII-52 were readily distinguished, thereby demonstrating that Friend cells produce two separate and distinct forms of type II cAMP-binding subunits. The relative rate of synthesis of RII-52 increased 12- to 14-fold during erythroid differentiation and treatment with 8-bromo-cAMP, while the rate of RII-54 synthesis either declined slowly or was unchanged. Thus, two homologous forms of RII are subject to different modes of physiological (differentiation) and pharmacological (chronic 8-Br-cAMP) regulation, and the accumulation of total RII observed in the present and previous (Schwartz, D. A., and Rubin, C. S. (1983) J. Biol. Chem. 258, 777-784) studies results from a selective increase in the rate of biosynthesis of RII-52.

1-Methyl-3-isobutylxanthine↗
PubMed1987

Effects of anagrelide on platelet cAMP levels, cAMP-dependent protein kinase and thrombin-induced Ca++ fluxes.

Anagrelide (BL-4162A, 6,7-dichloro-1,5-dihydroimidazo[2, 1-6] quinazolin-2[3H]one monohydrochloride hydrate) is a potent and broad spectrum inhibitor of platelet aggregation. Prior studies showed that anagrelide inhibited platelet cyclic AMP (cAMP) phosphodiesterase activity but did not appreciably elevate platelet cAMP levels. We examined the effects of anagrelide on washed human platelets and found that anagrelide caused significant elevation of cAMP levels. Anagrelide treatment also resulted in activation of the platelet cAMP-dependent protein kinase at anagrelide concentrations of 0.1 to 1 microgram/ml, which inhibited platelet aggregation but caused only small increases in platelet cAMP content. When whole platelets were incubated with radiolabeled phosphate, anagrelide increased phosphorylation of platelet proteins with relative molecular weights of 22, 26, 50 and 80 kilodaltons. The pattern of protein phosphorylation stimulated by anagrelide treatment was similar to that observed when the platelets were treated with forskolin. Anagrelide also inhibited the rise in intracellular Ca++ caused by thrombin, as measured using Fura-2-loaded platelets. The inhibition of increased intracellular Ca++ resulted from block of thrombin-induced mobilization of intracellular Ca++, as well as prevention of Ca++ influx through the plasma membrane. Anagrelide itself had no influence on inositol 1,4,5-trisphosphate-induced Caz5++ release from isolated platelet membrane vesicles. These studies suggest that anagrelide inhibits platelet phosphodiesterase activity in intact platelets resulting in an elevation in cAMP levels sufficient to activate the cAMP-dependent protein kinase and inhibit agonist-activated Ca++ fluxes.

Blood Platelets↗
PubMed1985

[Activity of cAMP-dependent protein kinases and cAMP-binding proteins from rat renal cytosol upon dehydration].

The activity of cAMP-dependent protein kinases, cAMP binding and the spectrum of cAMP-binding proteins in renal papillary cytosol of intact rats and of rats kept on a water-deprived diet for 24 hours were investigated. It was found that the stimulation of protein kinases by 10(-6) M cAMP in the experimental group was significantly higher than in the control one. On DEAE-cellulose chromatography, the position of peaks of the specific cAMP binding corresponded to those of the regulatory cAMP-dependent protein kinases type I and II. Under these conditions, more than 80% of the binding activity in intact animals was localized in peak II, whereas in rats kept on a water-deprived diet over 60% of the binding activity was localized in peak I. The total binding activity of cytosol in experimental animals remained unchanged is compared to intact rats. It is suggested that in renal papilla dehydration is accompanied by the induction of synthesis of regulatory subunits of cAMP-dependent protein kinase type I.

Animals↗
PubMed1985

Nerve growth cones isolated from fetal rat brain. II. Cyclic adenosine 3':5'-monophosphate (cAMP)-binding proteins and cAMP-dependent protein phosphorylation.

Cyclic adenosine 3':5'-monophosphate (cAMP)-binding proteins and cAMP-dependent protein phosphorylation were examined in growth cone particles (GCPs) prepared from fetal rat brain. Several major proteins which specifically bind a photoactivatable analogue of cAMP are observed in GCPs and correspond to isoelectric variants of the regulatory subunits of the cAMP-dependent protein kinase described in adult brain. We found no evidence for differential compartmentalization of specific cAMP-binding proteins in subcellular fractions of fetal brain or within GCPs. cAMP-stimulated phosphoproteins of GCPs are similar to cAMP-dependent protein kinase substrates characterized in nerve terminals (synaptosomes) of adult brain and include the nerve terminal-specific protein, synapsin I. However, as shown in the companion paper (Katz, F., L. Ellis, and K. H. Pfenninger (1985) J. Neurosci. 5: 1402-1411), this synaptic phosphoprotein is not the major kinase substrate in the GCP fraction. The finding of synapsin I in a subcellular fraction prepared from fetal brain suggests that components of the mature nerve terminal are already present in fetal brain during neuronal sprouting and prior to synaptogenesis.

Animals↗
PubMed1992

The cAMP-CRP/CytR nucleoprotein complex in Escherichia coli: two pairs of closely linked binding sites for the cAMP-CRP activator complex are involved in combinatorial regulation of the cdd promoter.

Transcription initiation at CytR regulated promoters in Escherichia coli is controlled by a combinatorial regulatory system in which the cAMP receptor protein (CRP) functions as both an activator and a co-repressor. By combining genetic studies and footprinting analyses, we demonstrate that regulated expression of the CytR controlled cdd promoter requires three CRP-binding sites: a high affinity site (CRP-1) and two overlapping low affinity sites (CRP-2 and CRP-3) centred at positions -41, -91 and -93, respectively. In the absence of CytR, cAMP-CRP interacts at one set of sites (CRP-1 and CRP-2) and both of these binding sites are required for full promoter activation. In the presence of CytR, however, the two regulators bind cooperatively to cddP forming a nucleoprotein complex in which cAMP-CRP binds to CRP-1 and CRP-3 and CytR occupies the sequence between these sites. Thus, association of the two regulators involves a repositioning of the cAMP-CRP complex. Moreover, mutant cdd promoters in which CRP-2 and CRP-3 have been deleted are partially regulated by CytR, and cAMP-CRP and CytR still bind cooperatively to these promoters. These findings provide clues to an understanding of how cAMP-CRP and CytR interact at a structurally diverse set of promoters.

Bacterial Proteins↗
PubMed1995

Okadaic acid, cAMP, and selected nutrients inhibit hepatocyte proliferation at different stages in G1: modulation of the cAMP effect by phosphatase inhibitors and nutrients.

The protein phosphatase inhibitor okadaic acid (> 100 nM) caused an abrupt and complete cessation of primary rat hepatocyte cell cycle progression at the restriction point in late G1. A decline in the G1/S transition rate was observed in response to elevated cAMP, excess selected nutrients, and okadaic acid (< 100 nM). Excess nutrients (40 mM glucose +/- 5 mM dihydroxyacetone) acted by imposing an incomplete block in early G1. The cAMP action was potentiated by the phosphatase inhibitor microcystin, which in itself did not affect DNA replication. This suggests that cAMP acted by phosphorylating substrate(s) that is dephosphorylated by a microcystin-sensitive phosphatase. The additive effects of submaximal concentrations of okadaic acid and cAMP analogs indicated that okadaic acid and cAMP acted via different pathways. In conclusion, okadaic acid, cAMP, and excess nutrients, acting through distinct pathways, inhibited hepatocytes in different parts of the G1 phase.

Animal Nutritional Physiological Phenomena↗
PubMed2006

Effect of dibutyryl cAMP on the cAMP content, meiotic progression, and developmental potential of in vitro matured pre-pubertal and adult pig oocytes.

Pre-pubertal pig oocytes display reduced developmental competence compared with adult oocytes following in vitro maturation (IVM). Exposure to dibutyryl cyclic adenosine monophosphate (dbcAMP) for the first 20 hr IVM improves development of pre-pubertal oocytes, suggesting that their cAMP content may be inadequate. This study examined the effect of 1 mM dbcAMP treatment for the first 22 hr of IVM on the cAMP content, meiotic progression, and embryo development of pre-pubertal and adult oocytes. In control groups, a two-fold increase in cAMP was observed in adult oocytes after 22 hr IVM, with no change in pre-pubertal oocyte cAMP content. At 22 hr IVM, dbcAMP treatment resulted in two- and five-fold increases in pre-pubertal and adult oocyte cAMP, respectively. After 22 hr control IVM, a greater proportion of pre-pubertal oocytes occupied metaphase I (MI) compared with adult oocytes (69% vs. 49%). dbcAMP treatment reduced the proportion of pre-pubertal and adult oocytes in MI stage at 22 hr. Despite dbcAMP treatment, the proportion of pre-pubertal oocytes in the MI stage at 22 hr remained higher than that of adult oocytes. In control groups, adult oocytes displayed a greater ability to form blastocysts compared with pre-pubertal oocytes following either parthenogenetic activation (59% vs. 25%) or in vitro fertilization (IVF) (47% vs. 19%). dbcAMP treatment increased subsequent blastocyst formation rates of pre-pubertal oocytes, whereas blastocyst formation rates of adult oocytes remained unchanged. Our results suggest that the reduced developmental capacity of pre-pubertal oocytes may be a consequence of their reduced ability to accumulate cAMP during IVM.

Animals↗
PubMed1990

The interaction of adenosine analogues with cAMP-generating and cAMP-independent positive inotropic agents in rabbit left atrium.

The effects of adenosine receptor stimulation on the contractile force of rabbit isolated left atrial preparations in the absence and presence of cAMP-generating and cAMP-independent agonists were investigated. Adenosine and the stable adenosine analogues 5'-(N-ethyl)carboxamido adenosine (NECA) and (-)-N6-phenylisopropyladenosine (R-PIA) produced a concentration-dependent direct negative inotropic effect. Responses to NECA and R-PIA were insensitive to atropine and were shifted to the right by the adenosine receptor antagonist 3-isobutyl-1-methyl xanthine (IBMX). NECA and R-PIA were found to reverse positive inotropic responses of left atria to the beta-adrenoceptor agonist, isoproterenol, but were less effective at reversing positive inotropic responses to the adenylate cyclase activator, forskolin, and were almost ineffective at reversing positive inotropic responses to alpha-adrenoceptor stimulation. Neither NECA nor R-PIA had a significant effect on basal cAMP levels or on cAMP levels elevated by isoproterenol in rabbit left atria. Similarly, R-PIA had no significant effect on basal cAMP levels or isoproterenol-induced increases in cAMP in the presence of adenosine deaminase to remove the influence of endogenous adenosine. Pretreatment of rabbits with 1.75 micrograms/kg pertussis toxin attenuated both the direct negative inotropic response of left atria to NECA and responses to NECA in the presence of isoproterenol and forskolin to a similar extent. Pretreatment of left atrial preparations with the potassium channel antagonist 4-aminopyridine resulted in a dose dependent attenuation of responses to NECA alone and in the presence of isoproterenol and forskolin. These data suggest that adenosine receptors in rabbit left atria are not coupled to adenylate cyclase.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1992

Ablation of stimulation of a cAMP-responsive promoter in CHO cell lines defective in their cAMP-dependent protein kinase system.

We have studied the requirement for an intact cAMP-dependent protein kinase (PKA) system to regulate cAMP-mediated gene transcription in Chinese hamster ovary (CHO) cells. Wild-type CHO cells and mutant CHO cell lines selected for their resistance to the growth inhibitory effect of 8-Br-cAMP and defective in their PKA system were transiently transfected with reporter plasmids containing 2.5 and 3.0 kb of the 5'-flanking sequence of the rat tyrosine aminotransferase (TAT) gene promoter. This segment of DNA contains no CRE-like sequences, yet wild-type transfectants exhibited a specific increase in TAT promoter activity following growth in medium containing 8-Br-cAMP. In CHO cell lines defective in their PKA, the transfected TAT promoter failed to respond to cAMP treatment. We conclude that an intact PKA system is necessary for the cAMP-mediated increase in TAT promoter activity in CHO cells and that there is no requirement for a CRE to see this effect.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

Catalytic subunit of cAMP-dependent protein kinase is essential for cAMP-mediated mammalian gene expression.

Cyclic AMP-stimulated mRNA levels in cultured rat hepatocytes were inhibited by three different inhibitors of cAMP-dependent protein kinase activity: (i) Rp-cAMPS, a cAMP analog with a sulfur substitution at the equatorial oxygen of the cyclic monophosphate; (ii) H8, an isoquinoline sulfonamide derivative; and (iii) PKI, a 20-amino acid synthetic peptide of the Walsh protein kinase inhibitor. These inhibitors specifically blocked the cAMP-stimulated increase in mRNA for tyrosine aminotransferase and phosphoenolpyruvate carboxykinase; they had no effect on the level of albumin mRNA which is not cAMP regulated. These results provide functional evidence that kinase activity involving protein phosphorylation is required in cAMP-mediated gene expression in mammalian cells.

Animals↗